lipase and phospholipase protocols

1 For a PLC assay, add 10 pMN-pyrene-PEIBLPG polymerized mixed hposomes m 2 mL of 10 mM MES buffer, pH 6.0,O 1 nuI4 ZnCl,, 10 ti CaCl, to a quartz cuvet For PLD assays, use 10 @IN-pyrene

1

-

Phospholipase A2 and Phosphatidylinositol-

Specific Phospholipase C Assays by HPLC

and TLC with Fluorescent Substrate

H Stewart Hendrickson

1 Introduction

Llpolytic enzymes have traditlonally been assayed by radlometrlc and t&-l- metric methods (I) RadIometrIc methods are quite sensitive but require ex- pensive radlolabeled substrates and tedious separation of labeled substrate and products In addition, the safe use of radioactive materials 1s of mcreasmg con- cern Tltrlmetrtc assays are contmuous and quite straightforward and use natu- ral substrates but suffer from low sensltlvlty and are subject to condltlons that may alter the amount of free hydrogen Ions released Fluorescence-based assays have sensltlvltles that approach those of radtometrlc methods; although they require synthetic fluorescent-labeled substrates, they are often more conve- nient and rapid For a recent review, see Hendrickson (2)

We first used dansyl-labeled glycerol ether analogs of phosphatidylcholme

as substrates for the assay of enzymes of the platelet-actlvatmg factor (PAF) cycle m peritoneal polymorphonuclear leukocytes (3) This became a general method for the assay of phosphollpase A2 (PLA,) (see Fig 1; refs 4 and 5) This method can be modified to assay other enzymes of the PAF cycle such as lyso-PAF acyltransferase, lyso-PAF acetyltransferase, and PAF acetylhydro- lase (3) Smce the probe remains attached to the glycerol backbone, simulta- neous assay of all of these enzymes IS possible

The method described here for the assay of PLA2 uses thin-layer chroma- tography (TLC) to separate products from substrate, and quantltatlon by fluo- rescent scanning The use of high-pressure hquld chromatography (HPLC) with fluorescence detection IS included as an alternative to TLC The assay IS spe-

From Methods m Molecular &o/ogy, Vol 109 Lipase and Phospholipase Protocols

Edlted by M H Doohttle and K Reue 0 Humana Press Inc , Totowa, NJ

I

Fig 1 Reactron scheme for the assay of PLA, with dansyl-PC

cific for PLA2 since the substrate, with an ether lmkage at the sn-1 position of glycerol, ts not hydrolyzed by PLA, Hydrolysis of the substrate by phosphoh- pases C or D present m crude enzyme preparattons will be apparent as addi- tional products that will be seen by TLC or HPLC

Phosphattdylmosttol-specific phospholipase C (PI-PLC) from Bacdlus cerexs catalyzes the hydrolysis of PI to a diglycertde and 1 o-myo-inositol- 1,2- (cychc)phosphate (6) The latter is subsequently slowly hydrolyzed by the same enzyme to 1 o-myo-mosttol- l-phosphate This enzyme also catalyzes the release

of a number of enzymes linked to glycosylphosphatidylmosttol membrane an- chors (7)

Several years ago we synthestzed 4-(l-pyreno)butylphosphoryl-l-myo- mosttol (pyrene-PI) as a substrate for the assay of PI-PLC from B cereu~ (see Fig 2; refs 8 and 9) The method described here uses reverse-phase HPLC wtth fluorescence detectton to separate and quantitate the product released The methods described here are well suited to the assay of crude enzyme preparattons since the presence of other phospholtpase acttvmes will be appar- ent The methods are independent of the specific condtttons for the enzyme reaction, so a variety of detergents can be used These assays can also be auto- mated by the use of an autosampler for HPLC and larger plates wtth multiple

Fluorescence-Based Phospholipase Assays

, PI-PLC

I

H,CH2CH&H20H

H&

po3=

Fig 2 Reactlon scheme for the assay of PI-PLC wrth pyrene-PI

lanes for TLC; they can thus be used to screen many enzyme samples and potential mhibltors The TLC-based assays can done m a qualitative manner

by simply vlsuahzing the plates under an ultraviolet (UV) lamp

2 Materials

2.1 PL A2 Assay

1 PLA, buffer 0.395 M NaCl, 66 nuW Tns, 13.2 mi!4 CaCl,, pH 7 0 (adjust with HCl)

2 PLA, substrate dansyl-PC, 1 n-J4 In CHCl, Dissolve 1 mg of dansyl-PC (cat

no D-3765, Molecular Probes, Inc , Eugene, OR) m 1 23 mL of chloroform (see Note 1) Store m a brown bottle at -20°C (see Note 2)

3 Trlton X-100 solutlon, 10 mM Triton X-100 (cat no T9284, Sigma, St LOUIS, MO) m water

4 PLA, stock assay solution place 100 pL of PLA, substrate (1 mMdansyl-PC) m

a small test tube (10 x 75 mm) and dry under a stream of mtrogen and then under high vacuum for 10-15 mm to remove all traces of solvent Add 20 pL of Trlton X-100 solution (see Note 3) and 380 pL of PLAz buffer Vortex and somcate (bath-type somcator) repeatedly until the lipid 1s completely dissolved and the solution is optlcally clear

4 TLC solvent* CHC13/CH30H/conc ammoma/water (90.54 5.5:2 [v/v])

5 TLC plates 10 x 10 cm, HPTLC plates, (cat no 60077, Analtech, Newark, DE)

6 Fluorescence scanner: densitometer (model CS9000, Shlmadzu) with fluorescence accessory, or other sultable Instrument for fluorescence scanning of TLC plates

7 Quenching solvent* hexane/lsopropanol/acetlc acid (6:8: 1.6)

8 PLA, HPLC solvent hexane/lsopropanol/water (6 8.1.6)

4 Hendrickson 2.2 PI-PLC Assay

1 PI-PLC buffer 50 mM2-(N-morpholmo)ethane sulfomc acid (MES, Sigma), pH

7 0 (adjust with NaOH)

2 PI-PLC substrate 1 tipyrene-PI m CHCls/CH,OH (2.1) Dissolve 0 5 mg of racemic 4-( 1 -pyreno)butylphosphoryl- 1 -myo-mositol (cat no P-3764, Molecu- lar Probes) m 1 25 mL of solvent Store m a brown bottle at -20°C (see Note 2)

3 PI-PLC stock assay solution place 250 pL of PI-PLC substrate (1 mM pyrene- PI) m a small test tube (10 x 75 mm) and dry under a stream of nitrogen and then under high vacuum for 10-15 min to remove all traces of solvent Add 200 yL of PI-PLC buffer Vortex and sonicate (bath-type somcator) repeatedly until the lipid is completely dissolved and the solution is clear

4 PI-PLC HPLC solvent 5 mM tetrabutylammonmm dihydrogenphosphate (cat

no 26,8 10-0, Aldrich, Milwaukee, WI) in acetomtrile/methanol/water (70 10.20)

2.3 HPLC Analysis

1 HPLC column for PLA, assay 15 cm x 4.6 mm, 5 pm spherical silica gel (cat

no 85774, Waters, Milford, MAtprotect with a guard column

2 HPLC column for PI-PLC assay’ 25 cm x 4.6 mm 5 pm Spherisorb ODS (cat no

583 12, Supelco, Bellefonte, PA) protect with a guard column (see Note 4)

3 Fluorescence detector Kratos model 980 or other suitable detector

4 HPLC mstrument with autosampler (optional) and mtegrator/recorder

0 2 rnA4 Triton X-100, 5 mMCaC12, 25 mMTris-HCl, pH 7 0

2 At various times over a period of 30-60 mm, remove 5-pL aliquots Spot directly

on a TLC plate for TLC analysis, or add to 90 pL of quenchmg solvent m a 500~pL microcentrifuge tube for HPLC analysis (vortex)

3 For TLC analysis dry the spots and develop the TLC plates m TLC solvent After the solvent has evaporated, scan the plates with a fluorescence densitometer (set excitation at 256 nm; measure emission above 400 nm [cutoff filter]) The Rr val- ues for dansyl-PAF and lyso-dansyl-PAF are 0 35 and 0 15, respectively

4 For HPLC analysis centrifuge the quenched samples at top speed m a rmcrocenmfuge for several minutes to remove any precipitated protein Equilibrate the s&a gel HPLC col- umn in PLAZ HPLC solvent at a rate of 1 ml/r&n Set the fluorescence detector at excna-

hOn 256 nrn and emission >4 18 nm (cutoff filter) InJect 20 pL of sample onto the column, Elution times for dansyl-PC and lyso-dansyl-PC are about 6 and 19 min, respectively

5 Calculation of acttvtty the mol fraction of product released is determined by dividmg the area of the lyso-dansyl-PC peak by the sum of the areas of the dansyl-

Fluorescence-Based Phospholipase Assays 5

PC and lyso-dansyl-PC peaks Thts value times the mmal amount of substrate present in the assay (0 01 pmol) equals the amount of product released Plot the pmol of product released vs time to determine the mitral lmear rate (acttvtty, umol/min)

3.2 Pi-PLC Assay

1 Add 5 pL of PI-PLC (contammg the equivalent of 0 2-4 ng of pure enzyme (cat

no P-6466, Molecular Probes) (see Notes 5 and 6) to 20 pL of PI-PLC stock assay solutton m a 500~uL microcentrifuge tube (vortex) Incubate at room tem- perature Fmal concentratton of substrate, 1 mM

2 At vartous times over a period of l&30 mm remove 5-uL altquots and dilute with 95 uL of PI-PLC HPLC solvent in 500~pL microcentrifuge tubes (vortex) Centrifuge these samples at top speed in a microcentrtfuge for several minutes to remove any prectpttated protein (see Note 7)

3 Equilibrate the ODS reverse-phase HPLC column m HPLC solvent at a rate of

1 mL/mm (see Note 4) Set the fluorescence detector at excitation 343 nm and emission >370 nm (cutoff filter) Inject 20 PL of sample onto the column Elu- tion times for pyrene-PI and pyrenebutanol are 2 4 and 6.2 mm, respectively

4 Calculatton of activity the mol fraction of product released is determmed by dtvtdmg the area of the pyrenebutanol peak by the sum of the areas of the pyrene-

PI and pyrenebutanol peaks This value ttmes the untial amount of substrate present m the assay (0 025 pmol) equals the amount of product released Plot the pmol of product released vs time to determme the mttial linear rate (activity, pmol/min)

4 Notes

Dertvatives of dansyl-PC are useful in the assay of other enzymes of lipid metaboltsm Lyso-dansyl-PC and dansyl-PAF (cat no D-3766 and D-3767, respectively, Molecular Probes) can be used as substrates m assays of lyso-PAF acyltransferase, lyso-PAF acetyltransferase, and PAF acetylhydrolase (3) This solution 1s stable for a year or more tf protected from moisture and stored m

a brown bottle at -20°C Before use, warm to room temperature and make sure the lipid ts completely dissolved

Snake venom PLA, IS quite active in the presence of Trtton X- 100, but other PLA2s (parttcularly pancreattc PLA2) may be less active with this detergent The pancreatic enzyme is best assayed using sodium cholate as a detergent Hexa- decylphosphocholme (cat no H6722, Stgma) IS also a good detergent for other phosphohpases The concentration of substrate and the ratto of substrate to deter- gent may be varied as destred The spectfic activity of pure snake venom PLA, in this assay with dansyl-PC is about 13 ymol/mm/mg

