Báo cáo khoa học: Characterization of structural and catalytic differences in rat intestinal alkaline phosphatase isozymes pdf

The coordinated metal at the active site was predicted to be a zinc triad in rIAP-I, whereas the typical combination of two zinc atoms and one magnesium atom was proposed for rIAP-II.. T

in rat intestinal alkaline phosphatase isozymes

Tsuyoshi Harada1, Iwao Koyama1, Toshiyuki Matsunaga1, Akira Kikuno1, Toshihiko Kasahara1, Masatoshi Hassimoto1, David H Alpers2and Tsugikazu Komoda1

1 Department of Biochemistry, Saitama Medical School, Saitama, Japan

2 Division of Gastroenterology, Washington University School of Medicine, St Louis, MO, USA

Alkaline phosphatases (EC 3.1.3.1) (APs) are dimeric

metalloenzymes that catalyze the hydrolysis of

phos-phate monoesters into inorganic phosphos-phate [1] The two

Zn2+and one Mg2+ligand combination at the catalytic site of APs is largely conserved from Escherichia coli (E coli) to humans and is essential for enzymatic

Keywords

3D modeling; intestinal alkaline

phosphatase; isozyme; rat; zinc

Correspondence

T Komoda, Department of Biochemistry,

Saitama Medical School, 38 Morohongo,

Moroyama-machi, Iruma-gun, Saitama

350–0451, Japan

Fax: + 81 492 76 1155

Tel: + 81 492 76 1155

E-mail: tkalp1lp@saitama-med.ac.jp

(Received 4 December 2003, revised 15

February 2005, accepted 17 March 2005)

doi:10.1111/j.1742-4658.2005.04668.x

To understand the differences between the rat intestinal alkaline phospha-tase isozymes rIAP-I and rIAP-II, we constructed structural models based

on the previously determined crystal structure for human placental alkaline phosphatase (hPLAP) Our models of rIAP-I and rIAP-II displayed a typ-ical a⁄ b topology, but the crown domain of rIAP-I contained an additional b-sheet, while the embracing arm region of rIAP-II lacked the a-helix, when each model was compared to hPLAP The representations of surface potential in the rIAPs were predominantly positive at the base of the active site The coordinated metal at the active site was predicted to be a zinc triad in rIAP-I, whereas the typical combination of two zinc atoms and one magnesium atom was proposed for rIAP-II Using metal-depleted extracts from rat duodenum or jejunum and hPLAP, we performed enzyme assays under restricted metal conditions With the duodenal and jejunal extract, but not with hPLAP, enzyme activity was restored by the addition

of zinc, whereas in nonchelated extracts, the addition of zinc inhibited duo-denal IAP and hPLAP, but not jejunal IAP Western blotting revealed that nearly all of the rIAP in the jejunum extracts was rIAP-I, whereas in duo-denum the percentage of rIAP-I (55%) correlated with the degree of AP activation (60% relative to that seen with jejunal extracts) These data are consistent with the presence of a triad of zinc atoms at the active site of rIAP-I, but not rIAP-II or hPLAP Although no differences in amino acid alignment in the vicinity of metal-binding site 3 were predicted between the rIAPs and hPLAP, the His153 residue of both rIAPs was closer to the metal position than that in hPLAP Between the rIAPs, a difference was observed at amino acid position 317 that is indirectly related to the coordi-nation of the metal at metal-binding site 3 and water molecules These find-ings suggest that the side-chain position of His153, and the alignment of Q317, might be the major determinants for activation of the zinc triad in rIAP-I

Abbreviations

AP, alkaline phosphatase; BAC, base of the active site cleft; CB, carbonate-bicarbonate; DM, double mutant; ECAP, Escherichia coli alkaline phosphatase; GCAP, germ cell-type alkaline phosphatase; hPLAP, human placental alkaline phosphatase; IAP, intestinal alkaline

phosphatase; M1, metal-binding site 1; M2, metal-binding site 2; M3, metal-binding site 3; PLAP, placental alkaline phosphatase; rIAP, rat intestinal alkaline phosphatase; SAP, shrimp alkaline phosphatase; TNAP, tissue-nonspecific alkaline phosphatase.

