The action of snake venom phospholipase a and trypsin on purified myelin in vitro

Purified myelinwassuspended inwater.Thesuspended myelinwasincubatedwith crude snakevenom10-150,ug/mgof myelinprotein, lysophosphatidylcholine 20,cg-1.0mg/mgof myelin protein and phosphol

Biochem J.(1976) 159, 273-277

Printed In Great Bitain

The Action of Snake Venom, Phospholipase A and

Trypsin on Purified Myelln in vitro

Miriam Marks DepartmentofNeurochemistry,InstituteofNeurology, TheNationalHospital,

QueenSquare,London WC1N 3BG, U.K

(Received 3 May 1976)

1.Purifiedmyelinwasincubated with snakevenomorphospholipaseAinthepresenceof

orabsence oftrypsinat37°C,pH7.4,for different times.2.Analysisof themyelinpellet

obtained after centrifugation of the myelin sample incubated with snake venom or

phospholipaseAalone showedconversionofphosphatidylcholine,

phosphatidylethanol-amine andphosphatidylserineinto theircorresponding lysocompounds.Nosignificant

loss of myelinproteinwasobserved in thesesamples 3.A marked digestion of basic

proteinandproteolipid proteinwasobservedfromthemyelinpelletwhentrypsin was

presentin theincubation mixture 4 Thedigestionof basicprotein andparticularly of

proteolipid frommyelinsuggeststhatphospholipasesmaymakeproteinmoreexposed

toproteolyticenzyme for its digestion 5 Therelevance of the co-operativeeffect of

phospholipasesandproteinasesas amodelsystemof the mechanism ofmyelin

break-down indegenerativebrain diseasesisdiscussed

Radioisotopic studies of myelin constituents

indicate that atleast part of the structure is

meta-bolically rather stable (Davison, 1961; Smith,

1972; Sabri et al., 1974; Agrawal et al., 1976)

However,inmultiple sclerosis and other

demyelin-ating conditions there isprimary dissolution of the

myelinlamellae,withearly loss of basic protein As

this protein is susceptibletoproteolysis, proteinases

have been implicated in the demyelinating process

(Einsteinetal., 1972; Adamsetal., 1971) Previous

studies on isolated myelin showed that the basic

proteinwaspartially loston treatmentwithtrypsin,

but unexpectedly the myelin-sheath ultrastructure

appears to be unaltered (Raghavan et al., 1973;

Banik &Davison, 1974;Wood et al., 1974)

Since phospholipase A incubated with isolated

myelin causes changes in its lipid composition

(Coles et al., 1974), we have investigated the

possibilitythatphospholipases,togetherwith

proteo-lytic enzymes, may cause the more complete

destruction ofthemyelin sheath Thusthe purpose

of the present work was to study the co-operative

effect of phospholipases and proteinases on the

dissolution ofthemyelin membranein thehope that

it will provide an experimental model for the

degenerative process A preliminary report ofthis

work has appeared elsewhere (Banik & Davison,

1975)

* Present address: Neurological Unit, Veterans

Ad-ministration Hospital, Stanford University School of

Medicine, 3801 Miranda Avenue, Palo Alto, CA 94304,

U.S.A.

Vol 159

Experimental Materials Acetylated trypsin,lysophosphatidylcholine,crude snake (Naja naja) venom and purified phospho-lipase A were obtained from Sigma (London) Chemical Co (Kingston-upon-Thames, Surrey, U.K.) All other chemicals were AnalaR grade (BDH Chemicals Ltd., Poole, Dorset,U.K.)

Methods Preparation of myelin Adult Wistarratsofeither

sex were used throughout these experiments Rats

were anaesthetized with chloroform before

exsan-guination Brains were quickly removed, weighed and transferred into ice The tissue was homo-genized in 0.32M-sucrose and purified myelin was

preparedasdescribed by Norton (1971)