Do not leave the reverse-phase ODS column in PI-PLC HPLC solvent (wtth tetrabutylammonmm dthydrogen phosphate) for any length of time without sol- vent running through; if so, the column will become plugged After use, tmmedt- ately wash the column with CHsOH/H,O (80.20)

With pure B cereus PI-PLC, the specific activity m this assay wtth pyrene-PI is about 60 pmol/mm/mg, about 4 % hydrolysis IS observed m 1 mm wtth 4 ng of pure enzyme

R, values for TLC of pyrenebutanol and pyrene-PI on silica gel plates 0.75 and

0 0, respectively, m CHCl,/CH,OH (95 5), 1 0 and 0 25, respectively m CHCl,/ CH,OHIH,O (65 35 3)

4 Hendrickson, H S , Kotz, K J , and Hendrtckson, E K (1990) Evaluation of fluorescent and colored phosphatidylcholme analogs as substrates for the assay of phosphohpase A, Anal Bzochem 185,80-83

5 Hendrickson, H S (1991) Phosphohpase A, assays with fluorophore-labeled lipid substrates Methods Enzymol 197,9&94

6 Bruzik, K S and Tsar, M -D (1994) Toward the mechamsm of phosphomosttide- spectfic phospholtpase C Bloorg Med Chem 2,49-72

7 Low, M G and Saltiel, A R (1988) Structural and functional roles for glycosylphosphattdylmositol m membranes Sczence 239,268-275

8 Hendrickson, E K , Johnson, J L., and Hendrickson, H S (1991) A fluorescent substrate for the assay of phosphattdylmosnol-specific phosphohpase C 4-( l- pyreno)butylphosphoryl- 1-myo-inositol Bloorg Med Chem Lett 1,6 19-622

9 Hendrickson, H S , Hendrickson, E K , Johnson, J L , Khan, T H , and Chlal, H

J (1992) Kinetics ofB cereus phosphatldylmositol-specific phospholtpase C with thiophosphate and fluorescent analogs of phosphatidylmosnol Bzochemlstry 3 1, 12,169-12,172

2

Fluorometric Phospholipase Assays Based

on Polymerized Liposome Substrates

Wonhwa Cho, Shih-Kwang Wu, Edward Yoon

and Lenka Lichtenbergova

Among the vartous assays used for the different classes of phospholt- pases (2), those based on fluorometry (3-7) serve as the most sensttrve type

of contmuous assay However, a maJority of current fluorometrtc methods rely on the use of synthetic phospholiptds containmg a fluorophore at the

~2-2 posrtion of the phospholtptd that restricts then use to the assay of PLA* In order to measure the activity of many types of phospholtpases, we have recently developed a novel fluorometrrc assay utlhzmg polymertzed mixed lrposomes (7)

Polymerized mixed hposomes are composed of a pyrene-labeled phospho- hptd monomer Inserted mto a polymertzed phospholtptd matrix, the pyrene- labeled phospholiprd constitutes only a small mole percentage of the total polymerized mixed hposome substrate In this system, the polymerized matrix

is essentially inert, and only the monomer (unpolymerrzed) inserts are selec- tively hydrolyzed by the phosphohpase (8) For example, the PLA2 actrvtty

From Methods m Molecular Biology, Vol 109 Lfpase and Phospholfpase Protocols

Edlted by M H Dooltttle and K Reue D Humana Press Inc , Totowa, NJ

7

Cho et a/

Fig 1 SchematIc lllustratlons of fluolometrlc assays usmg polymerized mixed hposomes for PLA, (A) and PLC and PLD (B) F indicates a fluorescent label, such as pyrene, attached to either the acyl cham (A) or the polar head group (B)

assay utlhzes a polymerized mixed hposome substrate that IS composed of l- hexadecanoyl-2-( I-pyrenedecanoyl)-srt-glycero-3-phosphoglycerol (pyrene-PG) inserted into a polymerized matrix of 1,2-bls[ 12-(hpoyloxy) dodecanoyll-sn- glycero-3-phosphoglycerol (BLPG) The pyrene-PG Insert contams a pyrenedecanoyl fatty acid at the sn-2 posltlon, with the fluorescent pyrene molecule located at the methyl-termmal end of the fatty acid (see Fig 1A); the fluorescence emtsslon Intensity of the pyrene moiety ts largely quenched by the lipolc acid moieties of the BLPG molecules Using this substrate, PLA2 hydrolysis can be contmuously monitored by measuring an increase m pyrene fluorescence, since the hydrolyzed pyrene labeled-fatty acids are no longer quenched as they are removed from the llposome sur- face by serum albumin m the reaction mixture (see Fig 1A) Alternatlvely, for measurement of PLC and PLD hydrolysis, a new Insert IS used where the phosphollpld monomer contams a pyrene-labeled head group; agam, quenchmg of the pyrene moiety by the polymerized phosphollpld matrix IS lost as the hydrolyzed pyrene moiety readily diffuses away from the llpo- some surfaces (see Fig 1B) Thus, polymerized mixed llposomes serve as

an extremely versatile assay system for different phosphollpases, since one can readily modify the structure of the polymerized matrix, and the pyrene- labeled phospholipid inserts, to create an ideal combmatlon of llposome surfaces and hydrolyzable substrates for a specific phosphollpase Herein,

we describe the synthesis of polymerlzable phosphollplds and pyrene- labeled phospholiplds, the preparation of polymerized mixed llposomes from these synthesized products, and the use of the prepared llposomes as substrates to fluorometrlcally monitor phosphollpase hydrolysrs

Polymerized Liposome Substrates 9

2 L-a-glyceropl~ospl~orylchohne, 1 I cadmmm chloride adduct (Sigma, St Louts, MO)

3 Cabbage PLD IS prepared as follows The Inner ltght-green leaves of a Savoy cabbage (1 Kg) IS homogemzed wtth 200 mL of cold water for 5 mm The homo- genate IS filtered and the filtrate IS centrifuged at 20,OOOg for 30 mm The super- natant IS kept at 55 “C for 5 mm and placed on tee for 5 mm The prectpttate 1s removed by centrtfugatton (20,OOOg for 30 min), the supernatant 1s cooled to 0°C and 2 vol of acetone cooled to -15°C is added to the supernatant After 10 mm, the prectpttate containmg the active PLD IS collected by centrifugatton (20,OOOg for 30 mm) and used for transphosphattdylatton The acetone power can be stored

at -20°C for several months

4 PLD reaction buffer 0 1 M sodium acetate buffer, pH 5 6, 0 1 A4CaC12

2.2 Synthesis of Pyrene-Labeled Phospholipid Monomers (In- serts) Specific for the Measurement of Cytosolic PLA2 Activity

1 lo-(1-Pyreno)decanotc actd (a k a l-pyrenedecanotc actd) (Molecular Probes, Eugene, OR)

2 Ltthtum alummum hydrtde, pyridme, mesyl chloride, sodtum hydrtde, 2,3- tsopropyltdene-sn-glycerol, phosphorus oxychlortde, trtethylamme (Aldrrch, Mtlwaukee, WI)

3 Cholme tosylate, arachtdomc actd (Sigma, St Louis, MO)

2.3 Preparation of the Polymerized Mixed Liposome Substrate

1 Macro extruder Ltposofast (Avestm, Ottawa, Canada) with 0.1 -mm polycarbon- ate filter (M&pore, Bedford, MA)

2 Polymenzatton buffer, 10 mMTris-HCI, pH 8.4,O 16 M KC1

3 Sephadex G-SO (Pharmacta, Uppsala, Sweden)

4 Mobile phase for Sephadex G-50, 10 mM HEPES buffer, pH 7.4 , 0.16 KC1

2.4 Kinetic Measurements

1 Spectrofluorometer equtpped with a thermostated cell holder and a magnettc stnrer

2 1-Hexadecanoyl-2-( 1 -pyrenedecanoyl)-sn-glycero-3-phosphocholme (pyrene-PC), -phosphoethanolamme (pyrene-PE),-phosphoglycerol (pyrene-PG) (Molecular Probes)

3 N-( I-pyrenesulfonyl)-egg phosphattdyl ethanolamme (N-pyrene-PE) (Avanti, Alabaster, AL)

4 Fatty acid-free bovine serum albumin (BSA) (Bayer, Kankakee, IL)

70 Cho et al

5 Secretory PLA2 assay buffer 10 mMHEPES, pH 7 4,0 16 M KCI, 10 mMCaCl,, and 10 mMBSA

6 Cytosohc PLAz assay buffer 10 mMTns-HCI, pH 8.0,1 mA4CaCl,, and 10 mMBSA

7 PLC assay buffer 10 mMMES buffer, pH 6 0,O 1 n&‘ZrCl,, and 10 mA4CaClz

8 PLD assay buffer 10 mA4HEPES, pH 7 4,0 16 M KCl, and 10 mM CaClz

3.7.1 Synthews of 12-(Tetrahydropvranyloxy)Dodecanoic Acid (TCA)

1 Suspend 10 mmol (2 16 g) of 12-hydroxydodecanolc acid m 20 mL of dry tetrahydrofuran

2 Add 17 mm01 (1 39 g) of 3,4-dihydro-2H-pyran and stir the mixture for 10 mm at room temperature

3 Add 0 1 mm01 (20 mg) of crystallme p-toluenesulfomc acid monohydrate and stir the mixture for an addItIona 2 h at room temperature

4 Evaporate the solvent and purify the product by slllca gel column chromatogra- phy usmg chloroform/methanol (20 1, v/v) as eluent Check the product by thm- layer chromatography (TLC) (R,= 0 5 with the same solvent) Completely remove the solvent zn vacua

3.72 Synthesis of 1,2-Bis(lZ-Hydroxydodecanoyl)-sn-

Glycero-3-Phosphocholine

1 Dissolve the purified 12-(tetrahydropyranyloxy)dodecanolc acid (TCA) m dry dtchloromethane (15 mL) and add DCC (2.6 mmoU4 mm01 of TCA) dissolved m dry duzhloromethane

2 Stir the reactlon mixture at room temperature until it gets cloudy (approx 5 mm), then add L-a-glycero-phosphorylcholme (1 mmol/4 mmol of TCA) and DMAP (2 6 mmol/4 mm01 of TCA) Stir the mixture at room temperature overnlght

3 Filter the reactlon mixture to remove dlcyclohexylurea, evaporate the solvent and purify the product by slhca gel column chromatography using chloroform/ methanol (5 1) first and then chloroform/methanol/water (65 25.4) Check the product by TLC Rf= 0 35 with chloroform/ methanol/water (65 25:4)

4 Dtvlde the product mto about 200 mg fractions, dissolve each fraction m metha- nol (2 mL) and add equlmolecular crystallme p-toluenesulfomc acid monohy- drate Stir the mixture at room temperature for approx 1 5 h while checking the progress of the reactlon every 15 mm by TLC usmg chloroform/methanol/water (4 5 1) as developmg solvent (Rf = 0 3 for the product) The reaction must be completed within 2-2 5 h, otherwlse the hydrolysis of acyl groups of the phos-

Polymerized 1 iposome Substrates Ii

phohptd ~111 occur to a constderable degree Use the crude product lmmedrately for the next step

