Purification of circulating liver plasma membrane fragments using a monoclonal antileucine aminopeptidase antibody

Purification of Circulating Liver Plasma MembraneFragments Using a Monoclonal Antileucine

Aminopeptidase Antibody

JING T. DENG,1 MARC F. HOYLAERTS,1 ETIENNE J. NOUWEN,1 MARC E. DE BROE,1 AND VIVIANE O. VAN HOOF2

gesting some heterogeneity in the membrane composi-Membrane-bound liver alkaline phosphatase (Mem-tion of these fragments. (HEPATOLOGY 1996;23:445-454.)LiALP, EC 3.1.3.1.) is a high-molecular-mass liver

alkaline phosphatase (ALP) present in metastatic, infil-trative and cholestatic liver disease. Shedding of hepato- High molecular mass liver alkaline phosphatasecyte plasma membrane fragments (LiPMF) is thought to (ALP, EC 3.1.3.1.) was first described in 1972 by Shin-be responsible for the appearance of Mem-LiALP in the kai and Akedo1 in serum of hepatic cancer patients as acirculation. Several other membrane-bound enzymes, multi-enzyme complex containing various liver plasmasuch as g-glutamyltransferase (g-GT), leucine aminopep- membrane bound enzymes. The investigators sug-tidase (LAP), and 5*-nucleotidase (5*-Nu) are present in

gested that this multi-enzyme complex representedthe membrane of the shedded LiPMF. By means of im-liver plasma membrane fragments (LiPMFs), a hypoth-munohistochemical and immunoassay procedures, weesis substantiated by De Broe et al. in 19852 based onpresently show that AD-1, a specific monoclonal anti-

body originally produced against MemliALP, reacts with physical, morphological, histochemical, and immuno-LAP, a constituent of the human liver plasma mem- logical evidence. Shedding of hepatocyte plasma mem-brane. Using AD-1 as an immunosorbant, we isolated cir- branes is thought to be involved in the release of mem-culating LiPMF from cholestatic sera to a high level of brane-bound liver alkaline phosphate (Mem-LiALP)purity and separated it from other high-molecular-mass into the circulation.3 Because of its structural charac-material, such as liver ALP Ç lipoprotein-X complexes. teristics, high molecular mass liver ALP was recentlyThese purified membrane fragments retained their bio- renamed Mem-LiALP.4 Several studies using differentchemical characteristics. Glycosyl-phosphatidylinositol

supporting media during electrophoresis, such as agar,anchor bearing liver ALP (Anch-LiALP) could be re-agarose, starch, polyacrylamide gel, and cellulose ace-leased from the LiPMF by Triton X-100. Whereas ALPtate, showed that Mem-LiALP was present in meta-was released upon treatment of AD-1 purified LiPMF

with phospholipase C, phospholipase D only cleaved the static, infiltrative, and cholestatic liver diseases.5,6 Be-glycosyl-phosphatidylinositol anchor following deter- cause of its sensitivity and specificity, Mem-LiALP hasgent solubilization of the enzyme. Serum LiPMF from been advanced as a serum marker of hepatic metasta-patients with different kinds of cholestatic liver disease sis.4,7,8

were bound onto AD-1 coated nitrocellulose disks and Several other membrane-bound enzymes, such as g-the activity of four membrane-bound enzymes (LAP, glutamyltransferase (g-GT), leucine aminopeptidaseALP, 5*Nu,g-GT) was analyzed. A considerable interindi- (LAP), and 5*-nucleotidase (5*-Nu) are present in thevidual variation of enzyme activities was observed, sug-

membrane of the shedded vesicles. Both ALP and 5*-Nu are attached to the plasma membrane by meansof a glycosyl-phosphatidylinositol anchor (GPI-anchor),Abbreviations: ALP, alkaline phosphatase; LiPMF, liver plasma membraneand can be released in vitro by the action of GPI-specificfragments; MemLiALP, membrane-bound liver alkaline phosphate; g-GT, g-

glutamyltransferase; LAP, leucine aminopeptidase; 5*-Nu, 5*-nucleotidase; phospholipase-C (GPI-PLC).9,10 The precise mechanismGPI, glycosyl-phosphatidylinositol; GPI-PLC, GPI-specific phospholipase-C; of the in vivo release of these enzymes and the role ofGPI-PLD, GPI-specific phospholipase-D; AD-1, monoclonal antibody; phospholipases in this process is still unclear. It hasFrom the 1Department of Nephrology-Hypertension, University of Antwerp,

been speculated that GPI-specific phospholipase-DWilrijk, Belgium, and 2Department of Clinical Chemistry, University HospitalAntwerp, Edegem, Belgium. (GPI-PLD), abundantly present in serum, might be re-

