Nucleoside phosphatase activities on pig pancreas zymogen granule membranes analyzed by nondenaturing polyacrylamide gel electrophoresis

Pergamon

ht. J. Biock-m. Cd Bid. Vol. 27, No. 10, pp. 1043-1054, 1995 Elsevier ScienceLtd. Printed in Great Britain

1357-2725(95)00076-3

Nucleoside Phosphatase Activities on Pig Pancreas Zymogen Granule Membranes Analyzed by Nondenaturing Polyacrylamide Gel Electrophoresis MARCO SORIANI, M. CARINA SPAANS, MARKUS TOBLER, ANDREAS U. FREIBURGHAUS* Department of Internal Medicine, University Hospital, H LAB 8, CH-8091 ZG-ich, Switzerland The membrane of the pancreatic zymogen granule plays an important part in the sequenceof storage, transport and exocytosis of digestive enzymes. While much is known on stimulussecretion coupling, very little is understood about how the storage organelles move in the cytoplasm to the luminal plasma membrane and why and how they fuse with it to release the contents. It is assmned that nucleoside phosphatases are involved in these energy consuming processes.Pancreatic zymogen granule membranes contahr one major glycoprotein, GP-2, and a few minor proteins all with unknown functions. In order to identify functions we have purified zymogen granule membranes from pig pancreas, solubilized the proteins under nondenaturhtg conditions with the detergent CHAPS and characterized the extracted protein by polyacrylamide gel electrophoresis,histochemistry and leetins. Three major protein bands, often fused in one broad band, revealed enzymatic activity for adenoshte-, cytidine-, inositol- and guamdinedi- and triphosphates by the precipitation of liberated phosphate by Pb(NO& This activity was sensitiveto known ATP diphosphohydrolase inhibitors. The band with activity arises from a 92 kDa glycoprotein. A different narrow band showed mooophosphatase activity for AMP, GMP, IMP and CMP. Some of the activities were inhibited by different &ins, indicating glycosyl groups near the active site. Electroo microscopical cyto&emiitry cotdinned a nucleoside phosphatase activity on granule membranes. Our results show for the first thne that the nucleoside phosphatase activity of the zymogen granule membranes is carried by a 92 kDa glycoproteio, probably the known self-associating form of GP-2. The hydrolysis of tri- and diphosphate nucleotidescould provide the energy required by exocytosis. Keywords: Pancreas Zymogen granule membrane Nucleoside phosphatase Lectins CP-2 Gel electrophoresis Electron microscopy. Int. J. Biochem.

Cell Biol. (1995) 27, 1043-1054

INTRODUCTION

Digestive enzymes destined for regulated secretion (zymogens) are stored in the exocrine pancreatic acinar cells in intracellular granules. These zymogen granules (ZG) must approach the apical plasma membrane in order to fuse with it and release their contents into the luminal space. While detailed knowledge has accumulated on the stimulus-secretion aspect of pancreatic exocytosis, the molecular events *To whom all correspondence and reprint requests should be addressed. Received 14 February 1995; accepted 12 June 1995.

leading to the formation, maturation, movement, targeting and fusion of the ZG still lie in the dark. These functions may be energy requiring. The zymogen granule membrane (ZGM) contains only few integral proteins, which are assumed to be involved in these events (Palade, 1975; Young and Young, 1984). Nucleoside (tri-) phosphatases are known to be part of energy consuming processes like secretion. Several experiments with pig exocrine pancreas have revealed the presence of a protein with a native molecular mass of 130 kDa presenting an ATP-diphosphohydrolase activity capable of catalyzing the