activity [2] In E coli, the Zn2+ ion at metal-binding

site 2 (M2) activates the hydroxyl group of Ser102,

which performs a nucleophilic attack on the phosphate

moiety of the substrate, resulting in a covalent

phos-phoseryl intermediate [3] A water molecule, activated

by the Zn2+ ion at metal-binding site 1 (M1),

hydro-lyzes this intermediate via a noncovalent

enzyme–phos-phate complex This phosenzyme–phos-phate moiety is then released

from the complex and the enzyme returns to its free

state Thus, of the three metal ions found at the active

site, the two Zn2+ions are thought to play important

roles in catalysis The role of the Mg2+ ion at

metal-binding site 3 (M3), however, is unclear A recent

study indicated that this Mg2+ion affects the

orienta-tion of Ser102, which alters the protein conformaorienta-tion

near the Zn2+ion at M2 [4]

A mutated E coli AP (ECAP), containing a triad of

Zn atoms at its active site, has been shown to be less

active than the wild-type AP [5], and green crab and

bovine kidney AP activity were reported to be

inhib-ited by sufficient concentrations of Zn2+, possibly by

the displacement of Mg2+ from its active site [6,7]

Bovine milk AP activity markedly decreased when the

Mg2+ ion at its active site was replaced by Zn2+ [8],

and a number of APs require the addition of Mg2+to

achieve maximum activity

Others have proposed that the first duplication of

the ancestral AP gene during the evolution of the AP

gene family produced a tissue-nonspecific type AP

(TNAP) and that subsequent duplications gave rise to

further modifications, resulting in tissue-specific APs

such as intestinal-type AP (IAP), placental-type AP

(PLAP), and germ cell-type AP (GCAP) [9,10] No

tissue-specific APs have, to date, been found in

inver-tebrates IAP was a late development in the AP gene

family, appearing for the first time in mammals [10];

IAP is represented by a single protein in most

mam-mals, although two IAP genes have been isolated in

the rat and four IAP genes have been isolated in the

cow [11,12] Neither PLAP nor GCAP isozymes have

been detected in rodents, and rat IAPs (rIAPs)

appear to be the sole tissue-specific isozymes in rats

[10,13] However, no significant preference for

differ-ent substrates has been noted between the rIAP

iso-zymes

Recently, the presence of a Zn triad at the active site

of shrimp AP (SAP) was confirmed by the analysis of

its 1.9 A˚ crystal structure [14] Furthermore, the

sensi-tivity of rat AP isozymes for Zn2+ has been reported

to differ, in particular, the AP activity from the small

intestine is not decreased by exogenous Zn2+ [15]

However, the presence of a Zn triad at an AP active

site has never been demonstrated in mammals

In this study, we used the swiss-model program to construct 3D models of rIAP-I and rIAP-II; we then analyzed the active site of the enzymes and studied the effect of various combinations of metals at the active site The models showed the possibility of a Zn triad

at the metal-binding positions of the active site in rIAP-I We also investigated whether the activity of metal-complemented rIAPs was stimulated by Zn2+

Results

The sequence alignment of rIAP-I and human PLAP (hPLAP) acquired using blast showed 75% identity and 85% homology, with no insertions or deletions relative to hPLAP The sequence alignment of rIAP-II and hPLAP revealed 77% identity and 87% homo-logy, with no insertions but the deletion of one residue relative to hPLAP

The structure of rIAP-I was predicted based on the structures of hPLAP, SAP, and the ECAP mutant (PDB entry codes: 1EW2, 1K7H, and 1KHJ, respect-ively) using the swiss-model program, and the struc-ture of rIAP-II was predicted based on the strucstruc-tures

of hPLAP, SAP, and two ECAP mutants (PDB entry codes: 1EW2, 1K7H, 1KHK, and 1KHL, respectively) The stereochemical parameters, main-chain parame-ters, and side-chain parameters (checked using pro-check) were within the allowable range for both rIAP-I and rIAP-II A Ramachandran plot showed that 86.0% of the residues lay in the most favoured regions, 11.8% in additionally allowed regions, 1.7%

in the generously allowed regions, and 0.5% in the disallowed regions of rIAP-I, with values of 87.2%, 11.4%, 1.2%, and 0.2% for the corresponding regions