Received December 30, 1994; accepted October 3, 1995. sponsible for the in vivo release of GPI-anchored pro-Supported by grants from the Sportvereniging tegen Kanker and the Vere- teins.11 Shedded plasma membrane vesicles are easily

niging voor Kankerbestrijding, Brussels, Belgium. isolated from cholestatic serum by chromatography onAddress reprint requests to: Viviane Van Hoof, M.D., Ph.D., Departmentsepharose 4B, but other high-molecular-mass material,of Clinical Chemistry, University Hospital Antwerp, Wilrijkstraat 10, B-2650

Edegem, Belgium. such as ALP-lipoprotein-X complexes, coelutes with theDr. Hoylaerts’s current address is: Center for Molecular and Vascular Biol- vesicles during this procedure.12,13 In a previous re-

ogy, University of Leuven, Leuven, Belgium. port,14 we described the production of a monoclonal an-Copyright q 1996 by the American Association for the Study of Livertibody (AD-1) against human Mem-LiALP that did notDiseases.

0270-9139/96/2303-0008$3.00/0 cross-react with liver ALP. We have already used this

445

5p0b$$0009 02-20-96 17:52:18 hepa WBS: Hepatology

446 DENG ET AL. HEPATOLOGY March 1996

LAP bands were visualized by incubating the gel for 30 min-antibody to develop a sensitive and accurate methodutes at room temperature with a 2 g/L Fast Garnet GBCfor the quantification of Mem-LiALP in serum by bind-(Sigma, St. Louis, MO) solution in 0.25 mmol/L Tris-maleateing it to AD-1–coated nitrocellulose membrane disks.14

buffer, pH 6.2.In this study we have presently characterized the AD-Isolation of Mem-LiALP Bearing Vesicles (LiPMF) From1 antibody in more detail and have clarified that LAP

Cholestatic Serum. Serum Mem-LiALP was partially puri-(EC 3.4.11.1), an enzyme constituent of the plasma fied by gel filtration on Sepharose 4B (Pharmacia, Uppsala,membrane, is the true entigen recognized by AD-1. The Sweden) in 50 mmol/L Tris-HCl buffer, pH 8.0, containingavailability of pure LiPMF would allow, among several 100 mmol/L NaCl and 0.05% NaN3. The ALP fractions elut-other possibilities, the study of the releasing mecha- ing in the void volume were collected and used for further

purification either by immunoaffinity chromatography onnism of enzymes from liver cell plasma membrane.AD-1 columns, or by binding on nitrocellulose disks coatedTherefore, we have used AD-1 as an affinity adsorbentwith AD-1. To set up the AD-1 MoAb immunoaffinity column,to purify LiPMF from the serum of different individualsa quantity of activated CH-Sepharose 4B (Pharmacia LKBwith cholestasis. Then the effect of detergents and ofBiotechnology, Uppsala, Sweden) was swollen in 0.5 mol/Lphospholipases (GPI-PLC and GPI-PLD) on the releaseNaCl and washed on a sintered glass filter. AD-1 was addedof enzymes from the purified LiPMF was studied, as to the beads and incubated for 2 hours at room temperaturewell as the plasma membrane enzyme composition. with gentle mixing. Remaining active-free carboxyl groupson the beads were blocked by incubating them with 0.1 mol/PATIENTS AND METHODSL Tris-HCl buffer, pH 8.0, for another 2 hours at room tem-perature. Unbound proteins were removed by washing withSerum samples were obtained from patients with chole-

static liver disease and from healthy individuals by using three alternating cycles of 0.1 mol/L sodium acetate (pH 4.0)and 0.5 mol/L NaCl, followed by a bicarbonate buffer. Thevenipuncture. The samples were analyzed within 1 week of

storage at 47C or were analyzed within 1 month when stored beads were packed into a column (21 18 cm) and equilibratedwith the starting buffer (50 mmol/L Tris-HCl buffer, pH 8.0,at 0807C.