Incubationofmyelin Purified myelinwassuspended

inwater.Thesuspended myelinwasincubatedwith crude snakevenom(10-150,ug/mgof myelinprotein), lysophosphatidylcholine (20,cg-1.0mg/mgof myelin protein) and phospholipase A(80,cg/mg of myelin protein) in the presence or absence of acetylated trypsin (10-25pg/g of myelin protein) in 50mM-Tris/HCl buffer, pH7.4 (Colesetal., 1974),at37°C

with constantshaking Myelin withorwithout

tryp-sin, lysophosphatidylcholine or snake venom or

phospholipase A at zero time served as controls After the incubation the experimental and control tubes werequickly chilled in ice and centrifuged at

273

N L BANIK, K GOHIL AND A N DAVISON

12000gfor 10min Afirmmyelin pellet and

super-natant were obtained on centrifugation and were

analysed

Determination ofprotein and adenosine 2':3'-cyclic

monophosphate 3'-phosphodiesterase (EC 3.1.4.16)

activity Protein wasdetermined by the method of

Lowry et al (1951), with albumin as standard,

and adenosine 2':3'-cycic monophosphate

3'-phosphohydrolase activity was measured by the

methodof Banik & Davison(1969)

Lipid extraction andseparation Lipid was extracted

by the method ofFolch et al.(1957)and wasseparated

by t.l.c as described previously (Banik &Davison,

1971).Lipidswereseparated byt.l.c in thesolvent

system chloroform/methanol/aq 12% (w/v) NH3

(17:7:1,byvol.).Inthissystemthelysoethanolamine

phosphoglyceride was found to co-migrate with

sphingomyelin, lysophosphatidylcholine and

phos-phatidylinositol;lysophosphatidylserine movedasa

separateband Whenplateswerestained with iodine

vapour the loss of phosphoglyceride and

con-comitant appearance of darkly stained bands

for-corresponding lyso compounds were observed

(see Plate 2) Lysophosphatidylcholine was also

separatedbyt.l.c.bythemethod of Colesetal.(1974)

Gel electrophoresis Electrophoresis of the de.

lipidized samples in a sodium dodecyl sulphate

medium was carried out by the method ofBanik

et al (1974) Gels were stained with Coomassie

Brilliant Blue overnight andde-stained asdescibed

byAgrawaletal.(1972) After de-staining gelswere

scannedinau.v.spectrophotometerat595nmfitted

withascanner

Electron microscopy The pelleted fractions were

fixed overnight in 4.0% (whv) glutaraldehyde in

0.1M-potassium phosphate buffer, pH7.4, then

washed three times in the same buffer and fixedin

1.0%(w/v) 0S04for 2h

Results

Effect oflysophosphatidylcholine, snake venom and

phospholipaseA inthepresenceorabsence oftrypsin

onincubated myelin

In our experiments, when myelin preparations

wereincubatedfor60min inTris/HCIbuffer at37°C,

some digestion of both basic proteins occurred,

suggestingthepresenceofanendogenousproteinase

Allourexperimentswerethereforerepeatedin

dupli-cate anddata were corrected forchangesin control

preparations No apparent loss of membrane

protein occurred when myelin was incubated for

different time-intervals separately with either

lyso-phosphatidylcholine A.A9%/ loss of protein from

myelin was observed when it was incubated with

snake venom alone However, there wasa marked

loss of protein (17%) compared with controls

whenmyelin was incubatedwithcrude snake venom

in the presence of acetylated trypsin (Table 1)

Digestion,particularlyofbasic protein,wasobserved

in thesesamplesinthepresence of trypsin, andthe

appearanceoffaster-movingproteinbandswasnoted

This loss of protein was greater (25%) when the

concentration of snake venom and trypsin was increased or the time of incubation extended

(Table1) An extensivedigestionof high-molecular-weight Wolfgram protein was evident from the electrophoreticpatternofincubatedsamplestreated witheitherphospholipaseA or snake venom When

trypsin was incubated for 30min with myelin previously exposed to snake venom, the loss of

protein was 25% In experiments in which both

phospholipase A and trypsinwerepresent,extensive

loss ofproteolipid protein and basic protein from

myelin preparations resulted Thelossofbasic and

especiallyproteolipidproteinappearedtobegreater when myelin preincubated with snake venom or

phospholipaseAwasfurtherincubated with trypsin Thedigestionofproteolipidprotein comparedwith

controlswas60%,andbothhigh-and low-molecular-weight basic proteinswereextensivelydegradedwhen myelin was incubated with either phospholipase A

orsnakevenomin thepresenceoftrypsin.Theextent

ofdigestionofhigh-molecular-weight basic protein

washigherin the presence ofphospholipaseAthan

with snake venom (Table 2) A similaramount of low-molecular-weight basic protein was digested in thepresence ofeither snake venom orphospholipase A