of rIAP-II

After the models had been cleaned up by using pro-check, most of the overall structure of rIAP-I and rIAP-II was consistent with the structure of hPLAP (Fig 1A) [16] Each model showed a typical a⁄ b topol-ogy, with a central 10 b-sheet sandwiched between a set of helices The rIAP-I isozyme was composed of 23 a-helices and 14 b-sheets, whereas rIAP-II was com-posed of 22 a-helices and 15 b-sheets In addition to the a-helices of PLAP, residues 469–471 (468–470 in rIAP-II, because of the deletion of a single residue at 401) at the carboxy terminus formed a helix in both rIAPs, but the a-helix between residues 277–279 formed a turn in rIAP-II The b-sheet (423–425) in the crown domain of PLAP formed a coil in rIAP-I (Fig 1A)

Examination of the electrostatic potential of the act-ive site revealed that the base of the actact-ive site cleft (BAC) was clearly positively charged on the surface of

the rIAPs (Fig 1B) The edge of the BAC in hPLAP

was constructed from Phe107, Gln108, Arg166,

Asn167, and Tyr367 Residue differences at the BAC

of the rIAPs consisted of Tyr107 and Lys108, with the

addition of Asp167 in rIAP-I

The rIAP-II model contained a Zn ion at

posi-tions M1 and M2, respectively, and a Mg ion at

M3, as in hPLAP, whereas that of rIAP-I contained

a Zn ion at M1, M2, and M3 As rIAP-I was not

suited to interact with the Mg ion at M3, the active site of the rIAP-I model was completely occupied by

Zn ions A comparison of the residues serving as the ligand for the M3 metal ion is shown in Table 1 The residues of the direct ligands to the metal were the same as in hPLAP, but those of indirect ligands, which bind the metal through water molecules, were different at residue 317 (Gln in rIAP-I and Arg in rIAP-II)

Fig 1 Comparison of the overall structures of monomeric rat intestinal alkaline phosphatases (rIAPs) (A) The overall structure of the mono-meric rIAPs and human placental alkaline phosphatase (hPLAP) is represented by ribbons, with the a-helix in red and the b-sheet in light blue Differences between the APs are indicated by asterisks The a-helix of the carboxyl terminal is denoted by the orange asterisk; the crown domain region is shown by the yellow asterisk, and the embracing arm is shown by the pink asterisk (B) Representation of surface potential, prepared using GRASP The potentials range from negative, in red ( )25), to positive, in blue (+ 25) The active site is circled in yellow.

rIAP-I and rIAP-II are expressed in the duodenal

mucosa, but the jejunal mucosa only expresses the

rIAP-I isozyme [17,18] We assayed the AP activity of

duodenum and jejunum mucosal extracts in a standard

buffer supplemented with 1 mm Zn (Fig 2A) The

exo-genous excess of Zn2+had no effect on the AP activity

in the jejunal extract A buffer containing 1 mm Zn

and 1 mm Mg inhibited the duodenal AP activity by

41% and the hPLAP activity by 33% Under

metal-free conditions, with no metals in the samples or in the

assay buffer, the AP activity of the duodenal and

jejunal extracts and of hPLAP was 90% lower than

the activity in nonchelated samples (Fig 2B)

Metal-chelated samples from the small intestine, but not of

hPLAP, were activated by 1 mm Zn under Mg-free

conditions, but duodenal activation was only 60% of

the degree of activation seen using jejunal extracts

The metal-chelated sample of hPLAP was not

activa-ted by the addition of exogenous Zn2+ alone The

presence of Mg2+ was required to obtain maximum

activity in all the metal-chelated samples When the

metal-chelated samples were assayed in an assay buffer

containing 1 mm Mg and 20 lm Zn, the activity levels

recovered to 60–70% of the level in nonchelated

extracts (data not shown)

The percentage of rIAP isozymes in both duodenum

and jejunum was examined by Western blotting (Fig 3)