Enzyme Assays. ALP activity was determined in microti- containing 200 mmol/L NaCl and 0.05% NaN3). ALP fractionscollected from the Sepharose 4B column or unfractinated se-terplates according to the method recommended by the Inter-

national Federation of Clinical Chemistry, Copenhagen, Den- rum were used to purify Mem-LiALP bearing vesicles. Thesamples were loaded onto the immunoaffinity column at amark,15 by using 1 mol/L diethanolamine buffer, pH 9.8,

containing 1 mmol/L ZnSO4, 2 mmol/L MgCl2, and 16 mmol/ rate of 24 mL/hr, and the column was washed extensivelywith the starting buffer. The LiPMF were eluted either by aL p-nitrophenyl phosphate as substrate at 377C. After incuba-

tion, absorbance measurements were performed on a microti- linear gradient of 0 to 3 mol/L NaSCN to find the optimalconcentration of NaSCN or stepwise with 3 mol/L NaSCNterplate reader (EAR easy reader EAR 400, SLT Lab Instru-

ments, Grdig, Austria). The ALP activities were expressed as dissolved in the washing buffer. NaSCN in the eluted sam-ples was immediately removed by dialysis.optical density at 405 nm or calculated with help of standard

dilutions. The activities of g-GT, 5*-Nu, and LAP were mea- Antibody-conjugated Nitrocellulose Membrane Disks. Themonoclonal antibody AD-1 was bound to membrane diskssured at 377C in 96-well plates, using the following reagent

kits: Enzyline 5*-Nu optimise unitaire (Biomerieux, Marcy as previously described.13 Shortly, nitrocellulose membranedisks, diameter 6 mm, (Hybond-C, Amersham, Bucks, En-l’Etoile France) for 5*-Nu, g-GT New (Boehringer, Mann-

heim, Germany) for g-GT and 3359 LIVER LAP (Merck, gland) were agitated for 3 hours at room temperature withpurified rabbit antimouse-Ig antibody(RAM) at 80 mg/cm2 ofDarmstadt Germany) for LAP.

Treatment with monoclonal antibody AD-1 was performed nitrocellulose surface in incubation buffer (50 mmol/L Tris-HCl buffer, pH 8.0, containing 0.15 mol/L NaCl and 0.05%by incubating 1 to 5 mg antibody/mL serum for 2 hours at

377C before electrophoresis. NaN3). After blocking with a 10% saturated casein solution,disks were washed with 10 mmol/L Tris-HCl buffer, pH 8.0,Electrophoresis. Agarose gel electrophoresis of ALP was

performed on Isopal gels (Beckman/Analis, Namur Belgium). containing 0.15 mol/L NaCl and 0.05% Tween 20, and furtheragitated overnight at 47C with AD-1 containing hybridomaElectrophoresis was performed either in the presence of de-

tergents16 or in the absence of detergents.17 For the later supernatant. The coated disks were stored at 47C until use,in a small volume of incubation buffer. Binding of the LiPMFelectrophoresis, the agarose gels were rehydrated in electro-

phoresis buffer-lacking detergents, instead of the equilibra- was achieved by incubating one disk in each well of a 48-wellplate with 150 mL of incubation buffer and 50 mL of serumtion buffer that contains a mixture of detergents (Analis,

Namur, Belgium). Triton X-100 polyacrylamide gel electro- or partially purified test sample, after which the disks wereagitated at 377C for 3 hours. In some experiments, the disksphoresis of LAP was performed as follows: nontreated and

AD-1–treated samples were incubated with an equal volume were then treated with different concentrations of Triton X-100 (0% to 2%) (Boehringer Mannheim GmbH Diagnostica,of buffer (0.5 mol/L Tris-HCl, pH 6.8, containing 1% Triton X-

100, 20% sucrose, 0.005% Bromophenol blue) for 10 minutes Mannheim, Germany) for 30 minutes at room temperature,or with a 20 U/mL solution of either GPI-PLC (EC 3.1.4.10,before application to the gel. Electrophoresis was performed

in 7.5% polyacrylamide gels containing 0.375 mol/L Tris-HCl from Bacillus cereus, Sigma, St. Louis, MO) or GPI-PLD (EC3.1.4.50, from bovine serum, Boehringer Mannheim, Mann-buffer, pH 8.8, and 0.5% Triton X-100 with a 3.75% stacking

gel containing 0.125 mol/L Tris-HCl, pH 6.8, and 0.5% Triton heim, Germany) in Tris-HCl buffer, pH 7, for 2 hours at 377C.Finally, the enzyme activities of ALP, 5*-Nu, g-GT, LAPX-100. Gels were run at 100 volts for 2 hours in 0.38 mol/L

Tris-borate buffer, pH 8.6, containing 0.5% Triton X-100. LAP bound on the AD-1–coated nitrocellulose membrane diskswere measured as described above.activity was detected by incubating the gel for 2 hours at