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Marco Soriani et ul

hydrolysis of triphosphoand diphosphonucleosides to yield nucleoside monophosphate and inorganic phosphate (Traverso-Cori et al., 1965; Le Be1 et al., 1980; Picher et al., 1994). This enzyme is activated by Ca’+ and Mg*+ but insensitive to the absence of Na+ and K+. In membranes of rat pancreatic ZG a Ca2+/Mg2+ requiring activity towards many nucleoside di- and triphosphates has been found (Meldolesi et al., 1971). Cytochemical studies have localized Mg*+-ATPase activities on the apical plasma membrane and on isolated ZGM (Harper et al., 1978). The enzyme appeared to be intrinsic with its active site localized on the internal face of the granule membranes. The enzyme S-nucleotidase, mostly taken for an AMPase, hydrolyses various ribonucleoside S-monophosphates (Zekri et al., 1988). It is widely distributed in various mammalian tissues (Drummond and Yamamoto, 1971), appears to circulate between the cell surface and an intracellular pool (Wilcox et al., 1982) and possibly interacts with elements of the cytoskeleton (Carraway et al., 1979). Most tissues contain two isoenzymes of S-nucleotidase, an intrinsic membrane form (Newby et a/., 1975) attached to the plasma membrane either as an integral membrane protein (Baron et al., 1986) or through a glycosyl-phosphatidylinositol lipid anchor (Low, 1987); the other is a soluble cytosolic form (Montero and Fes, 1982). Strong competitive inhibitors of the membrane-bound 5’-nucleotidase are ADP and ATP (Zekri et al., 1988). This enzyme has been detected on ZGM as well (Harper et al., 1978; Spaans et al., 1994). Despite many studies on both the 5’-nucleotidase and ATPdiphosphohydrolase, it is unknown how many enzymes are involved in the hydrolysis of the various nucleoside phosphates and whether the enzymes have similarities in substrate and inhibitor specificities. Also unknown is whether GP-2, the major protein component of the ZGM, has any enzymatic function. In the present study we attempted to further characterize the proteins of purified pig pancreas ZGM and to investigate their ability to hydrolyse various nucleoside phosphates. We also probed the presence of glycosyl residues near the active sites of the nucleoside phosphatases. We chose a non-denaturing polyacrylamide gel electrophoresis system in a low ionic strength medium containing the zwitterionic detergent CHAPS to separate the

proteins and probe them with nucleoside substrates and various lectins. We also cytochemitally visualized the activities in the gel and with electron microscopy on membranes, and probed with different lectins. MATERIALS

AND

METHODS

Reagents

Acrylamide, glycerol and PAGE-Biue G90 were purchased from BDH. Adenosine-5’diphosphate (ADP), Ricinus Communis lectin (RCA120), Soybean lectin (SBA), Wheat germ lectin (WGA), Galanthus Nivalis Agglutinin (GNA), Datura stramonium agglutinin (DSA) digoxigenin labelled, Sambucus nigra agglutinin (SNA) digoxigenin labelled, Maackia amurensis agglutinin (MAA) digoxigenin labelled, Phaseolus vulgaris lectin (PHA-L) digoxigenin labelled, oligomycin, g-strophanthin (ouabain), the DIG Glycan Differentiation Kit and the cc-amylase PNP Monotest were from Boehringer Mannheim. Osmium tetroxide was from EMS. Zwittergent 3-8 and okadaic acid were from Calbiochem. Epon-812, glycine, sodium fluoride and N,N,N’,N’-tetramethylethylenediamine (TEMED) were from Fluka. GelBond PAG film was from LKB. Ammoniumsulfide, adenosine-5’-monophosphate (AMP), glutardialdehyde, p-nitrophenylphosphate, sodium carbonate, sodium bromide, sodium azide, magnesium nitrate and nitric acid were from Merck. 3-((3Cholamidopropyl)dimethyIammonio) - 1 - propanesulfonate (CHAPS) was from Serva. Ammonium persulfate, calcium nitrate, benzamidine hydrochloride, ethacrynic acid, levamisole, conc;navalin A (Con A), cytochrome c (Type 6 C7752), phenylmethylsulfonyl fluoride (PMSF), soybean trypsin inhibitor, TRIZMA base, adenosine-5’-triphosphate (ATP), cytidin-5’-monophosphate (CMP), cytidine-5’diphosphate (CDP), cytidine-5’-triphosphate (CTP), guanosine-5’-monophosphate (GMP), guanosine-5’-diphosphate (GDP), guanosine5’-triphosphate (GTP), inosine-5’-monophosphate (IMP), inosine-5’-diphosphate (IDP), inosine-5’-triphosphate (ITP), cc&methylene ADP and prestained SDS-PAGE standard solution (mol.wt 34123 kDa) were from Sigma. Sucrose was from Schwarz/Mann Biotech. p-Methylaminophenol sulfate (ELON) and ammonium molybdate were purchased from Eastman Kodak Company and Aldrich respectively. Filters (0.2 pm) were

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Nucleoside phosphatase activities on ZGM

from Schleicher & Schuell. ProSieve Gel from FMC. System was purchased were Immobilon-P transfer membranes obtained from Millipore. Isolation of zymogen granule membranes