Morphology Electron-microscope observations of the washed myelin pellet after treatment with snake venom

or phospholipase A did not reveal any structural

difference compared with controls, and the myelin lamellae remained tightly packed However, the

washed myelin residues after treatment with

trypsin together with phospholipase A or snake venomrevealed lessdensely packed myelin There was extensive splitting ofmyelin lamellae at the

intra-periodline andnumerousdissociated singlelamellae

or free strands were also present (Plate 1) The

periodicity of the myein lamellae, trypsin- and

phospholipase A-treated and control samples re-mained unaltered

Effectonmyelin 2':3'-cyclicphosphohydrolaseactivity The total phosphohydrolase activity remained unchangedwhenmyelin was incubated with lysophos-phatidylcholine, snake venom or phospholipase A However, a 15-20% loss of enzyme activity was

observed when trypsi' was incubated with these

reagents (Table 1)

1976 274

The Biochemical Journal, Vol 159, No 2 Plate 1

EXPLANATION OF PLATE I

Electron micrograph ofthe myelin pellet obtained after incubation of myelin withphospholipase A in the presence of

acetylated trypsin Extensivesplittingand dissociation of themyelinlamellae can be seen after incubation with trypsin andphospholipaseA.

In normal ratmyelinfractions after incubation in buffer alone,splittingof the lamellae is minimal and few single membrane

vesiclesare present Sections were 70-80nm thick The horizontal bar represents 0.5,m.

The Biochemical Journial, Vol 1 59, No 2

Plate 2

-ch ol

'.

.:i:;:.Y.r,::?.s:: 8!::&::i:ra

'.n.':'^# -t' t j:w | | _.'.

rs |

s :x, i3 111 # <S.: X : s :.' ?:

' PC

.:.,£, ?lLi Ig - ?,.:.

'gL X | | |

W'aS l' | | i ::'.': j

L y s o P L

Lyso-pc

N L BANIK, K GOHIL AND A N DAVISON

DISSOLUTION OF MYELIN

Table 1 Lossofproteinand2':3'-cyclicAMPphosphohydrolaseactivity onincubationofpurifiedmyelinwithsnakevenom,

phospholipaseAandlysophosphatidyicholineinthepresence orabsenceofacetylated trypsin

Purified myelin alone incubated in buffer for 60min, and also myelin under various conditionsat zero time, served

as controls.*,Myelinpreincubated with snakevenomfor 60minwasfurtherincubatedwithtrypsin for 30min;t,myelin preincubated with snake venom for 60min was pelleted and the pellet wasincubated withtrypsinfor 30min

Conditions

Totalprotein

inmyelin

residue (mg/sample)

.~~~~~

Incubationtime(min) 0

Incubation of purifiedmyelin with:

1. Lysophosphatidylcholine(20,ug/mg of myelin 2.56

protein)

Lysophosphatidylcholine (20,ug/mg)+trypsin 2.63

(lOpg/mgofmyelin protein)

2. Snake venom(lOO1ug/mg of myelin protein) 2.26

Snakevenom(lOO,ug/mg)+trypsin (15#ug/mg 2.36

ofmyelin protein)

3. PhospholipaseA(80,cg/mgofmyelinprotein) 2.44

Phospholipase A (80jug/mg)+trypsin (I5,pg/ 2.38

ofmyelinprotein)

4. Snakevenom (1504ug/mgofmyelinprotein) 1.10

Snakevenom(lSO,g/mg)+trypsin (25pug/mg 1.17

ofmyelin protein)

*Snakevenom(150ug/mg)+trypsin(154ug/mg 0.92

ofmyelinprotein)

tSnakevenom+trypsin(l50.ug/mg)+trypsin 0.92

(lSgg/mg of myclin protein)