In the jejunum, one rIAP band, with an apparent

molecular mass of 70 kDa, was present, but two bands

of 88 kDa and 75 kDa were detected as rIAP-II and

rIAP-I in the duodenum The molecular mass values

of the rIAPs were similar to those of previous reports

[19,20] When the quantitative ratio of the rIAPs was

determined using densitometry, 96% of the IAPs in

the jejunum were identified as rIAP-I In the

duo-denum, the proportion of rIAPs was 55% and 45%

for rIAP-I and rIAP-II, respectively The 55%

contri-bution of rIAP-I to total IAP activity correlates almost

exactly with the observation that Zn2+

supplementa-tion in metal-chelated condisupplementa-tions activates duodenal

AP to a level that is 60% of that seen with jejunal extracts (Fig 2B) These data suggest that the finding

of Zn2+ inhibition without metal chelation and of

Zn2+ activation with metal chelation (Fig 2) can be explained by the proportion of rIAP-I in the duodenal and jejunal extracts

Most of the active-site residues in APs have been perfectly conserved throughout evolution [21] In view

Fig 2 Effect of zinc on alkaline phosphatase activity Each of the extracts was adjusted to an activity level of between 200 IUÆmL)1 and 400 IUÆmL)1by dilution with carbonate-bicarbonate (CB) buffer prior to use in the assay The assay was performed using non-chelated (A) and non-chelated (B) extracts The relative activity is the proportion of the residual activity compared with that in nonche-lated extracts assayed in a CB buffer containing 1 m M Mg Each value for the rat duodenal and jejunal extracts represents the mean ± SD of data from four animals.

Table 1 Distances (A ˚ ) between the residues and the metal at

metal-binding site 3 (M3) hPLAP, human placental alkaline

phos-phatase; rIAP, rat intestinal alkaline phosphatase.

a Distances were calculated between OE1 and the metal in hPLAP,

and between OE2 and the metal in the rIAPs The metal position

was based on the M3 position of hPLAP.

of the Zn2+ activation of rat APs, we further

exam-ined the M3-related residues (Table 2) No differences

among residues that directly interact with the metal at

the M3 site were found in mammalian tissue-specific

APs, but a specific difference in ligands with an

indi-rect interaction was observed: Gln317 in rIAP-I, and

Arg317 in rIAP-II The His317 of hPLAP is known to

be indirectly associated with the M3 site and to

main-tain the Mg ion at this site via a water molecule [16]

Comparative stereoviews of the M3 site residues in

the active site are shown in Fig 4 Mg2+ at the M3

site is coordinated octahedrally with three residues and

three water molecules, i.e the OD2 oxygen atom of

Asp42, the OG2 oxygen atom of Ser155 (Thr155 in

E coli), and the OE1 oxygen atom of Glu311 in

hPLAP [16,22] The positions of Asp42, Ser155, and Glu311 did not vary significantly from their positions

in the side-chains of hPLAP His153 was clearly closer

to the metal position in rIAP than in hPLAP A com-parison of the distances between the residues and the metal revealed that His153 was the closest residue to the metal at 2.40 A˚ in rIAP-I and was closer to the metal than Glu311 in rIAP-II (Table 1) Because of these differences in the side-chain structure, the posi-tion of the water-interacting atom, i.e a nitrogen atom

of Gln317 in rIAP-I and a nitrogen atom of Arg317 in rIAP-II, was also different from that in hPLAP

Discussion

The rat duodenal mucosa contains two types of rIAP mRNA (a 2.7 kb mRNA and a 3.0 kb mRNA) that encode different rIAP genes – rIAP-I and rIAP-II, respectively [11,23] The rIAP-I gene is expressed in both duodenal and jejunal mucosa, whereas the

rIAP-II gene is expressed only in duodenal mucosa [17,18] Differences in substrate preferences and kinetic param-eters have been observed between these two isozymes [17,18] The expression of these isozymes responds differently to fat feeding, 1,25-dihydroxyvitamin D3, cortisone, and lipopolysaccharide [18,24–27] However,

Table 2 Residues constituting metal-binding site 3 (M3) The resi-due numbers correspond to the human placental alkaline phospha-tase (hPLAP) sequence number H153 and H317 in hPLAP are homologous to D153 and K328 in Escherichia coli and to H149 and H316, respectively, in shrimp AP The alkaline phosphatase (AP) abbreviations are as follows: ECAP, E coli AP; hGCAP, human germ cell AP; hIAP, human intestinal alkaline phosphatase; hTNAP, human tissue-nonspecific AP; mIAP, mouse IAP; mTNAP, mouse tissue-nonspecific AP; rIAP, rat intestinal alkaline phosphatase; rTNAP, rat tissue-nonspecific AP; SAP, shrimp AP.