3.1.3 Synthesis of 1,2-Bls[lZ-(lipoyloxy)Dodecanoyl]-sn-Glycero-3-

Phosphocholine (BL PC)

1 Dtssolve 6,8-drthrooctanotc acid (5 mmol/l mmol of TCA) m dry dtchloro- methane and add DCC dtssolved in dry dichloromethane (3 mmoli5 mmol of 6,8- dtthtooctanorc acid)

2 Stir the reactton mtxture at room temperature untrl tt gets cloudy (approx 5 mm), then add the crude product of the above reactton drssolved m dry dtchloromethane and DMAP (2 mmol/l mmol of TCA) Star the mixture overmght m the dark at

-20°C m the dark

3.7.4 Synthesis of BLPG

1 Suspend 50-100 mg of BLPC m ethyl ether (1 5 mL) m a small glass vial

2 Add 0 6 g of glycerol to 3 mL of 0 1 M sodium acetate buffer, pH 5 6, 0 1 A4 CaCl,, combme tt with the above BLPC solutton and vortex the mixture brrefly

3 Add approx 100 mL of cabbage PLD (approx 1 mg/ml) to the reactton mtxture and stir the mixture vrgorously for 1 5 h at room temperature

4 Evaporate ether from the reaction mixture and extract the aqueous layer wtth

chloroform Dry the orgamc layer over anhydrous sodmm sulfate and evaporate

the solvent In vacua

S Purify the product by sthca gel column chromatography using CHCl,/CH,OH (3 1) first and then chloroform/methanol/water (40 10.1)

6 1,2-bts[ 12-(lipoyloxy)-dodecanoyl]-sn-glycero-3-phosphatldlc acid (BLPA) can

be synthesized by performing the PLD reaction m the absence of glycerol 3.2 Synthesis of Pyrene-Labeled Phospholipid Monomers

(Inserts) Specific for the Measurement of Cytosolic PLA2 Activity

Cytosolrc PLA, (cPLA,) has htgh specificity for the arachidonyl motety m the m-2 posrtron of phospholtptds (20, II) Furthermore, cPLA, has both PLA, acttvtty and lysophosphohpase acttvrty and, as a result, tt hydrolyzes both sn- 1 and m-2 acyl esters tf 1,2-dtacyl-sn-glycero-3-phosphohptds are used as a sub- strate Thus, a pyrene-labeled substrate for cPLA, must contain ~12-2 arachtdonyl ester and a nonhydrolyzable group at sn- 1 posttton l-O-( 1 -pyrenedecyl)-2- arachrdonyl-sn-glycero-3-phosphocholine (PAPC) IS a synthetic phospholtptd

12 Cho et al that meets this reqmrement PAPC IS prepared from lo-(I-pyreno)decanolc acid by a five-step synthesis

3.2.1 Synthesis of IO-( 1 -Pyreno)Decanol

Convert IO-( 1-pyreno)decanotc acid to an ethyl ester by refluxmg tts ethanol solutton for 4 h wtth 1 drop of concentrated HCI

Dilute the mtxture with chloroform and wash the organic layer with 1 M solution

of sodmm bicarbonate

Dry the organic layer over anhydrous sodmm sulfate and evaporate the solvent Dtssolve the product m dry tetrahydrofuran, place the mixture on ice, add llthlum alummum hydride and stir the mtxture at 0°C for 30 mm and then at room tem- perature for 2 h

Stop the reaction by coolmg the mrxture on Ice and adding water to the mtxture Extract the mixture wnh chloroform and purtfy IO-( 1 -pyreno)decanol by stltca gel column chromatography using hexane/ethyl acetate (4.1, [v/v])

3 2 2 Synthesis of 1 O-( I- Pyreno) Decanyl Mesyiate

1 Drssolve IO-( I-pyreno)decanol m 5 mL of dry pyrtdme and 5 ml of hexane Stn the mixture m a 25°C water bath and slowly add mesyl chloride (2 mmol/l mm01

4 Dry the organic phase over anhydrous sodmm sulfate and evaporate the solvent Purify the product by sthca gel column chromatography usmg hexaneiethyl acetate (2.1, v/v)

Polymerized Liposome Substrates

Add choline tosylate (1 8 mmol) and stir the mixture at 0°C for another 30 mm Check the progress of reaction by TLC, R, = 0 5 for the product with chloroform/

methanol/water (40 10 1, [v/v/v])

Quench the reaction by adding water and stirring the mixture at room tempera- ture for IO mm

Evaporate the solvent and purify the product on a sdlca gel column Elute first

with chloroform/methanol (5 I, [v/v]) and then with chloroform/methanol/water

1 mmol of the above product) Stir the mixture at loom temperature overnight

3 Filter the reaction mixture to remove dlcyclohexylurea, evaporate the solvent and purify the product by slltca gel column chromatography Elute first with chlo- roform/methanol (5 I, [v/v]) to completely remove DMAP and unreacted fatty

acid and then chloroform/methanol/water (65 25.4, [v/v/v]) Check the final prod-

uct by TLC; R, = 0 5 with chloroform/methanol/water (65 25 4, [v/v/v]) The purified product IS dissolved m dlchloromethane and stored at -20°C

3.3 Preparation of the Polymerized Mixed Liposome Substrate

Polymerrzed mixed hposomes are prepared by polymerlzmg pre-formed mixed llposomes of BLPG (or its denvatlves) and a pyrene-labeled phospho- llpld Although the composition of pyrene-labeled phosphollpld m polymerized mlxed hposomes can vary from 1 to 10% without affectmg the kinetic propertles

of phosphollpases, a mmlmal composltlon (1 “A) 1s maintained for all polymer- ized mixed hposomes for kinetic consistency and economical reasons

1 To prepare 2 mL of 1 mMpolymerized mixed Iiposomes, mix 1 98 pmol of BLPG and 0 02 pmol of pyrene-labeled phosphohpld in a glass vial, evaporate the organic solvent with the stream of nitrogen gas and add 2 mL of IO mMTns-HCI

buffer, pH 8 4, to the hptd film and vortex the dispersion for about 5 mln

2 Prepare large umlamellar hposomes by multiple extrusion (15X) of phospholipld

dlsperslon through 0 1 -mm polycarbonate filter m a micro extruder Liposofast

14 Cho et al

3 Prepare polymerized mlxed hposomes by Incubating the mixed hposomes at 37°C fol24 h m the presence of 10 mM dlthlothreltol The degree of polymeflzatlon can be monitored by TLC and by the loss of the UV absorption at 333 nm (9) (see Note 1)

4 Separate the polymerized mixed hposomes from the reaction mixture on a short Sephadex G-SO column (1 x 10 cm) eqtuhbrated with 10 mMHEPES buffer, pH 7 4 contammg 0 16 KC1 Elute the hposomes with the same buffer, monitor the absor- bance at 280 nm and collect the first peak elutmg at the void volume of the column

5 Determine the total phosphohpld concentration of hposomes by phosphate analy-

SIS (2.5) (see Note 2)

3.4 Kinetic Measurements

All kmetlc measurements are performed at 37°C The change m fluores- cence mtenslty of pyrene-labeled phosphohplds m polymerized mixed hpo- somes IS measured as a function of time using a fluorescence spectrometer The excltatlon wavelength 1s set at 345 nm and the emlsslon wavelength at 380 nm Spectral band width IS set at 5 nm for both excltatlon and emlsslon The sample

IS contained m a stirred, thermostated 1 -cm path length quartz cuvet

3.4.1 Secretory PLA,

1 Add 2 mL of 10 mMHEPES buffel, pH 7 4, contammg 10 pMpolymerlzed mixed hposomes (e g , pyrene-PGiBLPG), 10 FM BSA, 0 16 M KCl, 0 1 mM EDTA to

a quartz cuvet

2 Place the cuvette m the spectrometer and monitor the fluorescence intensity at

380 nm until the baseline 1s stabilized (approx 2 mm)

3 Add an ahquot of enzyme solution and incubate the mixture for a few minutes

4 Initiate the reaction by adding 10 FL of 2 MCaCl, solution to the final concentra- tionof lOmA

3 4.2 Cytosok PLA2

In contrast to secretory PLA,s, cPLA,s shows extremely low actlvlty toward polymerized mixed hposomes, such as PAPUBLPG polymerized mixed hpo- somes This 1s presumably owing to the fact that cPLA2 must penetrate mto the core of phosphohpld bilayer to pull a substrate molecule mto its active site, which

IS largely prohibited m the polymenzed mixed hposome system On the other hand, cPLA, shows high activity toward PAPC m unpolymerized mixed hposomes of PAPUBLPG (see Chapter 4) Thus, PAPUBLPG ( 1:99, mol/mol) mixed hposomes are used as a substrate for cPLA, As IS the case with the polymerized mixed hpo- some system, PAPC 1s selectively hydrolyzed m this system because cPLA2, owing

to its high sn-2 specificity, shows essentially no detectable activity toward unpolymerized BLPG To ensure the completely selective hydrolysis of PAPC, one can employ PAPC/D-BLPG mixed hposomes D-BLPG can be synthesized from D-a-glycerophosphocholme as described for BLPG (see Note 3)

Polymerized Liposome Substrates 15

1 Add 2 mL of 10 mM Tns-HCI buffer, pH 8 0, contammg 1 mM CaCI,, 10 @I PAPUBLPG mixed hposomes and 10 @I BSA to a quartz cuvet

2 Place the cuvette m the spectrometer and momtor the fluorescence mtenslty at

380 nm until the baselme IS stablhzed (approx 2 mm)

3 Initiate the reactlon by addmg an ahquot of enzyme solution

3.4.3 PLC and PLD

With commercially available N-pyrene-PE in polymerized mixed liposomes, one can measure the actlvlty of phosphatldylcholme-specific PLC and PLD For PLC, N-pyrene-PEIBLPG polymerized mlxed hposomes are used as a sub- strate m which N-pyrene-PE 1s selectively hydrolyzed For PLD reactions, N- pyrene-PE/BLPA polymerized mixed liposomes are used as a substrate owing

to relatively high activity of PLD toward polymerized BLPG molecules For both assays, BSA IS not included

1 For a PLC assay, add 10 pMN-pyrene-PEIBLPG polymerized mixed hposomes

m 2 mL of 10 mM MES buffer, pH 6.0,O 1 nuI4 ZnCl,, 10 ti CaCl, to a quartz cuvet For PLD assays, use 10 @IN-pyrene-PEIBLPA polymerized mlxed llpo- somes m 10 mA4 HEPES, pH 7 4,0 16 MKCl, 10 mM CaCl,

2 Place the cuvet m the spectrometer and monitor the fluorescence mtenslty at 380 nm until the baseline is stabdlzed (approx 2 mm)

3 Initiate the reactlon by addmg an allquot of enzyme solution

3.4.4 Determination of Rate Constants

1 The actlvlty (pmol/mm) of PLA2 for polymerized mixed hposomes IS calculated according to the followmg equation (7)

Actlvlty = [pyrene-PL], x (AF- A.FO)

AF,nax where [pyrene-PL], mdlcates the total concentration of pyrene-labeled phospho- hpld (in pmol); AF, and AF, the fluorescence change per minute for nonenzy- matic and enzymatic hydrolysis, respectively, AF,,,,, the maxlmal fluorescence change that IS measured by addmg an excess amount of enzyme (see Note 4)