377C in a substrate solution containing 20 mg of L-leucyl-b- Lipoprotein-X was detected with the Rapidophor kit (Im-muno Diagnostics, Brussels, Belgium), according to the in-naphthylamide (dissolved by mixing it with 2 mL of 10 mmol/

L HCl and 50 mL of 0.02 mol/L phosphate buffer, pH 7.0). structions of the manufacturer. When the precipated arc was


HEPATOLOGY Vol. 23, No. 3, 1996 DENG ET AL. 447

visualized, as a result of overlaying the gel with a polyanionicsolution containing heparin and polyanions, gels were photo-graphed. After quickly washing the gel to eliminate excessreagents, the gel was dried and incubated with a 2-amino-2-methyl-1-propanol buffer, pH 10.4, containing 1.89 mmol/L5-bromo-4-chloro-3-indolylphosphate as substrate for 30 min-utes at 377C to visualize ALP activity.

Immunohistochemical Staining of LAP With AD-1. Normalhuman liver and kidney tissue were frozen in liquid nitrogenand cut into 5-mm-thick sections. The sections were mountedon poly-L-lysine (0.1 mg/mL) coated glass slides and dried atroom temperature for 1 hour, then fixed in 100 mL of a 1:1chloroform/acetone solution for 5 minutes. After equilibrationin tris buffer saline and treatment with normal horse serum(1:5) for 20 minutes, the AD-1 solution (4 ng/mL) was applied FIG. 1. Agarose gel electrophoresis (in the absence of detergents)and the glass slides were incubated overnight. The sections of Sepharose 4B gel filtered Mem-LiALP (lane 1 and 2) and after its

further purification on AD-1 Sepharose (lane 3 and 4). Mem-LiALPwere then washed and treated with biotinylated affinity-puri-is shown nontreated (lane 1 and 3) and after preincubation with AD-fied horse antimouse immunoglobulins for 30 minutes, fol-1 for 1 hour (lane 2 and 4).lowed by the avidin/biotin/peroxidase complex (Vector labora-

tories Inc., Burlingame, CA) for 1 hour. All dilutions weremade in TBS. After extensive washing, peroxidase wasstained with 0.02% 3-amino-9-ethyl carbozole and 0.002% avidin/biotin/peroxidase complex. Peroxidase staining wasH2O2 in 20 mmol/L acetate buffer (pH 5.2) containing 9.5% performed in 0.1 mol/L Tris-HCl buffer, pH 7.6, with 0.5 mg/dimethyl sulfoxide. Control staining was performed on adja- mL diaminobenzidine and 0.03% hydrogen peroxide in thecent sections by replacing AD-1 with culture supernatant presence of 0.01 mol/L imidazole. The stained disks werecontaining a mouse monoclonal antibody of the same immu- fixed in 2% glutaraldehyde for 1 hour and 2% osmium tetrox-noglobulin class (IgG1) but of irrelevant specificity. The sec- ide for 2 hours and were embedded in SPURR.19

tions were counterstained with methyl green and mountedin glycerin/gelatin mounting medium. RESULTS

Histochemical Staining of LAP. The adjacent sections ofthe previous experiment were used to localize LAP histo- Isolation of Mem-LiALP (LiPMF)chemically by the method of Nachlas et al.,18 with L-leucine-

Serum Mem-LiALP was separated from low-mo-4-methoxy-b-naphthylamide HCl as substrate and 0.1% fastlecular-mass ALPs by Sepharose 4B gel filtration.blue as color reagent, in 0.1 mol/L Na-Acetate buffer, pH 6.4,During the subsequent immunoaffinity chromatogra-containing 0.85% NaCl, 1 mmol/L KCN. The sections werephy, Mem-LiALP bound quantitatively to the insolubi-counterstained with methyl green and mounted in glycerin/

gelatin mounting medium. lized AD-1 antibody. On elution with NaSCN, the dia-Electron Microscopical Study of Isolated LiPMF. LiPMF, lyzed Mem-LiALP retained their size, evident from

isolated from 4 mL serum by gel filtration on Sepharose 4B rechromatography on Sepharose 4B (not shown), ascolumns, were incubated overnight at 47C with 80 mg/mL of well as their electrophoretic mobility on agarose gelsAD-1 in 0.1 mol/L tris-HCl buffer, pH 8.0. Antibody bound (Fig. 1). This electrophoresis in the absence of deter-LiPMF were immunoprecipitated by rabbit antimouse immu- gents identified that the purified Mem-LiALP behavednoglobulins, as was described earlier for the precipitation of