5. Trypsin(15gg/mgofmyelin protein) 0.92

60 2.52 2.32 2.06 1.96 2.36 2.00 1.00

0.87

0.70 0.71

0.81

2':3'-CyclicAMP

Lossof phosphohydrolase protein (pmolofproduct/

(Y.of h persample)

controlvalue)

12.0 9.0 17.0 3.3 16.0 9.0 25.6 24.0

4576 4068

4219 3872

4366 3360

3986 3740

4135 3167

1907 1812

2138 1590

Lossof

enzymeactivity

(Y.of controlvalue)

5 12 8 23 6 14 5

25 26 18

Table 2 Loss of myelin proteins on incubationwithsnake venom, phospholipase andlysophosphatidylcholine in the presence

orabsence ofacetylatedtrypsin

Resultsare expressed as percentagelossofdifferent protein compared with control The symbols * and t are as in Table 1.

Lossof myelinproteins(Y.of control value)

Conditions

Myelin incubatedwith:

1 Snake venom(l00pg/mg of myelin protein)

Snakevenom(l00g,g/mg)+trypsin(15,g/mg

ofmyelin protein)

2. PhospholipaseA(80,ug/mgofmyelinprotein)

Phospholipase A (80pg/mg)+trypsin (15,g/

mg of myclin protein)

3 Snake venom(l50g/mgof myelin protein)

Snake venom(150,ug/mg)+trypsin(lSpg/mg

ofmyelinprotein)

4. Lysophosphatidylcholine(20,ug/mg of myelin

protein)

Lysophosphatidylcholine(20pg/mg)+trypsin

(lOpg/mgofmyelin protein)

Vol.159

time Wolfgram Proteolipid Basic protein Basicprotein

(min) protein protein (large) (small)

60

60

<5 <5 <5

275

N L BANIK, K GOHIL AND A N DAVISON

Effectofphosphatidylkholineonmyelin lipids

Lysophosphatidylcholinehadaless marked effect

than crudevenom enzyme or purephospholipaseA

onthecomposition ofmyelin lipids Onincubation

withlysophosphatidylcholinenosignificantchange in

the turbidity of myelin was found compared with

controls Although thehigher amountof

lysophos-phatidylcholine (1mg/mg of myelin protein,

incu-batedfor 14h) hadaneffectonmyelin proteins,the

effect waslessthan that obtained with crude snake

venom or purephospholipaseA

Action of crude snake venom andphospholipase A

onmyelinlipids

Although therewas bomeloss oflipidfound in the

samples treated with trypsin alone, no formation

oflysocompoundswasdetected

When myelin preparations were incubated with

either crude snakevenomorphospholipaseA inthe

presence orabsenceoftrypsin, themyelin

phospho-lipids,ethanolamine-containingphospholipids,

phos-phatidylcholine and phosphatidylserine were found

tohave been convertedintothecorrespondinglyso

compounds (Plate2).Therelativeratesofhydrolysis

ofphosphoglyceridestolysophosphoglyceridesin the

membrane were phosphatidylserine>

phosphatidyl-choline>ethanolaminephospholipid.The treatment

of myelin (1mg of myelin protein) with snake

venom (100,cg) showed that 74%of

phosphatidyl-choline,58%ofethanolaminephosphoglycerideand

83% ofphosphatidylserine were cleaved, and with

phospholipaseA(80,ug),57%ofphosphatidylcholine,

40% of ethanolamine phosphoglyceride and 63%Yo

of phosphatidylserine were hydrolysed compared

with the control.Most of thelysophosphoglycerides

were present in the pellet obtained after

centri-fugationoftheincubatedmyelin sample, and onlya

negligible amountoflysophosphatidylcholine could

bedemonstrated in thesupernatantfractionont.l.c

Thus phospholipase A present in the crude snake

venom was activefortheconversion of myelin

phos-phoglycerides into their lyso derivatives, whereas

galactolipid and cholesterol contents remained

un-changed The change observed in cholesterol and

cerebroside concentration afterincubation with either

snakevenom(100,ug)orphospholipaseA(80,cg)was

less than5% compared with thecontrol

No complete hydrolysis of myelin

phospho-glycerides was obtained even when the amount of

crude snakevenomwasincreasedto100-150,ccg/mg

of myelin protein Under these experimental

conditions theextentofhydrolysiswas greater than

that found with lesser amount of crude venom

(20,ug/mgofmyelin protein).The rateof hydrolysis

of phosphoglycerides was obtained by incubating

myelin at different times either with snake venom

(20pg)orphospholipase A.Phosphatidylserine was hydrolysed more rapidly than phosphatidylcholine andethanolamine phospholipid, and phosphatidyl-choline was hydrolysed faster than ethanolamine phosphoglyceride