2 The specific activity (pmol/mm/mg) IS determmed by divldmg the enzyme actlv- Ity by the amount of protein (mg)

3 Owing to the umque structural property of polymerized mixed hposomes m which only a small percentage of phospholipid m the hposome IS hydrolyzed, the kinetic pattern of hydrolysis of these hposomes IS, m general, simpler than that of the hydrolysis of conventlonal hposomes (13) Yet various factors, such as enzyme- to-phosphohpld ratio, can slgmficantly affect the kinetic pattern for different phospholipases Therefore, It 1s recommended that the range of the enzyme con- centration wlthm which the activity IS directly proportlonal to the enzyme con- centration be estabhshed first for each phosphohpase and Its best polymerized mlxed hposome substrate at a given concentration (1 e., 10 @4)

16 Cho et a/

4 Notes

1 BLPG (and BLPC) molecules are prone to polymerlzatlon even m the absence of dlthlothreltol and at low temperature For this reason, a fresh BLPG solution IS prepared m every three months

2 Owmg to their chemical and mechanical stablhty, polymerized mlxed llposomes can be stored at room temperature for several weeks under argon and in the pres- ence of 0 0 1% sodium azlde It has been notlced, however, that some phosphoh- pases show considerably altered activity toward these hposomes after elongated storage It 1s therefore recommended that polymerized mlxed hposomes are used within several days of preparation for the reproduclblllty of kinetic data

3 To design an assay system using polymerized mlxed hposomes for a newly dls- covered phosphohpase, one must first find a polymerized matrix that 1s reslstant

to the hydrolysis by the enzyme and a pyrene-labeled insert that 1s rapidly hydro- lyzed by the enzyme The derivatives of BLPG and other pyrene-labeled phospho- lipids (I e , pyrene-PA and pyrene-PS) (24) can be prepared by the PLD-catalyzed transphosphatldylatlon Specific actlvlty of phosphohpases for (polymerized) mlxed hposomes ranges from 0 5 to 1,000 mmol/mm/mg depending on the na- ture of enzyme (7)

4 Some phosphohpases show a short lag period (approx 10 s) before they 1 each the steady state For these enzymes, the sequence of adding enzyme and cofactors can be varied to mmlmlze the lag If the lag persists, the mltlal velocity 1s mea- sured after the reactlon reaches a steady state

References

Waite, M (1987) The Phospholzpases, Plenum, New York

Reynolds, L J , Washburn, W N , Deems, R A and Dennis, E A (199 I ) Assay strategies and methods for phosphohpases Methods Enzymol 197, 3-23

Radvanyl, F , Jordan, L , Russo-Mane, F , and Bon, C (1989) A sensltlve and contmuous fluorometrlc assay for phosphohpase A, using pyrene-labeled phos- phohplds m the presence of serum albumm Anal Bzochem 177, 103-I 09

Thuren, T , Vlrtanen, J A , Vamlo, P , and Kmnunen, P K J (1983) Hydrolysis

of 1 -tnacontanyl-2-pyrene-l-yl)hexanoyl-sn-glycero-3-phosphochollne by human pancreatic phosphohpase A, Chem Phy Llplds 33,283-292

Hendnkson, H S and Rauk, P N (198 1) Contmous fluorometrlc assay for phos- phollpase A2 with pyrene-labeled leclthms as a substrate Anal Blochem 116, 553-558

Kmkald, A R and Wllton, D C (1993) A continuous fluorescence dlplacement assay for phosphohpase A, using albumm and medium chain phosphollpld sub- strates Anal Blochem 212, 65-70

Wu, S -K and Cho, W (1994) A contmuous fluorometric assay for phosphoh- pases using polymerized mlxed hposomes Anal Blochem 221, 152-l 59

Wu, S -K and Cho, W (1993) Use of polymerized llposomes to study mterac- tlons of phosphollpase A2 with blologlcal membranes Bzochemzstry 32, 13,902-13,908

Polymerized Liposome Substrates 17

9 Sadowmk, A , Stefely, J , and Regen, S L (1986) Polymerized hposomes formed under extremely mild condltlons J Amer Ckem Sot 108,7789-7792

10 Clark, J D , Lm, L -L., Knz, R W., Ramesha, C S., Sultzman, L A., Lm, A Y , Mllona, N., and Knopf, J L (1991) A novel arachldomc acid-selective cytosohc PLA, contains a Ca2’-dependent translocatlon domam with homology to PKC and GAP Cell 65, 1043-105 1

11 Sharp, J D , White, D L, Chlou, X G., Gooden, T, Gamboa, G C , McClure, D , Burgett, S , Hoskm, J., Skatrud, P L., Sportsman, J R , Becker, G W , Kang, L H , Roberts, E F , and Kramer, R M (199 1) Molecular clonmg and expresslon of human Ca*+-sensltlve cytosohc phosphohpase A, J Blol Ckem 266, 14,85@-14,853

12 Kates, M (1986) Techmques of Llpldology, Elsevler, Amsterdam

13 Jam, M K and Berg, 0 G (1989) The kinetics of mterfaclal catalysis by phos- phohpase A, and regulation of mterfaclal actlvatlon hopping versus scootmg Blocklm Bzopkys Actu 1002, 127-156

14 Smtko, Y , Yoon, E T , and Cho, W (1997) High Speclficlty of Human Secre- tory Class II Phosphohpase A, for Phosphatldlc Acid Blockem J 321,737-74 1

3

Triglyceride Lipase Assays Based on a Novel

Fluorogenic Alkyldiacyl Glycerol Substrate

Albin Herrnetter

1 Introduction

Lipases are responsible for extracellular degradation and mtracellular metabolism of lipids m animals, plants, and microorganisms The conformational motif of all lipases whose three-dimensional structure has been determined IS characterized by an alp hydrolase fold (1) Most hpases utilize the catalytic triad Ser-His-Asp, with the serme residue actmg as the nucleophile in the mmal step

of carboxyl ester hydrolysis, this active site serme is contamed withm the Gly- Xaa-Ser-Xaa-Gly consensus sequence common to most hpases (2,2)

In humans and animals, triacylglycerols (and to a lesser extent diacyl- and monoacylglycerols) are the biological substrates of lipases Commensurate with their function, different triglyceride lipases are found m a variety of extra- cellular and intracellular locations For example, lingual hpase, gastric hpase, pancreatic lipase, and bile salt-activated lipase are present m the gastromtesti- nal tract Lipoprotein lipase and hepatic lipase are bound to the capillary endot- helium and hydrolyze triacylglycerols m cnulatmg hpoprotems Lipoprotem lipase and bile salt-activated hpase are also present m milk Finally, lysosomal lipase and hormone-sensitive lipase are intracellular enzymes

Importantly, the different lrpases exhibit specific preferences for then triacylglycerol substrates, depending on chain length, optical and positional isomers, and the characteristics of the lipid/water interface (3,4) For instance, lipoprotem hpase and hepatic lipase express maximum activity only against certain lipoprotem classes ($6) Pancreatic hpase acts on mixed micelles con- taming bile salt, although only in the presence of colipase (7,s) Moreover, enzyme activity may be enhanced by specific cofactors such as apohpoprotem CII for lipoprotein lipase (9) or colipase for pancreatic lipase

From Methods fn Molecular L?/o/ogy, Vol 109 Llpase and Phosphohpase Protocols

Edited by M H Doohttle and K Reue 0 Humana Press Inc , Totowa, NJ

19

20 Herrnetter Impaument of hpase functton due, for example, to the absence of enzyme activity as a consequence of mutattons m the genes coding for the enzyme or its protein cofactor(s), may be associated with severe pathological phenotypes For instance, elevated plasma triglycerides associated wtth metabolic disorders such

as type I hyperhpoprotememta results from the deficiency of hpoprotem hpase activity (10,12) The consequence of a deficiency m lysosomal actd ltpase IS cholesterol ester storage disease (12) Moreover, under pathological condmons, hpases may be found m an envnonment m which they usually do not occur and,

as such, may represent markers of destructive organ processes For instance, as a consequence of pancreatttts, high levels of pancreatic hpases are often present n-t the blood (13)

Determmatton of hpase acttvlty 1s routmely used for monitormg enzyme pro- tem durmg tsolatton, purtficatlon, detectton, and characterization of natural and recombinant proteins, for studies of biologtcal enzyme functton, and for the screenmg of biologtcal samples for diagnosis m medtcme A number of methods are available for the determmation of hpase acttvmes (#J&16), and include a variety of different substrate types (natural or synthetic triacylglycerols, radtoac- ttve or chromogenic llptds), as well as methods of substrate preparation (mono- disperse and polydisperse substrates, phospholtptd vesicles or detergent micelles) Both substrate type and its preparation are cructal wtth respect to reproducibility and relevance of the measured data

A htgh-performance lipase assay 1s expected to meet several criteria that are important for facility, quality, and selecttvity of the procedure (see Table 1) We have developed new fluorogemc trtacylglycerol analogs as substrates for a con- tmuous llpase assay that fulfills these requirements (1618) These substrates can be solubthzed m water containing fatty acid-free albumin Alternatively, stable lyophihsates of substrate/albumm complexes can be obtained commerctally and represent ready-to-use hpase substrates after dlssolutton m an appropnate aque- ous buffer (17) In addmon, these fluorogernc substrates can be substituted for

triacylglycerol m many types of standard substrate preparations, mcludmg deter- gent mtcelles, phosphohptd vesicles, emulsions, and orgamc solvents (18)

The structure of these novel fluorogemc lipase substrates 1s an alkyldtacyl- glycerol m an enanttomerically pure form, as shown m Fig 1 In posttions sn-1

or ~2-3, a hexadecyl residue is linked to glycerol by an ether bond, whtch pre- vents esterolysts of this bond by the hpase In posttton 92-2, glycerol is estertfied with the fluorescent molecule pyrenedecanotc acid Finally, a dodecanoyl chain,

substituted at its omega-end with a fluorescence quencher (a trlmtrophenylammo

residue), IS bound by an ester linkage to posttion sn-3 or sn- 1 of the glycerolipld

In then Intact form, these substrates show only low fluorescence intensity due to mtramolecular resonance energy transfer In the presence of a hpase, the fatty acid ester containing the quencher 1s hydrolyzed and the fluorescent product, an

Lipase Assays Usmg Fluorogemc Substrates 21

Table1

Requirements for a High-Performance Lipase Assay

Category I facllltatlon of the assay

General apphcablhty to different reaction media

Determmatlon of enzyme actlvlty under native condltlons

No interference with nonspeclfic esterases

Fig 1 Chemical structure of the fluorogemc hpase substrate l-tnmtrophenyl- amlnododecanoyl-2-pyrenedecanoyl-3-0-hexadecyl-sn-glycerol

alkylacylglycerol, IS formed Thus, from the contmuous mcrease m fluores- cence mtenstty, substrate hydrolysts can be monitored as a measure of ltpase activity, using a calibratton curve obtamed by plotting fluorescence mtenst- ties vs concentratton of an unquenched standard (pyrenedecanotc acid) (see

Figs 2 and 3) Under condtttons of nonltmttmg substrate concentrattons whtch depend on the reaction medium, enzyme actrvrty IS lmearly propor- tional to enzyme concentratton The detetectton limit for pure lipases IS >5 ng depending on the lipase type, the stabthty of the hpase, and the quality of the enzyme preparation