as a homogeneous electrophoretic entity. Followingduodenal vesicles.17 Precipitates were processed for electrontreatment with AD-1, the Mem-LiALP band was re-microscopy as was described for tissue specimen.2 They weretarded (Fig. 1). However, the addition of 0.1% Tritonfixed for 2 hours at room temperature with 2% glutaralde-X-100 to purified Mem-LiALP before electrophoresis, orhyde in 0.1 mol/L Na-cacodylate-HCl buffer, pH 7.4, con-

taining 1% sucrose. After rinsing with 0.1 mol/L Na-cacodyl- electrophoresis in the presence of detergents, abolishedate buffer, pH 7.4, containing 4% sucrose, the pellet was this antibody induced change in electrophoretic mobil-postfixed at room temperature, first for 2 hours with 2% os- ity (not shown), confirming that ALP is not the truemium tetroxide in 0.1 mol/L Na-cacodylate buffer and then antigen of AD-1.treated for 1 hour with 2% uranyl acetate in distilled water.Finally, the pellet was embedded in EPON, cut into 70-nm Identification of True AD-1 Antigenultrathin sections, and the morphology of the LiPMF wasanalyzed by electron microscopy. To identify the antigen recognized by AD-1, Mem-

Electron Microscopical Immunohistochemical Staining for LiALP originating from cholestatic sera was bound onLAP on Immobilized LiPMF. LiPMF were bound on AD-1– AD-1 coated nitrocellulose membrane disks, and thecoated nitrocellulose disks as described above. The immuno- activity of four membrane associated enzymes (ALP,histochemically staining for LAP using AD-1 was performed LAP, g-GT, and 5*-Nu) was measured after incubationas described for the light microscopic immunohistochemical of the disks in buffers containing increasing concentra-staining of LAP in tissue sections. In short, after treatment tions of Triton X-100 (0% to 2%). We found a detergentwith normal horse serum for 20 minutes, the LiPMF-loaded

concentration-dependent decrease of nitrocellulosenitrocellulose disks were incubated with AD-1 (4 ng/mL) indisk associated activities for g-GT, 5*-Nu, and ALP,tris buffer saline at 377C for 2 hours. The disks were thenwhich was complete at 0.4% of Triton X-100. The activ-washed and treated with biotinylated affinity-purified horse

antimouse immunoglobulins for 30 minutes, followed by the ity of nitrocellulose disk-bound LAP on the other hand



retarded during electrophoresis by being pretreatedwith AD-1. (Fig. 3C).

Immunohistochemistry and Histochemistry

Because the above experiments suggested that AD-1 reacted with LAP, immunohistochemical stainingsusing this antibody were compared with histochemicalLAP enzyme stainings both in consecutive human liverand kidney sections. In the normal human liver, thecanalicular membrane showed specifical and intensivestaining with AD-1 antibody. The apical epithelialmembrane of the larger bile ducts was also LAP posi-tive (Fig. 4A). In the normal human kidney, AD-1 stain-ing was present only on the brush border of proximaltubule cells in the cortex (S1 and S2 segments) and inthe outer stripe of the outer medulla and in the medul-lary rays (S3 segments) (Fig. 4C). Both for liver andkidney sections, identical patterns were obtained forAD-1 and LAP enzyme stainings (Fig. 4).

Electron Microscopical Study of ImmunoprecipitatedLiPMF. The morphology of partially purified LiPMFwas analyzed by means of electron microscopy afterimmunoprecipition with AD-1. As shown in Fig. 5A theprecipitated vesicles varied in size between 20 and 240nm, (Fig. 5A) and displayed a bilayer structure typicalfor plasma membranes (Fig. 5B). Morphological analy-

FIG. 2. Triton X-100 induced dissociation of liver plasma mem- sis of bound Mem-LiALP on AD-1 coated nitrocellulosebrane fragments isolated from serum by binding onto AD-1 coated membrane disks, after immunocytochemical stainingnitrocellulose disks. Residual enzyme activities bound to AD-1 are of LAP with AD-1, revealed that AD-1 is distributedrepresented as a function of the detergent concentration for ALP, 5*- homogeneously over the plasma membrane fragmentNu, g-GT, and LAP.

surface. In addition, AD-1 bound onto the nitrocellulosemembrane disks is equally visualized on the surface(Fig. 5C).was unaffected by the detergent (Fig. 2), indicating that

LAP might be the true antigen of AD-1. This suggestion Distinction Between Mem-LiALP and Lipoprotein-X. The electron microscopical analysis suggested thatwas supported by the observation that incubation of

AD-1 coated nitrocellulose membrane disks with Mem- other organelles membrane fragments were absenct inthe purified Mem-LiALP. To further exclude the pres-LiALP negative serum resulted in binding only LAP

(not shown). Further confirmation of the anti-LAP en- ence in the purified Mem-LiALP of lipoprotein-X, lipo-protein-X positive serum (detected by the Rapidophortity of AD-1 consisted in an affinity chromatography

experiment using either Mem-LiALP positive serum or electrophoretic system) was run on Sepharose 4B col-umns. A diffuse ALP peak with a turbid appearance,negative serum on AD-1 coupled Sepharose 4B column.