Residues at M3

a Residues indirectly associating with the metal via water mole-cules.

A

B

Fig 3 Immunological detection of rat intestinal alkaline

phospha-tase (rIAP) isozymes in rat small intestine Western blotting was

performed, using an antiserum against rat IAP, in duodenal and

jej-unal mucosa extracts (A) The same extracts (2 lL) used for the AP

activity assay in Fig 2A were applied to SDS⁄ PAGE and blotted, as

described in the Experimental procedures This representative

pho-tograph shows the results for one of the four animals that were

examined (B) The abundance of rIAP isozymes in rat small

intes-tine was determined by zone densitometry The data represent the

mean ± SD of four experiments.

the physiological role and expression of two isozymes

in the same organ, particularly when their

physiologi-cal substrates differ, is not clear

rIAPs, as well as other mammalian APs, possess

additional secondary structural elements compared

with ECAP, such as: N-terminal a-helices; an

embra-cing arm region (residues 208–280) that connects with

other monomers; a noncatalytic metal-binding site;

and the ‘crown domain’ (residues 366–430) [16] A site

for collagen attachment has been localized in the loop

comprising residues 405–435 in the crown domain of

TNAP [28] When the TNAP gene is inactivated and

the crown domain is lost, the disorder that results

(hypophosphatasia) is characterized by poorly

mineral-ized bone [29–31] This crown domain allows

mamma-lian APs, which act as monoester phosphohydrolases,

to display a substrate preference The embracing arm region has been reported to be important in joining the two AP monomers of AP together [32] In a vari-ety of human cancer cell lines and sera, the Kasahara

AP isoform has been found to consist of heterodimers

of hIAP and hPLAP [33,34], and the human postnatal intestine also contains heterodimers of hIAP and hPLAP [35] Ovarian cancer cells and cell lines derived from these cells express heterodimers of hPLAP and hGCAP [36–38] While no heterodimers of rIAP-I and rIAP-II have been detected in rats, they must have an important role in the identification of each isozyme to form a homodimer in the same organ The structural differences between rIAPs observed in the crown

A

B

Fig 4 Stereoviews of the residues near metal-binding site 3 in (A) rat intestinal alkaline phosphatase-I (rIAP-I) and (B) rIAP-II The white resi-dues represent those seen in human placental alkaline phosphatase (hPLAP), and the orange sticks with red tips represent PO 4 The posi-tions of the metal, represented by a brown sphere, and of PO4, were constructed based on data for hPLAP (PDB code: 1EW2).

domain and the embracing arm are clues to the

physiological and functional differences between these

two rIAP isozymes

The base of the active site in rIAPs is a more clearly

positively charged patch than that in hPLAP, and

ECAP does not exhibit this positivity As a result,

neg-atively charged substrates, such as phosphomonoesters,

are drawn strongly towards the active site Mammalian

APs have 10–100-fold higher kcatvalues than bacterial

APs, and IAP has the highest specific activity of all

human and rat APs [39,40] The surface charge of this

region must be partly responsible for its activity A

similar observation has been made in regard to SAP,

and the surface charge of SAP is thought to optimize

the direction of the substrate to the active site in cold

(5
C) environments [14]