For all animal hpases so far mvesttgated, there IS a very high stereopref- erence for the sn-1 posttton of the fluorogemc alkyldtacylglycerols, espe- cially tf solubthzed m the presence of albumin (2618) Therefore, only the preferred enantromer, namely, 1 -trinitrophenyl-ammo-dodecanoyl-2-pyren- edecanoyl- 3-O- hexadecyl-sn-glycerol, will be described as the hpase sub-

Fig 2 Caltbration plot for an unquenched standard fluorophore Fluorescence mtensities at 400 nm are plotted agamst concentrations of pyrenedecanoic acid as a complex with albumm m PBS

Lipase Assays Using Fluorogenic Substrates 23

The features of this novel fluorogemc substrate that provide advantages com- pared with existmg techmques may be summarized as follows*

1 The substrate can be prepared m detergent-free form, or at submtcellar detergent concentration, and wtth a defined particle size m solutton (approxtmately 40 A) that does not disrupt membranes or affect hpoprotem structure Thus, hpase acttvmes can

be measured under native condmons

2 The fluorogemc substrate/albumm complex IS avarlable as a water-soluble lyophtbsate that represents a ready-to-use hpase substrate after reconstttutlon m aqueous buffer The lyophlltsate IS very stable and easily stored

3 The use of radioactive substrates is avoided

4 The assay IS continuous and much more sensitive than the continuous tttrtmetrtc methods using ahphatic short-cham triglycerides as substrates

5 The fluorogemc substrate is lipase specific Other commonly used short-chain trtglycertdes (e g, trtbutyrm, or carboxyhc acldp-mtrophenol esters) are not spe- cific smce they are also hydrolyzed by esterases

6 A large number of samples can be analyzed in a short ttme when a fluorescence plate reader IS used

In this chapter, detatled protocols are provided for the preparation of the substrate and the appropriate standards used for quantitation of the hydrolyzed product Also mcluded are methods for the determmation of hpase activtty from purified enzyme preparations or from biological samples (e g , serum or post-heparm serum.) Selective conditions are discussed for the detection of hpoprotem lipase and hepattc lipase based on measurements of enzyme active- ties m buffers of different iomc strengths A buffer system for the selective determmatton of pancreas ltpase activity m serum has recently been established

m our laboratory (see Note 1)

2 Materials

2.1 Fluorescence Standards

Tetrahydrofuran (analytical grade)

Pyrenedecanorc acid, availatble from Molecular Probes (Eugene, OR, USA, or

2333 AA Leaden, The Netherlands), or from Lambda Probes (Krrchbach/ Stelermark, Austria)

Bovine serum albumm, fatty-acid-free, fraction V (Boehrmger-Mannhelm, Mannhelm, Germany, or Sigma, Munchen, Germany)

Buffer for hpase assay (see Subheading 2.3.)

Trtton X-100, especially purified for membrane research (Boehrmger-Mannhelm) Alternatively, fluorescence standards can be purchased from PROGEN Biotechntk (Heidelberg, Germany) as stable lyophtltzed preparations of

pyrenedecanoic acid/albumin complexes containing 0, 0.6, 1.2, and 2 4 nmol

pyrenedecanorc acid, respectively, with 16 mg of fatty actd-free albumin each,

2 4 nmol Trtton X- 100, and 300 mg starch as a stabilizer

24 Hen-netter 2.2 Lipase Substrate

1 Buffer for hpase assay (see Subheading 2.3.)

2 Bovine serum albumm, Trtton X- 100, and tetrahydrofuran (see Subheading 2.1.)

3 Ltpase substrate (fluorogemc alkyldtacylglycerol) 1 -truutrophenylammodo-decanoyl- 2-pyrenedecanoyl-3-0-hexadecyl-sn-glycerol (mol wt 1078) IS eastly soluble tn meth- ylene chloride and tetrahydrofuran and soluble in ethanol, its absorptton spectrum IS the sum of the mdtvtdual absorptton spectra of pyren-edecanotc acid (&,,, = 345 nm, E = 50,000) and tnnttrophenylammododecanotc acid (h,,, = 415 nm, E = 7000, E at 345 nm

= 16,000), solvent methylene chloride) Accurate substrate concentratrons m organic soluttons are determmed from the absorbance at 415 nm (whtch momtors trmt- trophenylamme absorptron) Purity of the alkyldtacylglycerol substrate can be deter- mmed by thin-layer chromatography (I&= 0 7), using sthca gel plates and chloroform/ acetone/acetrc acrd (96 4 1, by vol ) as a solvent The alkyldracylglycerol substrate m methylene chloride solutton or m solvent-tree form IS stored at -20°C The chemtcal syntheses of the hptd 1s described In ref 16

4 Alternattvely, the alkyldtacylglycerol substrate can be purchased from PROGEN Btotechmk as a lyophthzed substrate/albumin complex A typical preparatton con- tams 60 nmol fluorogeruc alkyldracylglycerol, 60 nmol (approximately 4 mg) fatty actd-free albumm, 2 4 nmol Tnton X- 100, and 300 mg starch as a stabrhzer (accord-

mg to Zulkowsky, from Merck, Darmstadt, Germany), store m refrigerator (4°C) 2.3 Lipase Activity Assay

1 A fluorometer, equipped wrth a cell holder (preferably with four cuvet posmons) that 1s water Jacketed for temperature control Suitable mstruments mclude the Perkm-Elmer LSSOB, Perkm Elmer, Uberlmgen, Germany, Shtmadzu RF-5301

PC, Shtmadzu, Kyoko, Japan, and Hitachi F-2000, Ntsset Sangyo, Toyko, Japan

2 Cuvets for fluorescence spectroscopy Suitable cuvets Include quartz cuvettes, 4 mL,

d = 1 cm (Hellma, Mullhelm, Germany), or polyacrylate cuvets, 4 mL, d = 1 cm (cat

no 2,3893, Merck)

3 Ltpase assay buffer Ltsted are recommended buffers for determmmg the enzymatic actrvrty of some hpases For purtfied preparattons of hpoprotem hpase, hepatrc hpase, and pancreattc hpase, use 0 1 MTns-HCI, pH 7 4, or phosphate-buffered sahne (PBS),

pH 7 4,0 2 g KCVL, 0 2 g KH,PO,/L, 8 g NaCl/‘L, 1 15 g Na2HP04/L These buffers also work well to assay total hpase acttvtty m serum or post-hepann serum or tn cell culture supematants (see Note 2) Addttton of 1 0 A4 NaCl to these buffers ~111 rnhrbrt hpoprotetn hpase acttvrty but ~111 not reduce hepatrc hpase actrvtty A buffer suttable for the specific assay of pancreatic hpase acttvtty has recently been developed (see Note 1)

3 Methods

3.1 Preparation of Fluorescence Standards and Lipase Substrate

3 1 1 Preparation of the Fluorescence (Pyrenedecanoic Aad) Standards The followmg procedure can be used to prepare 30 standard samples con- taming defined concentrations of unquenched fluorophore These standards are

Lipase Assays Using Fluorogenic Substrates 25 used to create a cahbratlon plot to quantltate the amount of substrate hydro- lyzed during the hpase assay (see Fig 2)

1 Dissolve 480 nmol fatty acid-free bovine serum albumin (approx 32 mg) m 240

mL PBS or the same buffer used for the hpase assay (see Subheading 2.3 for examples of different hpase buffers)

2 Prepare four concentrations of pyrenedecanotc acid by dlssolvmg 0,O 6, 1 2 and

2 4 nmol of pyrenedecanotc acid m 200 uL tetrahydrofuran With constant stu- rmg at 37°C add each concentration of pyrenedecanolc acid to 60 mL of the albumm solutton prepared m step 1, The final concentration of pyrenedecanotc acid m these standards will be 0, 10, 20,40 pmol/mL; 2 mL of each standard 1s used to construct the calibration plot (see Subheading 3.3 and Fig 2) These standards can be stored for 1 wk at 4°C

3 If detergents are present m the hpase assay, prepare the standards as descrtbed m step 2, except either supplement or replace the albumm solutton wtth the appro- priate detergent (e.g , Trtton X- 100, alkylsulfobetam, octylglucostde, sodmm cholate, or deoxycholate) For supplementation, add 200 uL of the pyrene- decanolc actd/tetrahydrofuran solution to 60 mL of an albumm solution as pre- pared m step 1 that contains 0 04 mMTriton X-l 00 (see Note 4) For replacement, add 200 uL of the pyrenedecanolc actdftetrahydrofuran solutton to 60 mL of

0 2 mM Triton X- 100 m PBS

4 Alternattvely, lyophthzed pyrenedecanotc actd/albumm standards can be pur- chased commercially (see Subheading 2.1.) These preparations represent ready- to-use standards after reconstitution in 2 mL of PBS or the same buffer used for the ltpase assay The final concentrattons of these standards are 0, 10, 20, and

40 pmol/mL or higher

3.1.2 Preparatron of the Lipase (Alkykiiacylglycerol) Substrate

The followmg procedure can be used to prepare enough llpase substrate for

30 assays Once prepared, the substrate can be stored overmght at 4°C or 3 h at room temperature

1 Dissolve 120 nmol fatty acid-free bovme serum albumin (approx 8 mg) m 60 mL PBS or the same buffer used for the lipase assay (see Subheading 2.3 for examples of dtfferent hpase buffers)

2 To prepare a detergent-free substrate, dissolve 120 nmol of the fluorogemc alkyldtacylglycerol substrate rn 200 pL of tetrahydrofuran wtth constant sttrrmg

at 37°C Add the substrate/tetrahydrofuran solution to the 60 mL albumm solu- tion prepared in step 1 to gave a final concentration of 2 nmol/mL alkyl- dtacylglycerol Each hpase assay utilizes 2 mL of this substrate solutton

3 To prepare substrate supplemented with detergent and albumm, follow the same procedure descrtbed m step 2 except dissolve 240 nmol fluorogemc alkyl- dtacylglycerol substrate m 200 uL of tetrahydrofuran and add this solution to 60-mL albumm solution contammg 0 04 pA4 Trtton X- 100 The assay using this detergent supplement substrate is much more sensmve

26 Herrnetter

4 To prepare substrate wtth detergent but no albumm, prepare the substrate

as descrtbed tn step 2, except replace the albumm solution with the appro- prtate detergent (see Subheading 3.1.1 for examples of detergents that can be used)

5 Alternatively, a lyophilrzed preparation of the alkyldiacylglycerol/albumm substrate can be purchased commercrally (see Subheading 2.2.) This prepa- ration represents a ready-to-use substrate after reconstttutton m the same buffer used for the ltpase assay For 30 ltpase assays, add the lyophtltsate contammg 240 nmol alkyldracylglycerol to 60 mL of lrpase buffer to gave a final concentratton of 4 nmol/mL for the albumm/detergent assay as descrtbed under step 3

3.2 Measurement of Fluorescence Standards and Calibration

of the Fluorometer

Set monochromators to the followmg parameters excttatton wavelength, 342 nm, emtsston wavelength, 400 nm; exttatton and emmtssron sht wtdth, IO nm Brmg the cuvet holder to 37°C by setting the temperature of the ctrculatmg water bath (see Note 5)

F1112 mL of each standard solutton mto cuvets and allow the standards to equib- brate to 37°C by keepmg them m the cuvet holder for 10 mm

Check fluorescence mtenstty of the standard contammg the htghest fluorophore concentrattons at 37°C Attenuate or improve fluorescence signal by adJusting m- strument sensitivity if fluorescence intensity IS too high or too low, respectively