As shown in Fig. 3A, during gradient elution with suggesting the presence of lipids, eluted in the voidvolume (not shown). This fraction was collected andNaSCN of Mem-LiALP positive serum, both LAP and

ALP activities were detected in the bound fraction. The run on the AD-1 immunoaffinity column, after whichthe flow-through and the bound (eluted) fractions wereLAP activity could be separated in two entities. The

first LAP peak coincided with ALP activity eluting at concentrated. The flow-through fraction remained tur-bid. Both fractions were resubjected to Rapidophor gel2 mol/L NaSCN, suggesting that this LAP entity is

associated with LiPMF, and the second one, most likely electrophoresis. As shown in Fig. 6A, after incubationof the gels with a lipoprotein-X precipitating polyan-soluble LAP, eluted at 2.5 mol/L NaSCN. After affinity

chromatography of Mem-LiALP negative serum, on the ionic solution, a precipitating arc (arrowheads), indi-cating the presence of lipoprotein-X, was observed bothother hand, LAP was quantitatively retained on the

column, the ALP eluted in the flow-through fraction for the native serum and the flow-through fraction ofthe AD-1 Sepharose, but no such precipitation was(Fig. 3B). The mixture of soluble LAP and LiPMF-asso-

ciated LAP isolated by AD-1 Sepharose chromatogra- present for the LiPMF isolated by AD-1 chromatogra-phy. After a further incubation of the treated gel withphy of Mem-LiALP positive serum was subjected to the

Triton X-100 PAGE and revealed both the presence of ALP substrate, a diffuse circle of ALP activity aroundthe application well was detected for all fractions. How-soluble and LiPMF-derived hydrophobic LAP isoforms.

When compared, the LAP isoform of cholestatic serum ever, the flow-through fraction and control serumshowed some ALP activity coinciding with the lipopro-and the immunoaffinity purified LAPs, the same iso-

forms were observed in both conditions, which were tein-X dependent precipitation arcs (arrowhead). In



FIG. 3. (A) Immunoaffinitychromatography of LiPMF posi-tive serum on AD-1 Sepharose4B column. The elution patternboth of LAP and of ALP by a gra-dient of NaSCN (0-3 mol/LNaSCN) is shown. (B) Immu-noaffinity chromatography ofLiPMF negative serum on AD-1Sepharose. The arrow indicatesthe start of the elution by 3 mol/L NaSCN. Both the elution prof-its for LAP and ALP are shown.(C) Triton X-100 PAGE and LAPstaining of Mem-LiALP positiveserum (lane 1 and 2), and step-wise eluted (3 mol/L NaSCN) im-munoaffinity purified LAP (lane3 and 4), before (lane 2 and 4) orafter (lane 1 and 3) preincuba-tion with AD-1 for 1 hour.

agreement with the absence of ALP Ç lipoprotein-X in 5*-Nu, g-GT, and LAP of purified plasma membranefragments for a series of serum samples derived fromthe isolated Mem-LiALP, no ALP activity was found in

the Rapidophor gels at the corresponding position of patients with cholestatic liver disease by a two-stepprocedure. First, membrane-bound enzymes were sepa-the precipitated ALP Ç lipoprotein-X complexes (Fig.

6B). rated from their soluble isoforms by gel filtration onSepharose 4B, then the membrane-bound enzymesThe Enzyme Composition of LiPMF is Heteroge-

neous. We analyzed the relative distribution of ALP, were adsorbed onto AD-1 coated nitrocellulose disks



FIG. 4. Immunohistochemi-cal (A and C) and enzyme histo-chemical (B and D) stainings forleucine aminopeptidase on con-secutive sections of human liver(A and B) and kidney (C and D)tissues. Immunohistochemicalstainings were with AD-1; andhistochemical staining for LAPwith L-leucyl-b-naphthylamide(A and B, original magnification1140; C and D, original magnifi-cation 1225.) In the liver, LAPand AD-1 staining was presentin canalicular membranes andthe apical cell surface in largebile duct. In the kidney, AD-1and LAP staining was restrictedto the brush border of proximaltubule cells.