One of the striking findings of this study was that

exogenous Zn2+ can restore the activity of

metal-chelated rIAP, but not that of hPLAP, as predicted

using a 3D-model A number of mammalian APs are

thought to require the presence of Mg2+ to achieve

maximal activity An excess of Zn2+inhibits the

activ-ities of AP in all tissues, and this inhibition is

attrib-uted to the displacement of Mg2+ from its M3 site by

Zn2+[1,7,8] Hung & Chang [41] showed that the

con-formation of Zn2+ at the M3 site is unfavorable for

catalysis in hPLAP and that both Mg2+ activation

and Zn2+ inhibition of AP are reversible processes In

the rat, Andeniyi & Heaton [15] reported that the

addition of Zn2+ (0.01 mm) decreased the activity of

TNAP isolated from the liver and the kidney, but

increased the activity of AP isolated from the small

intestine These findings are consistent with the

con-cept that the IAP isozyme binds Mg2+at the M3 site

more strongly than TNAP and hPLAP [1,15,21] Based

on our results, we speculated that the rIAPs,

partic-ularly rIAP-I, are activated by Zn2+, even if Zn2+

occupies all three metal-binding sites Thus, Zn2+ can

act as a catalytic coordinator at the M3 site of rIAP-I

Catalytic differences in metal preferences between

rIAPs and hPLAP may reflect the subtle coordination

of amino acids constructing the M3 space No

differ-ences in amino acid alignment have been observed

between SAP and rTNAP, yet Zn2+ and Mg2+ are

preferentially selected at the M3 sites, respectively [14]

The wild-type hPLAP favors Mg2+at the M3 site, but

the Chelex-treated mutant Gly429 hPLAP can be

acti-vated by low concentrations (0.5–20 lm) of Zn2+

[41,42] Mg2+ coordination at the M3 site can be

des-cribed as a slightly distorted octahedron involving the

OD2 oxygen atom of Asp42, the OG2 oxygen atom of

Ser155, the OE1 oxygen atom of Glu311, and three

water molecules The structure of hPLAP indicates

that His153, while coordinated with a water molecule

at the M3 site, is too far from the Mg2+at the M3 site

to function as a ligand for the M3 metal [16] Metal specificity can be altered by a single amino acid substi-tution, and the X-ray structure of a mutant ECAP, the D153H mutant enzyme, showed that the replacement

of Asp153 with histidine changed the metal at the M3 site from an octahedrally coordinated Mg2+ in the wild-type structure to a tetrahedrally coordinated zinc; the K328H mutant and the double mutant D153H⁄ K328H (DM) enzyme also contained a Zn triad at their active sites [5,43,44] Sequence alignments showed that this histidine residue is conserved in mammalian sequences, including hPLAP, although a Mg ion is reported to occupy this site [16] The side-chain of His153 in D153H and DM_ECAP is sufficiently close

to be a direct ligand of the metal; thus, the metal coordination changes from octahedral to tetrahedral [5]

In addition to the distance between the metal posi-tion at M3 and His153, His317 is also important for coordinating the water molecule that interacts with

Mg2+ at the M3 site and with the phosphate group When His317 in hPLAP was mutated to Ala317, the mutant enzyme had higher kcat and Km values than the wild-type hPLAP [21] This finding indicates that the disruption of the water molecule results in an enzyme with a lower affinity for both its substrate and its products and that affects the interaction between the phosphate group and the water molecule All mammalian APs discovered to date, except for the rIAPs and mouse IAP (mIAP), possess His317 (rIAP-I contains Q317, while rIAP-II and mIAP contain R317) [11] We confirmed that a jejunal homogenate, rIAP-I, had a higher sensitivity to Zn2+ activation Therefore, the Q317 of rIAP-I may affect the water molecule in a manner that enables the water molecule to interact with the phosphate residue and catalyze the substrate

in the presence of a Zn triad

In conclusion, this structural analysis of rIAPs revealed important differences that may provide clues regarding the physiological role of phosphate monoest-erases in the small intestine We confirmed that when a

Zn triad is present at the active site, rIAP-I can be activated under Mg-free conditions We speculate that the dephosphorylation targets of rIAP-I and rIAP-II may differ Furthermore, we demonstrated that rIAP-I, which is a nonmutated mammalian AP containing His153, is functional and able to coordinate Zn2+ at the M3 site, as in the ECAP mutant, D153H These rIAP models suggest that the distance between the metal position at M3 and His153 is important for the selective affinity of the metals Moreover, these models indicate that Q317 is the key amino acid responsible

for coordinating the water molecules and metals in the

process of enzyme activation However, to prove that

M3 is actually occupied by a metal in rIAPs, further

study would be needed to define the precise molecular

structure by X-ray crystallography

Experimental procedures

Modeling and structural analysis of rIAP-I and

rIAP-II

The rIAP-I and rIAP-II sequences were aligned to the

hPLAP sequence, which was elucidated by analysis at a

1.8 A˚ resolution [16] using the blast program [45]

The rIAP-I and rIAP-II monomeric models were

con-structed and optimized by using the swiss-model and

swiss-Pdb Viewer programs [46] The quality of the model

geometry was checked and cleaned-up by using the

pro-checkprogram [47] The protein surface and the

combina-tions of metals and ligands were analyzed by using the

grassprogram [48] The residue accessibility was calculated

and visualized by using the DSTM ViewerPro program

(Accelrys)