Measure the fluorescence mtenstttes of all four standards under the same

instument settings

Generate a caltbratton curve by plotting fluorescence intensity versus pmol fluorophore/mL (see Fig 2)

3.3 Determination of Lipase Activity

3 3.1 General Method for the Assay of Lipases

Using the Alkyldiacylglycerol Substrate

The sensitivtty of the lipase assay using the substrate/albumm complex is highest when the fluorogemc alkyldtacylglycerol IS freshly injected into an albumin solutron followed by equihbration as described m Subheading 3.1.1 The detection hmtt for pure (e.g., recombmant) hpases 1s >5 ng depending on the type and quality of the enzyme smce many of these proteins are unstable (e.g., hpoprotem hpase or hepatrc hpase m pure form are unstable; m contrast, a microbial hpase from Chromobactenum vlscosum was found to be very stable)

In most cases, hpase actrvttres as determined by the fluorescence method can be correlated with acttvmes obtained with radioactive trracylglycerols (M Duque, I Wrcher, R Zechner, F Paltauf, and A Hermetter, unpublished results) Very low hpolyttc activmes that are undetectable using radiolabeled trracylglycerols can often be measured usmg the fluorogemc substrate

1 lpase Assays Using Fluorogenic Substrates 27

1 Fdl 2 mL of substrate solutions into cuvets and equlhbrate to temperature (see Note 4) by mcubatlon m the water-Jacketed cuvet holder for 10 mm

2 Before adding hpase, measure fluorescence intensity (that equals the blank actlv- Ity) for 1 mm under Instrument settings as optimized for the standards

3 Under the same instrument and temperature condttlons, add the hpase sample (between 10 and 50 pL volume), mix thoroughly for a few seconds and measure contmously the increase m fluorescence mtenslty (AI) for 5-10 mm (see Fig 3 and Note 3)

4 From the recorded AI/mm of the hpase sample, subtract the AI/mm of the blank The resulting difference represents the fluorescence increase (AI/mm) due to the hydrolysis of the substrate This value can be converted to pmol of substrate hydrolyzed per mm by usmg the cahbratlon curve described m Subheading 3.2 and Fig 2

3 3.2 Considerations for the Selectwe Assay of Lipoprotein lipase and Hepatlc Lipase

In aqueous buffer solutions, activities of isolated lipoprotein hpase and

hepatlc hpase are additive when usmg the fluorogemc substrate High sodmm chloride concentrations (1 IV) inhibit lipoprotein hpase actlvlty, but not hepatlc

lipase activity, similar to that observed using the more traditional racholabeled

trlolein substrate preparations However, whereas the total hpase activity of postheparm serum 1s also due to both hpoprotem hpase and hepatic hpase, the

activities contributed by these two enzymes are not additive m this complex

blologlcal fluid m the absence of detergent (e.g., using 20 pL serum m 2 mL substrate solution) Although only hepatlc hpase activity is measured m the presence of 1 M NaCl (M Duque, R Zechner, F Paltauf, and A Herrnetter, unpublished results), the activity levels detected are often as high as the com- bined lipoprotem hpase and hepatlc hpase activity levels measured m the absence of salt This IS because hepatic lipase m serum is “activated” by salt under detergent-free conditions However, m the presence of detergent (e g , 0.04 ph4 Triton X- 100, see Note 4), this “actlvatlon” 1s not apparent so that the activity measured m 1 MNaCl 1s always less than the total hpase activity mea- sured without salt (unpublished observations) It should also be noted that mea- surement of total hpase activity and hepatic lipase activity m post-heparm serum 1s sensitive to the serum composltlon of the donor (e.g., lipids, hpopro- teins, and heparm concentration m post-heparin serum; also see Note 2) Thus,

routine analysis of serum trlglycerldes 1s recommended for interpretation of

the measured hpase activity as determined by the fluorescence assay

Finally, it should be emphasized that the fluorescence hpase assay described measures hpase actlvltles, even m complex blologlcal fluids, under nondlsturbmg condltlons Thus, the “actual” hpase activity is measured m its natural envlron- ment, and this environment may well affect the structural properties of the enzyme

28 Hermetter itself and contrlbute to modulations of enzyme actlvlty due to the presence of endogenous components such as lipids, heparan sulfate proteoglycans and other components present In complex blologlcal samples

4 Notes

1 Aativity of pancreatic lipase can be measured m the absence of cohpase (16)

However, the presence of the cofactor sigmficantly improves enzyme activrty (M Duque, F Paltauf, and A Hermetter, unpublished results) A high-sensitivity assay for the specific determmation of pancreatic hpase activity m the presence

of other hpolytic enzymes (for example in serum) has recently been developed m our laboratory

2 Heparm at high concentrations mhtbits activity of hpoprotem hpase and hepatic hpase In some cases, this might cause difficulties m assaying hpase activmes of heparmized cell culture supernatants (M Duque and A Hermetter, unpublished results), Identification of heparm-bmdmg domains of hpoprotem hpase have been reported m ref 19, and references therem

3 Sensitivity is lower when standards and substrate solutions are prepared by reconstmmon of lyophihsates m aqueous buffer, as described m Subheading 3.1

4 Sensitivity and reproducibility can be increased when the substrate (and stan- dard) samples are solubillzed m aqueous buffer contammg albumin and submicellar concentrations of detergent (e g , 0 04 mM Triton X- 100, which IS a nomomc and “mild” amphiphile) Conversely, sensitivity may be high, but reproducibihty is lower when the substrate (and standard) samples are solubi- hzed m detergents above thetr critical micellar concentrations (e g , 0 2 mM Tri- ton X- 100) In addition, due to impurities, most detergents show high background fluorescence if excited at 342 nm (1 e , the excitation wavelength of the substrate)

5 The recommended temperature for the assay of most mammalian hpase activities

IS 37°C This temperature is especially important when assaying hpoprotem hpase and hepatic hpase activities m serum, since activity 1s greatly reduced at lower temperatures usmg the assay system described herem Conversely, most micro- bial hpases can be convemently assayed at room temperature

Acknowledgments

Financial support by the Fonds zur Forderung der wlssenschaftllchen Forschung m Osterrelch (FWF), project F0107, IS gratefully acknowledged

References

1 Ollis, D L , Cheah, E , Cygler, M , Dijkstra, B , Frolow, F , Franken, S M , Harel,

M Remington, S.J., Silman, I , Verschueren, K H G., and Goldman, A (1992) The o/p hydrolase fold Protein Eng 5, 197-211

2 Brenner, S (1988) The molecular evolutton of genes and proteins a tale of two sermes Nature 334, 528-530

3 Ransac, S , Carriere, F., Rogalska, E , Verger, R , Marguet, F , Buono, G , Plnho

Melo, E , Cabral, J M S , Egloff, M -P E , van Tilbeurgh, H , and Cambillau, C

(1996) The kmetrc:, spectticmes and structural features of hpases, in NA TO ASI Series, Molecular Dynamzcs of Blomembranes, vol 96 (Op den Kamp, J A F ,

ed ), Sprmger, Berlm, pp 265-304

4 Brockman, H L (1984) General features of lipolysis reaction scheme, mterfactal structure and exper mental approaches, m Lzpases (Borgstrom, B and Brockman,

H L , eds.), Elsevter, Amsterdam, pp 346

5 Ohvecrona, G and Oltvecrona, T (1995) Trtglycertde hpases and atherosclero- SIS Cur-r Open Llpldozal 6, 291-305

6 Goldberg, I (1996 ) Ltpoprotem ltpase and hpolysis central roles m hpoprotem metabohsm and atherogenests J Lipid Res 37, 693-707

7 Erlanson-Albertsson, C (1992) Pancreatic cohpase Structural and phystologlcal aspects Blochun t1zophy.s Acta 1125, l-7

8 Verger, R (1984) Pancreatic hpases, m Llpases (Borgstrom, B and Brockman,

H L , eds ), Elsevier, Amsterdam, pp 83-150

9 Smith, L C and Pownall, H J (1984) Lipoprotem hpase, m Lzpases (Borgstrom,

B and Brockman, H L , eds ), Elsevter, Amsterdam, pp 346

10 Eckel, R H (1989) Lrpoprotem hpase: a multtfuncttonal enzyme relevant to com- mon metabolic diseases N Engl J Med 320, 106G-1068

11 Brunzell, J D (1’989) m The Metabolic Baszs of Znherzted Dzsease, 6th ed (Scrtver, C R , Beaudet, Al , Sly, W S , and Valle, D , eds ), Mac Graw-Hill, New York, pp 1165-l 180

12 Amens, D , Brockman, G , Knobhch, R , Megel, M , Ostlund Jr, R E , Yang, J W , Coates, P M , Conner, J A , Femman, S V , and Greten, H (1995) A 5’ sphce- region mutation and a dmucleotide deletion m the lysosomal acid hpase gene m two patients with cholestetyl ester storage disease J Lzpzd Res 36, 241-250

13 Ttetz, N W and Shuey, D F (I 993) Ltpase m serum - the elusive enzyme an overview Clan Chem 39,746-756

14 Hendrtckson, H S (1994) Fluorescence-based assays of ltpases, phospholipases, and other hpolytic enzymes Anal Bzochem 219, l-8

15 Henderson, A D , Richmond, W., and Elkeles, R S (1993) Hepattc and ltpopro- tem bpases selectively assayed m postheparm plasma Chn Chem 39, 2 18-223

16 Duque, M , Graupner, M , Stutz, H , Wtcher, I , Zechner, R , Paltauf, F , and Hermetter, A (1996) New fluorogemc trtacylglycerol analogs as substrates for the determmation and choral discrtmmation of hpase activtttes J Lzpzd Res 37, 868-876

17 PCT apphcatton no EP 95/O 19 19

18 Zandonella, G., Haalck, L., Spener, F., Faber, K , Paltauf, F , and Hermetter, A (1995) Inversion of hpase stereospecificity for fluorogemc alkyldiacyl glycerols Effect of substrate solubiltsatton Eur J Bzochem 231, 50-55

19 Hata, A , Ridmger, D N , Sutherland, S , Emt, M , Shuhua, Z , Myers, R L , Ren,

K , Cheng, T , Inoue, I , and Wilson, T E (1993) Bmdmg of lipoprotein ltpase to heparm Identtficatton of five crittcal residues m two dtstmct segments of the amino-terminal domain J Biol Ckem 267,2 1,499-2 1,504

4

Purification and Assay of Mammalian Group I

and Group Ila Secretory Phospholipase A2

Wonhwa Cho, Sang Kyou Han, Byung-In Lee, Yana Snitko,

and Rajiv Dua

1 Introduction

Phospholipases A, (PLA,) are a family of ubiquitous lipolytic enzymes that are found both as intracellular and secreted proteins Secretory PLA2s are small (14 kDa), homologous proteins that require milhmolar Ca2+ for catalytic activ- ity They can be classified mto at least four groups based on minor structural differences (1,2) In particular, two major classes of highly homologous secre- tory enzymes (class I and Ha) have been found m mammahan tissues Mamma- lian class I PLA2s are synthesized in the pancreas as pro-enzymes and activated

by proteolytlc cleavage m the intestine (for review see ref 3 and references therem) All known mammahan pancreatic PLA2s show strong sequence homology The mam function of these pancreatic PLA2s 1s to digest dietary phosphohplds emulsified with bile juice (3) Recently, several lines of evi- dence have indicated that marnmahan pancreatic PLA,s are present m dlffer- ent tissues and might play other physiological roles, including cell surface receptor-mediated mflammatlon (4)