FIG. 5. Electron micrographsof AD-1 precipitated LiPMF (A),the higher magnification showingthe membrane bilayer structure(B) and immunocytochemicalstaining of LAP using AD-1 on thesurface of two vesicles immobilizedonto AD-1 coated nitrocellulosedisks (C). (A, original magnifica-tion1130,000; B, original magnifi-cation 1168,000; C, original mag-nification 172,000.)

and their activities were measured. The enzyme compo- Purified LiPMF. LiPMF from cholestatic sera werebound onto AD-1–coated nitrocellulose disks and weresition related to the activity of ALP varied considerably

between the samples (Table 1). Analysis of variance of used to study the release of ALP, g-GT, and 5*-Nu fromthese membrane fragments by incubating them withthese data showed a significant difference between the

patients for ALP/g-GT, for ALP/5*-Nu, and for ALP/ 0.1% Triton X-100, GPI-PLC (20 U/mL) or GPI-PLD(20 U/mL). After treatment, residual activities of nitro-LAP, with P Å .0001 for each combination. Further-

more, one-way Anova analysis showed that the largest cellulose disk-bound ALP, g-GT, and 5*-Nu were mea-sured. As shown in Fig. 7, ALP and 5*-Nu show a simi-difference was observed for ALP/5*-Nu (only patient 2

and 4 were not significantly different from each other lar behavior: both are released from the LiPMF byTriton X-100 and GPI-PLC, but both are resistant toat the 0.050 level), while the smallest difference be-

tween the patients was observed for ALP/LAP. GPI-PLD; g-GT on the other hand shows a differentpattern: it cannot be released by GPI-PLC or GPI-PLDEffect of Treatment With Triton X-100 and Phospholi-

pases on the Release of Membrane-bound Enzymes From and can be partially released by Triton X-100.



from hepatocytes expressing liver ALP on their surface.Hence, our MoAb was most likely directed against aspecific epitope of the liver membrane fragments whichwas not liver ALP. The present study suggests thatLAP is the true antigen reacting with AD-1. This con-clusion is supported by the following observations: (1)treatment of purified Mem-LiALP with Triton X-100abolished the reactivity with AD-1. (2) Triton X-100also induced a dose-dependent decrease of nitrocellu-lose membrane disk-bound activities for g-GT, ALP,and 5*-Nu, while the disk bound LAP activity was unaf-FIG. 6. Visualization of lipoprotein-X by the Rapidophor system.fected, suggesting that LAP was the true antigen of(A) Rapidophor agar gel after electrophoresis and precipitated arcsAD-1 in the plasma membrane fragments. The unaf-(arrowheads) formed by polyanion precipitation. (B) The same Rapi-

dophor gel stained with BCIP substrate for ALP activity. Lipopro- fected LAP activity bound to the antibody in the pres-tein-X positive serum (lane 1); flow-through fraction of this serum ence of Triton X-100 can possibly be explained by aduring AD-1 immunoaffinity chromatography (lane 2); purified Mem- reuptake of LAP released from the LiPMF by nonoccu-LiALP (bound fraction) of this serum (lane 3).

pied AD-1. (3) Affinity chromotography of Mem-LiALPpositive and negative serum resulted in the binding ofboth ALP and LAP in the positive serum, and of LAP

DISCUSSION only in the negative serum. (4) PAGE electrophoresisshowed a change in electrophoretic mobility of LAPIncreased activities of ALP, g-GT, LAP, and 5*-Nuafter treatment with the monoclonal antibody. (5) Re-have been described in all forms of cholestasis.1,6,20,21

sults for the immunohistochemical and the histochemi-These enzymes are bound to the plasma membrane ofcal staining of LAP in normal liver and kidney tissueliver cells and in physiological conditions they circulatesections were identical. In human liver, LAP wasat low activities mostly in a soluble form. In cholestaticmainly localized at the canalicular membrane, whichliver disease, they often circulate as high-molecular-is in agreement with previous results.2 In the humanmass forms, generated by shedding of LiPMF, whichkidney, LAP activity was distributed throughout theresults in vesicular structures expressing the differententire proximal tubules, as described by Guder andmembrane-bound enzymes at their surface, such asRoss.25 Detailed morphological analysis of the isolatedALP, g-GT, LAP, and 5*-Nu.2 Shedding of plasma mem-LiPMF by electron microscopy was indicative of theirbrane fragments is a well-described phenomenon inhigh purity and their membrane nature. Furthermore,many cells, especially in malignant cells and in hepato-antibody binding during their isolation, did not perturbcytes under pathological conditions.3,22-24 Therefore, thethe LAP distribution on their surface.quantification of these high-Mr forms in serum, and