Animals

All experimental protocol were approved by the Animal

Research Committee of Saitama Medical School prior to

the start of the experiments Specific pathogen-free male

[Crj:CD(SD)IGS] rats (340–380 g) were obtained from

Charles River Japan (Yokohama, Japan) and used in all

the experiments The rats were individually housed in

stain-less steel cages with self-watering systems under specific

pathogen-free conditions and were given access to sterilized

rat chow ad libitum The rats were allowed to acclimatize

for 1 week before the start of the experiment Rats were

killed by exsanguination following the intraperitoneal

administration of sodium pentobarbital anesthesia (50

mgÆkg)1of body weight): the abdomen was opened and the

duodenum and jejunum were removed

Enzyme preparation

After opening the intact bowel longitudinally, the mucosal

surface was washed with Tris⁄ HCl buffer (10 mm, pH 7.8)

containing 1 mm MgCl2 Immediately after the tissues had

been washed, they were homogenized in nine volumes of

the Tris⁄ HCl buffer at 4
C for 1 min using a Potter-type

homogenizer The homogenates were centrifuged at 9000 g

for 30 min, and the postmitochondrial fractions were

pooled as samples The hPLAP was obtained from

Sigma-Aldrich Chemicals Co (St Louis, MO, USA) and further

purified by ordinary method [49] The purity of hPLAP was

confirmed, by electrophoresis, as a single band, and the

enzyme was replaced in a 50 mm carbonate-bicarbonate (CB) buffer (pH 9.8)

Enzyme assay All solutions used in the assay were treated with

Chelex-100 resin (Bio-Rad Laboratories, Richmond, VA, USA) using the batch method, stored in Chelex, and filtered before use

The metal-chelated samples were prepared by the addi-tion of 100% (w⁄ v) Chelex-100 for 12 h, and the superna-tants were collected after a brief centrifugation

AP activity was assayed by measuring the amount of p-nitrophenol released from disodium p-nitrophenylphos-phate (p-NPP) The samples were preincubated for 5 min with 240 lL of 50 mm CB buffer containing the indicated amounts of MgCl2and⁄ or ZnCl2.The reactions were star-ted by the addition of 60 lL of CB buffer containing

50 mm p-NPP at 37
C and monitored spectrophotometri-cally at 405 nm

Western blotting

To remove the membranous moiety of AP isozymes bearing

a glycan-phosphatidylinositol anchor, an equal volume of n-butanol was added to the postmitochondrial samples The aqueous phase was collected and dialyzed against Tris⁄ HCl buffer These samples were then subjected to elec-trophoresis on an SDS⁄ PAGE (8% acrylamide) gel under reducing conditions The separated proteins were trans-ferred to Immobilon-P membranes (Millipore, Bedford,

MA, USA) at 0.4 mA for 1 h at 4
C and blocked over-night in Tris⁄ HCl-buffered saline, pH 7.8, containing 5% (w⁄ v) nonfat dry milk and 0.1% (v ⁄ v) Tween-20 The mem-branes were then washed with Tris⁄ HCl-buffered saline containing 0.1% (v⁄ v) Tween, and the rIAP bands were detected using a previously characterized rabbit anti-(rat IAP) serum that cross-reacted with both I and

rIAP-II, but not with rTNAP [19] The membranes were incuba-ted in a buffer containing this antiserum (at a dilution of

1 : 5000; v⁄ v) for 1 h at room temperature After washing with Tris⁄ HCl-buffered saline containing 0.1% (v⁄ v) Tween, the membranes were incubated for 1 h at room temperature with anti-rabbit horseradish peroxidase-linked antibody (diluted 1 : 10 000; v⁄ v) as the secondary anti-body The rIAP bands labeled by these antibodies were visualized using an enhanced chemiluminescence kit (Amer-sham Pharmacia Biotech, Bucks., UK) and a CCD camera (ATTO, Tokyo, Japan)

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