Class IIa secretory PLA2s are synthesized and secreted by a variety of inflammatory cells mduced by mflammatory cytokines, such as tumor necrosis factor (TNF) and mterleukin-1 (IL-l) (for review, see ref 5 and references therein) In particular, high levels of class IIa secretory PLA, have been found

m the synovial fluid of patients with inflammatory arthritis and in the plasma

of patients suffering from septic shock Although these findings have implicated the class IIa secretory PLA2s m mflammatlon, questions still remam as to the source of these enzymes detected at inflamed sites, their exact physlologlcal roles and the mechanism by which they interacts with certain types of cells

31

32 Cho et al

To fully understand the physiological functions and regulation of the two classes of mammahan secretory PLA2s, it is necessary to prepare a sufficient amount of pure protems and to quantttattvely assay then acttvtties The purifi- cation of these enzymes from natural sources 1s often hampered by the hmtted availabihty of tissues (1 e , human pancreas) and the low abundance of pro- teins, m particular class IIa PLA, To overcome these dtfficulttes as well as to perform systematic structure-function studies through mutattonal analyses, sev- eral forms of recombinant mammalian secretory PLA2s have been expressed m various cells (6-13), among which the expression m Escherzchza colz and baculovnus-infected insect cells has been most efficient Herem, we describe the expression of human pancreatic PLA, (hp-PLA,) and human class IIa PLA, (hIIa-PLA,) using our bacterial expresston vector (pSH-hp) (21) and baculo- virus expression vector (pYS-bv) (ZO), respectively We also describe two different assay methods for mammalian secretory PLA2s usmg amomc poly- merized mixed ltposomes (14,25) and mixed micelles (16), respectively

The hp-PLA, expression vector (avatlable from author)

Lurta broth 10 g of Bactotryptone, 5 g of yeast extract (both from Fisher, Pttts- burgh, PA) and 10 g of NaCl in 1 L of deionized water, pH 7 4

Ampictllm (Sigma, St Louis, MO)

Isopropyl P-n-thiogalactopyranoside (IPTG) (Boehrmger Mannhelm, Indtanapolis, IN) Cell lysts buffer 0 1 A4 Trts-HCl, pH 8 0,5 mA4 EDTA, 0 5% (v/v) Trtton X- 100 (Pierce, Rockford, IL)

5,5’-Dtthtobts(2-mtrobenzoic acid), sodmm sulfite (Aldrich, Milwaukee, WI) 2-Nttro-5-(sulfothto)-benzoate 1s synthesized from 5,5’-Dtthtobts(2-mtrobenzotc acid) as follows Dissolve 0 5 g of 5,5’-Dtthtobts(2-mtrobenzotc acid) m 50 mL

of 1 Msodmm sulfite solution, adjust the pH to 7 5, and oxtdtze the solution with

a slow stream of oxygen gas until the color of the solutton changes from orange

to yellow The solutton IS then divided mto altquots and stored at -20°C The 2- Nitro-5-(sulfothio)-benzoate solution IS stable for approx 6 mo

Refolding buffer 25 mM Trts-HCl, pH 8 0, 5 mM dodecylsucrose (Calbtochem, San Diego, CA), 10 mA4 CaCl,, 8 mM reduced glutathtone, 4 mM oxtdtzed glu- tathtone (both from Stgma)

Sephadex G-25, HtLoad 16/10 S Sepharose (Pharmacta, Uppsala, Sweden) Medium pressure protem chromatography system wtth dual pumps and a UV detector (1 e , Pharmacla FPLC system)

Mobile phase for Sephadex G-25 25 mM Trts-HCl buffer, pH 8 0, 5 M urea (Ftsher, Pittsburgh, PA), 5 mA4 EDTA

Mobile phase for HtLoad 16110 S Sepharose A, 25 mA4 HEPES buffer, pH 8 0,

B, 25 mM HEPES buffer, pH 8 0,O 5 M NaCl

Punficatmn and Assay of PLA, 33 2.2 Preparation of Recombinant Human Class Ila PLAP

1 The pYS-bv expression vector (available from author)

2 Plasmid preparation kit, hpopolysacchande extraction kit (QIAGEN, Chatsworth, CA)

3 St9 cells, serum-free TNM-FH medium (Invitrogen, San Diego, CA)

4 BaculoGoldrM Transfectton krt (PharMmgen, San Diego, CA)

5 27’C Incubator

6 SP-Sepharose FF, Mono S 5/5 (Pharmacia, Uppsala, Sweden)

7 Mobile phase for SP-Sepharose A, 10 mA4 borate buffer, pH 9 0, B, 10 mM borate buffer, pH 9 0, 1 MNaCl

8 Mobile phase for Mono S A, 25 mM HEPES buffer, pH 8 0, B, 25 mM HEPES buffer, pH 8 0,2 M NaCl

2.3 Assay of Mammalian Secretory Class I and Class Ila PLA2 1-Hexadecanoyl-2-( 1 -pyrenedecanoyl)-sn-glycero-3-phosphoglycerol (pyrene- PG) (Molecular Probes, Eugene, OR)

1,2-b~s[l2-(l~poyloxy)-dodecanoyl]-sn-glycero-3-phosphoglycerol (BLPG) IS synthesized as described m Chapter 2

Polymertzed mixed hposomes are prepared as descrtbed m Chapter 2

Fatty acid-free bovine serum albumm (BSA, Bayer, Kankakee, IL)

Assay buffer 10 mM HEPES, pH 7 4,0 16 M KCl, 10 mA4 CaCl*, 10 mM BSA Spectrofluorometer equipped with a thermostated cell holder and a magnetic stirrer 1,2-dioctanoyl-sn-glycero-3-phosphoglycerol (diCsPG, Avanti, Alabaster, AL), Trrton X-100 (Pierce, Rockford, IL), sodium deoxycholate (DC, Sigma)

To prepare 10 mL of Triton X-100 (2 mM)IDC (1 mM)IdiCsPG (0 5 mA4) mixed micelles* add 2 6 mg of dtC,PG dissolved m about 10 mL of chloroform to a round-bottomed flask and evaporate the solvent zlt vucuo Prepare 10 ml of 0.1 mA4 MOPS containing 10 mM CaCl*, 2 mA4 Triton X-100, and 1 mM DC and adjust the pH to 7 4 Add this solution to the lipid film and gently vortex the mixture until a clear solution appears

9 pH Stat mstrument equipped with a thermostated vessel and a magnetic stirrei

3 Methods

3.1 Preparation of Recombinant Human Pancreatic PLA2

Mammalian pancreatic PLAzs were ortgmally purtfied from pancreatic homogenates either by heat treatment or by using alkaline glycerol solutton for solubrlrzatlon Currently, several pancreatic PLA,s, including bovine and por- cme enzymes, are commerctally available Also, highly efficient bacterial expresston vectors are available for human (II), bovine (7), and porcine (6) pancreattc PLA,s The expression of hp-PLA2 using the pSH-hp vector con- structed m our laboratory IS described m this subheadmg The pSH-hp plasmld has the coding sequence of hp-PLA, inserted m frame with the mmatlon codon

of the T7 promoter-based pET2 la vector Thus, fully mature hp-PLA, is

34 Cl70 et a/ expressed m E coli with Ala as its first ammo acid As 1s the case with other secretory PLA,s expressed m E colz, hp-PLA, 1s expressed as an mcluslon

body that IS subsequently sulfonated, solublhzed m 8 A4 urea, and refolded under the condltlon that promotes the formation of dlsulfide bonds E colz

stram BL2 1 (DE3) IS used as a host for the expresslon of hp-PLA2

Induce the cells with IPTG (final concentration, 0 5 mM) when the absorbance at

600 nm 1s 0 8

Incubate the culture for addltlonal 4 h at 37°C

Harvest the cells by centrlfugatlon (3,000g fol 10 mm) at 4°C

Resuspend the pellet m 50 mL of 0 1 A4 Tris-HCl buffer, pH 8 0 containing 5 mA4 EDTA, 0 5% (v/v) Trlton X-100

Lyse the resuspended cells by somcation with a pulse mode of 10 x 15 s Collect the pellet by centrlfugatlon (10,OOOg for 10 mm) at 4 OC

Resuspend the pellet with the same buffer and repeat the somcatlon and cen- trifugation

Solublllze the mcluslon body m 10 mL of 8 Murea solution contammg 0 3 M sodmm sulfite (pH 8 0) and stn the solution vigorously at room temperature for 30 mm Add 2 mL of 25 mM 2-mtro-5-(sulfothlo)-benzoate solution and stir the mixture for 30 mm

Remove any msoluble matter by centrlfugatlon (35,000g for I h) at room tem- perature

Load the solublhzed protem onto a Sephadex G-25 column (2 5 x 20 cm) equlh- brated with 25 mM Tns-HCl buffer, pH 8 0, containing 5 M urea and 5 mM EDTA, and collect the first major peak (total volume of about 45 mL)

Initiate refolding of the sulfonated protein by slowly adding 100 mL of 25 mM Tns-HCl, pH 8 0, contammg 5 tidodecylsucrose, 10 mA4CaC12, 8 mA4reduced glutathlone, and 4 mA4 oxldlzed glutathlone over 2 h Keep the solution at room temperature for 20 h (see Note 1)

Dialyze the refolded protem solution against 4 L of 25 mM Tris-HCl buffer, pH

Purification and Assay of PLA,

3.2 Preparation of Recombinant Human Class Ila PLA1

35

Human and rat class IIa PLA, have been purified from various sources tn small quantities More recently, recombtnant forms of these proteins have been over-expressed tn bactertal cells (8,9), baculovirus-Infected St9 cells and mam- mahan cells (13) Unlike the expression of hp-PLA,, which yields a fully mature protem, the bacterial expression of hIIa-PLA2 entails the mutatton of the wild type sequence tn order to remove either the ammo-terminal methione residue or the ammo-termmal extension Furthermore, the purification of expressed protein involves the solubthzation of mcluston body and the subse- quent refolding of solubthzed protein On the other hand, the expression of hIIa-PLA2 m baculovnus-infected Sf9 cells, albeit less eflictent tn terms of the level of protem productton, allows the expression of a fully folded mature pro- tein Our baculovtrus expression vector (pY S-bv) contains the signal sequence

and coding sequence of hIIa-PLA, tn the pVL 1392 baculovtrus transfer vector and, thus, the expressed protein 1s secreted into the growth medium Owing to tts htghly cattonic nature (isoelectric point > lo), hIIa-PLA2 can be easily enriched from the growth medium using a cation exchange column

1 Prepare the pYS-bv plasmtd by a standard protocol In order to improve transfec- non efficiency, purify the DNA using a lipopolysaccharide extraction ktt

2 Grow Sf9 cells as monolayer cultures m serum-free TNM-FH medium (see Note 3)

3 Co-transfect viral DNA with the pYS-bv vector and incubate cells for 4 d at 27°C

4 Collect the supematant and use tt to amplify the vuus with multiplicity of mfec- non of 0 5-l After three cycles of amphficatton use the high-titer virus stock solutton for the protein expression