The immunoreactivity between AD-1 and LiPMFmore in particular of Mem-LiALP, is helpful for theshowed that the circulating Mem-LiALP can be specifi-diagnosis and follow-up of these patients.4cally measured by binding of LiPMF onto AD-1 coatedPreviously, we produced a specific monoclonal anti-nitrocellulose membrane disks or microtiterplates (Fig.body, AD-1, against Mem-LiALP.13 This MoAb exhib-

ited no cross-reactivity with other ALP isoenzymes, noteven with liver ALP, although Mem-LiALP originates

TABLE 1. Relative Enzyme Composition for LiPMFAssociated ALP, LAP, 5*Nu, and g-GT

ALP/g-GT ALP/5*-Nu ALP/LAP

1 2.2 18.3 2.42 1.3 20.0 2.13 2.1 10.6 4.14 1.1 20.8 2.55 3.0 23.8 1.56 6.5 13.0 2.07 4.1 26.5 2.18 2.2 16.7 2.0x 2.79 18.5 2.36 FIG. 7. Release of ALP, LAP, g-GT, and 5*-Nu from AD-1 boundSD 1.64 4.97 0.77 LiPMF by treatment with 0.1% Triton X-100 in 50 mmol/L Tris/HClCV 59% 27% 33% buffer, pH 7.0, or with GPI-PLC and GPI-PLD (20 U/mL) in the same

buffer, either alone or in combination as indicated. Residual enzymeNOTE. Plasma membrane fragments were isolated from various activities are presented proportional to the activities of nontreated

cholestatic serum samples by Sepharose 4B gel filtration and by controls.��

GPI-PLD; j; GPI-PLC;

�GPI-PLC / Triton X-100;

h Triton X-100.subsequent binding onto AD-1 coated nitrocellulose membrane disks.



LiPMF, unless it is combined with 0.1% of Triton X-100. The GPI-PLD resistance of ALP bound on a HeLacell plasma membrane was also described by previousstudies,32 the mechanism of which is not known. In aprevious report, Brown and Rose33 concluded that mostGPI-anchored proteins, including ALP, are at least par-tially insoluble in Triton X-100. This apparent discrep-ancy can be explained by our finding that relativelyhigh concentrations (0.4%) of Triton X-100 are requitedto finally release enzymes from the membrane frag-ments, i.e., at a 4-fold higher concentration than thatused by Brown and Rose.33 Secondly, because of thehydrophobicity of Triton X-100 released ALP, when us-

FIG. 8. Schematic representation of the immunometric procedure ing particularly partitioning techniques, the hydropho-for quantifying Mem-LiALP through binding of LiPMF to AD-1bic GPI-anchor bearing ALP isozyme will appear in thecoated nitrocellulose membrane disks or to microtiterplate wells.detergent phase, which is easily confused with LiPMF.

CONCLUSION8). A preliminary study of 97 cancer patients showedthat the measurement of Mem-LiALP by using AD-1 The present study shows that AD-1 reacts with LAP,is a sensitive marker for liver metastasis, a clinical a component of the plasma membrane. This antibodycondition well known to go along with increased shed- has permitted us to isolate intact LiPMF from chole-ding of plasma membrane vesicles.26 static human sera with a high level of purity, thus

Two kinds of high Mr ALP particles circulate in pa- offering an interesting tool for the further pathophysio-tients with cholestatic liver disease,27-30 one represent- logical studies dealing with the release of ALP froming the above mentioned plasma membrane fragments the plasma membrane surface. The AD-1 antibody wasreleased from hepatocytes by shedding, the other con- also used in an immunoassay to quantify Mem-LiALPsisting of complexes of liver ALP with lipoprotein-X. via binding of liver membrane fragments to AD-1,These two are difficult to separate because of their high which resulted in a useful and simple laboratory testMr and hydrophobicity.2,29 The present experiments to diagnose and monitor cholestatic liver disease.confirmed that AD-1 isolated LiPMF did not contain

Acknowledgment: The authors thank Dirk De-lipoprotein-X, indicating further that AD-1 antibodyweerdt for the layout of the figures.has provided us with a powerful tool to purify LiPMF

without interference of lipoprotein-bound materials. REFERENCESWe analyzed the activities of LAP, g-GT, ALP, and

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Purification of circulating liver plasma membrane fragments using a monoclonal antileucine aminopeptidase antibody