Discussion

Since it has been proposed thatproteolyticenzymes areinvolved inthebreakdown ofthemyelin sheath in demyelinating diseases (Einsteinetal.,1969; Hallpike

etal., 1970; Ramsey et al., 1974; Smith & Rauch, 1974), we have previously taken the effect of a

proteolyticenzyme, trypsin,on myelinin vitro as a

possible model system (Banik & Davison, 1974; Wood et al., 1974) Although our studies with trypsin showed the loss oflipids, including neutral lipid, and basic encephalitogenic protein from myelin, therewasunexpectedlyno alterationin the ultrastructure of the myelin sheath Wood et al

(1974) had noted the same in their experiments

Wetherefore extended thisstudybyadding

phospho-lipase A or crude snake venom to our incubation medium in the presence oftrypsin, to evaluate the combinedeffectofthese enzymes onthedissolution

ofmyelin Whenisolated myelin is incubated with eitherlysophosphatidylcholineorphospholipase A,

thereisnoapparentloss ofprotein(small corrections

aremade forendogenousmyelinproteinaseactivity)

In thepresenceof trypsinthereisa15-30% lossof protein from the membrane After incubation of myelin with phospholipase Aorsnakevenomin the

presence of trypsin, this loss of myelin protein is showntobeduetodigestionnotonlyofbasicprotein

butalso ofproteolipid protein

The lipid profile ofthe pelleted myelin fractions showedaloss of all classes oflipidsandalso showed

theconversionof myelinphosphoglyceridesinto their corresponding lyso compounds Lysophospholipids were found to have remained with the pelleted

myelin membrane, and only small amounts were

detectable in the supernatant These results are in

agreement with Coles et al (1974), where they incubatedmyelin preparations withphospholipase A Thereis evidence fromthefindings of Poduslo &

Braun (1973) that basic protein is localized on the cytoplasmic side (dense period line) of the myelin and is therefore available in myelin preparations

totryptic digestion,whereasproteolipid proteinmay

be protected by itshydrophobiclipid environment (Folch, 1971) Once these lipids are removed the

proteolipid protein becomes exposed to proteolytic attack,leading,itis postulated,tothedisintegration

ofthemembrane

The disintegration of the myelin sheath was

observed in the electron micrograph of the incubated myelin sample, where splitting of the myelin lamellaewas evident (Plate1) After asplit

1976 276

DISSOLUTION OF MYELIN 277

of the intraperiod line or dense line, the peeled-off

myelinlamellae was found to have formed vesicular

structures This type of dissolution of the myelin

sheath has been demonstrated in experimental

allergic encephalomyelitis (Lampert & Carpenter,

1965; Lampert & Kies, 1967) Thevesicularmyelin

debris as well as the part of the intact sheath are

probably laterremoved byactivatedmacrophages in

thediseased condition

Elevated activities of phospholipase have since

been demonstrated in tissues from patients with

experimental allergic encephalomyelitis and also

in tissues from patients with multiple sclerosis

(Woelk & Kanig, 1974; Woelk & Peiler-Ichikawa,

1974) Increased proteinase has also been found in

experimental allergic encephalomyelitis and

de-myelinating tissues, both histochemically and

bio-chemically, by various investigators (Einsteineta!.,

1969;Hallpikeeta!., 1970;Cuzner&Davison, 1973;

Ramsey et al., 1974) In view of these findings,

phospholipase and proteinases may be jointly

involved in the degradation of the myelin sheath in

demyelinating diseases These hydrolases present

inactivatedmacrophages(David, 1975) may well be

responsible for the primary attack on the myelin

sheathinthedemyelinatingprocess

We thank the Multiple Sclerosis Society of Great

Britain andNorthernIreland forfinancial support, and

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Vol.159