5 Seed I x 1 O7 St-9 cells onto twenty 10 cm-culture plates (total volume of medium,

200 mL) and infect them with the high-titer viral solution with a multtphctty of infection of 10

6 Incubate the cells for 3 d at 27°C

7 Collect the supematant and remove any dislodged cells by centrtfugatton at 2,000g

8 Dialyze the supematant against deionized water (3 x 4 L) and then against 10 mA4 borate buffer, pH 9 0 (1 x 4 L) at room temperature

9 Equilibrate SP-Sepharose column (5 x 10 cm) with 10 &borate buffer, pH 9 0

10 Load the supernatant onto the column, wash the column with 10 mA4 borate buffer, pH 9 0, until no further peak emerges Elute the bound protem with 1 A4 NaCl m the same buffer

11 Dialyze the eluted protein solution against 25 mA4 HEPES buffer, pH 8 0

12 Equthbrate a Mono S 5/5 column with 25 mM HEPES buffer, pH 8 0

13 Load the protein solutton onto the column, and elute with a linear gradient of NaCl from 0 to 2 A4 m the same buffer

14 Collect the major protein peak eluted with approx 0 8 M NaCI, dialyze against water, and lyophilize The lyophtltzed hs-PLA, can be stored indefinitely at -2O’C without any detectable loss of acttvtty

36 Cho et al

3.3 Assay of Mammalian Secretory Class I and Class Ila PLA2 Most of mammalian secretory PLA2s strongly prefer as a substrate anionic phosphohptd aggregates to electrically neutral ones Among a wide variety of amomc phosphollpld aggregates used for the assay of mammalian secretory PLA2s, two convenient contmuous assays usmg polymerized mixed hposomes and mixed mlcelles, respectively, are described m this subheading As described m Chapter 2, the fluorometrlc polymerized mixed llposome assay IS more versatile and more sensitive than the mlxed mlcelle assay (see Note 4) The latter allows, however, more rapid preparation of the assay mixture from components that are all commercially available

3.3.1 Polymerized Mixed Llposome Assay

1 Add 2 mL of 10 mM Tns-HCl buffer, pH 8 0, containing 0.16 A4 KCI, 0 1 n&I CaCI,, 10 mk4 pyrene-PG/BLPG mlxed hposomes, and 10 mA4 BSA to a quartz cuvet

2 Place the cuvet m the spectrofluorometer and monitor the fluorescence intensity

at 380 nm until the baseline IS stablhzed (approx 2 mm)

3 Imtlate the reaction by adding an ahquot of enzyme solution

3.3.2 Mixed Ahcelles Assay

1 Place 2 mL of mixed mlcelles solution (Tnton X- 100 (2 n&Q/DC (1 mM)IdlCsPG (0 5 mM) m 0 1 n&I MOPS, pH 7 4, 10 mA4 CaCl,, and 0 16 A4 KCl) in a thermostated reaction vessel of a pH stat instrument

2 Once the baseline IS stabilized, initiate the hydrolysis by adding an ahquot of enzyme solution and monitor the release of fatty acid as a function of time 3.3 3 Determination of Rate Constants

The activity (mmol/mm) of PLA, for polymerized mlxed llposomes IS cal-

culated from the fluorescence change as described m Chapter 2 The PLA,

activity toward mixed mlcelles IS directly determined from the mltlal slope of

progress curve since the pH stat directly measures the concentration of product

released The specific activity (mmolimmlmg) IS determined by dlvldmg the enzyme actrvlty by the amount of protem (mg)

4 Notes

1 To prevent the protein preclpltatlon during the addition of the refolding buffer to the urea solution of sulfonated hp-PLA*, the refolding buffer must contam a non- lomc detergent, such as dodecanoylsucrose, and the addition be as slow as pos- sible (approx 2 h) Once the refolding solutions are mixed, the protein preclpltatlon

IS not slgmficant during the subsequent mcubatlon and dialysis The progress of refolding can be momtored by directly measuring the enzyme activity of ahquots

of refolding mixture using pyrene-PG/BLPG as m Subheading 3.3.1

Purification and Assay of PLA, 37

2 Durmg the chromatographrc separatton of refolded hp-PLA, using a HrLoad S Sepharose column, a minor shoulder peak IS observed Thts peak contains a mtx- ture of improperly processed protems which have Met, Val, and Trp, respec- tively, as a first ammo acid and exhtbtt lower PLA, activity Thus, these proteins must be carefully separated from the maJor peak protein Overall yield of puri- fied hp-PLA, should be approx 5 mg of protem/L culture

3 To facthtate the purrficatton of secreted hs-PLA, from the growth medium, it 1s essential to use serum-free medmm The secreted hs-PLA, can be enriched from the medmm, with high efficiency, using an tmmobthzed heparm column A less expenstve canon-exchange column is used as an alternative m this protocol A typical yteld of purified hs-PLA, IS approx 1 mg/L culture

4 Although pyrene-PGiBLPG polymertzed mixed hposomes are a good substrate for most mammahan secretory PLA,, some PLA,s might have htgher acttvtty toward other polymertzed mixed ltposomes, such as pyrene-PA/BLPG polymer- ized mtxed ltposomes Also, some mutant proteins might have altered substtate specrfictty thereby preferring different pyrene-labeled inserts m polymertzed mixed hposomes

Davidson, F F and Dennis, E A (1990) Evoluttonary Relatronshtps and Imph- cations for the Regulation of phospholtpases A2 from snake venom to human secreted forms J Mel Evol 31,228-238

Wane, M (1987) The Phosphollpases, Plenum, New York

Tohkm, M , Ktshmo, J , Ishtzakt, J and Artta, H (1993) J Bzol Chem 268,286>287 1 Kudo, I , Murakamt, M , Hara, S , and moue, K (I 993) Mammaltan non-pancre- atic phosphohpase A, Blochlm Brophys Acta 1170, 2 17-23 1

de Geus, P , van den Bergh, C J , Kutper, 0 , Hoekstra, W P M , and de Haas, G

H (1987) Expression of porcine pancreatic phosphohpase A2 Generation of active enzyme by sequence-specific cleavage of a hybrid protem from Escherlchla toll Nuclei Acids Res 15, 3733-3759

Deng, T , Noel, J P , and Tsar, M -D (1990) A novel expresston vector for hrgh- level synthesis and secretion of foreign proteins m Escherzchza colz Overproduc- tion of bovme pancreatic phospholtpase A, Gene 93, 229-234

Franken, P A , van den Berg, L , Huang, J., Gunyuzlu, P , Lugtighetd, R B , VerheiJ, H M., and de Hass, G H (1992) Purtficatton and charactenzatron of a mutant human platelet phosphohpase A, expressed m Escherlchza cok Eur J Blochem 203,89-98 Othman, R , Worral, A, and Wrlton, D C (1994) Some properties of a human group Ha phosphohpase A, expressed from a synthetic gene m Eschenchla co/l Bzochem Sot Trans 22,3 17s

Smtko, Y , Yoon, E T., and Cho, W (1997) High Specificity of Human Secretory Class IIa Phospholtpase A1 for Phosphatidtc Acid Bzochem J 321, 737-741

271, 30,041-30,05 1

Weiss, I, Inada, M , Elsbach, P., and Crowl, R M (1994) Structural determinants

of the actton against Eschewhla colr of a human Inflammatory fluid phosphoh- pase A, m concert with polymorphonuclear leukocytes J Blol Chem 269, 26,33 l-26337

Wu, S -K and Cho, W (1993) Use of Polymerized Llposomes to Study Interactions

of Phosphohpase A, with Blologtcal Membranes Bzochemlstry 32, 13,902-13,908

Wu, S -K and Cho, W (1994) A contmuous Fluorometrlc Assay for Phosphob- pases Using Polymerized Mixed Liposomes Anal Bzochem 221, 152-159 Dua, R and Cho, W (1994) Inhibitton of Human Secretory Class IIa Phosphoh- pase A, by Heparm Eur J Bcochem 221,481490

5

Determination of Plasmalogen-Selective

Phospholipase A2 Activity by Radiochemical

and Fluorometric Assay Procedures

Akhlaq A Farooqui, Hsiu-Chiung Yang, Yutaka Hirashima,

and Lloyd A Horrocks

1 Introduction

Plasmalogens are a umque class of glycerophospholipids (2) characterized

by the presence of a vinyl ether substrtuent at the m-l position of the glycerol backbone Found m all mammalian cells, these phosphohpids are especially abundant m brain and heart (I) However, while cholme plasmalogen IS enriched m heart tissue, brain ttssue is rich in ethanolamme plasmalogen Despite thetr ubtqurtous drstrrbutlon, little IS known about then role m mam- malian metabolism These liptds may act as a reservoir for arachtdomc acid, the precursor for prostaglandms and thromboxanes The vmyl ether lmkage of plasmalogen may also play an important role m protectmg cellular membranes against oxtdattve stresses (2,3) The breakdown of plasmalogen may be a receptor-mediated process related to stgnal transductron (4,s)

Heart (6,7), brain (5,8), and kidney (9) contam a novel phosphohpase A, (PLA,) activity that hydrolyzes arachidonic acid from the SM-2 position of plas- malogen Thrs novel PLA2 has been purified from canme myocardmm (6,7), bovine brain (8), and renal proximal tubule (9) From each source, the purified enzyme differs with regard to kmetrc properties, substrate specrficlttes and response to detergents and various PLA, mhrbrtors These diverse PLAz enzymes suggest that they may play dtstmctrve roles m response to various phystological and pathological conditions

The purpose of thus chapter IS to describe methods for determinmg plas- malogen-selective PLA, activity, which can be assayed by both radtochemical

39

Glacial ace& acid

N,N’-Dimethyl-4-ammopyrldme (DMAP) (Aldrich Chemical, Milwaukee, WI) Recrystallize DMAP from chloroform/dlethyl ether (1 /l, [v/v]) before using Arachldonate and [3H]arachldonate can be purchased from Nu Chek Prep , Elysian, MN and DuPont-NEN, Boston, MA, respectively

N,N’-Dlcyclohexylcarbodnmlde (DCC) (Aldrich Chem Co , Milwaukee, WI) Methanol/Water/Acetomtrlle (57123120, [v/v]), hexane/2-propanollwater (48 51

48 5/3, [v/v]), and hexane/2-propanol/water (46/46/8, [v/v])

Slllca gel G plates (20 x 20 cm) 50 pm, Analtech, Newark, DE

Dynamax Macro-HPLC Silica column (21 4 mm x 25 cm), Ramm Instrument, Woburn, MA

Econosll octadecyl silica column (10 mm x 25 cm), 10 mm particle size, Alltech Assoclatlon, Deerfield, IL

0 04 M Tns-HCl buffer, pH 7 6, containing 0 5 M NaCI; store at 4°C

0 04 M Tns-HCl buffer, pH 7 6, contammg 15 mg/mL sodium deoxycholate Store at 4°C

0 04 M Tns-HCl buffer, pH 7 6, containing bovine serum albumin (15 mg/mL), store at 4°C

0 04 A4 Tns-HCl buffer, pH 7 6, contammg 0 002 M CaCl, Store at 4°C

Rhlzopus arrhlzus (R A ) hpase, 50,000 U/mL (Boehrmger Mannhelm, Indla- napolis, IN)

Chloroform and methanol (Fisher Scientific Co, Pittsburgh, PA), including 2/l (v/v) and 4/l (v/v) chloroform/methanol mixtures