ZG were prepared according to a modified method by De Lisle et al. (1984) and Spaans et al. (1994): excised pig pancreas was trimmed free of fat and connective tissue and suspended in a 4°C buffer containing 250 mM sucrose, 5 mM MPS, 0.1 mM MgS04 and 0.5 mM PMSF. The suspension was homogenized and then centrifuged at 700g for 10 min to remove unbroken cells and nuclei. The supernatant was decanted and centrifuged at 1300s for 20 min to yield a crude ZG pellet. This pellet was resuspended in Percoll buffer (pH 5.2). A self-forming density gradient resulted from the centrifugation at 100,OOOgfor 30 min at 2°C in a Kontron T-2050 ultracentrifuge in a type TST 28.38 rotor. The brownish mitochondrial layer was removed from the top of the white ZG layer. The latter was resuspended in Percoll buffer and again centrifuged at 100,OOOgfor 30 min. The final ZG fraction was then washed free of Percoll by centrifugation at 2800g for 2 min. For preparing ZGM a method described by Hansen et al. (1983) was modified. ZG (max. 20 g/200 ml) were mixed with ice-cold 100 mM Na&Os for 30 min. Aliquots of granule lysate were centrifuged at 150,OOOgfor 1.15 hr at 2°C in a type TFT 50.38 rotor to separate soluble proteins from the secretory granule membranes. Benzamidine hydrochloride (0.1 mM), phenylmethylsulfonyl fluoride (PMSF) (10 mM) (added from a 0.2 M stock made up in ethanol), and soybean trypsin inhibitor (0.01 mg/ml) were included in the lysate to inhibit proteolytic degradation. ZG lysate, prepared as above, was layered on a step gradient consisting of equal volumes of 1 M sucrose and 0.3 M sucrose and was centrifuged at 100,OOOg for 2 hr at 2°C in a Kontron TST 28.38 rotor. The granule membranes, which accumulate at the 0.3 M/l M gradient step, were carefully collected and suspended in 0.375 M NaBr (NaBr-stock : membrane suspension = 2 : 1). The suspension was centrifuged for 1.5 hr at 150,OOOg at 2°C in the same rotor. The pellet was washed in small amounts of distilled water in floating tubes and again centrifuged at 100,OOOg for 1 hr at

2°C. Pellets were stored in liquid before use.

nitrogen

Enzyme and protein assays

Cytochrome C oxidase (EC 1.9.3.1) as an indicator of mitochondrial contamination was measured by spectrophotometry according to Bayerdorffer et al. (1985), Sinjorgo et al. (1986) and Azzi et al. (1981) with the following modifications: the reduced cytochrome c concentration was 10 mM and 0.5% (w/v) Sulfobetaine 3-8 (Zwittergent 3-8) was added to solubilize the particulate preparation and fully activate the enzyme. Protein was determined by the BCA-protein microassay from Pierce with bovine serum albumin as a standard. cc-Amylase (EC 3.2.1.1) as a marker enzyme for ZG content was measured with the PNP Monotest. Sample preparation for gel electrophoresis

ZGM were suspended at l-l.5 mg protein/ ml in 0.1 M Tris/HCl (pH 9.5) containing 2% CHAPS. The suspension was incubated at 37°C for 10 min, sonicated, and centrifuged (10 min, 10,500g) on a Beckman Microfuge11. The supernatant was filtered (0.2 pm pore size) and stored at -20°C. Aliquots of 250 mg protein in 0.5 ml were mixed with 0.1 ml glycerol and 0.1 ml of sample buffer containing 10.4% (w/v) glycerol, 2% (w/v) CHAPS, and 62.5 mM Tris/HCl (pH 9.5) and warmed up to 37°C. Before SDS-PAGE the samples solubilized in 2% CHAPS were mixed with 0.1 ml glycerol and 0.1 ml of sample buffer containing 10.4% (w/v) glycerol, 2% (w/v) SDS, and 62.5 mM Tris/HCl (pH 6.8). Polyacrylamide gel electrophoresis

CHAPS polyacrylamide gels (20% T, 5.4% C, 0.75 x 140 x 130 mm, polymerized onto GelBond PAG film) were prepared using the modified method of Davis (1964) and electrophoresis was performed in a LKB 2001-001 (Hoefer SE 600) vertical electrophoresis unit. Proteins of ZGM (200-250 pg/gel) were subjected to non-denaturing electrophoresis in 515% polyacrylamide gradient gels containing 0.2% CHAPS and 0.1 M Tris/HCl (pH 9.5). Samples were applied over the whole length so that after electrophoresis and after cutting in vertical stripes a clear assignment of bands was possible. Electrophoresis was carried out at 200 V, 4 mA and 1 W (upper limits) per gel at 18°C for 16 hr or at 500 V, 150 mA and

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20 W at 18°C for 3.0 hr. The electrode buffer contained 0.2 M glycine and 0.1% CHAPS in 25 mM Tris/HCl (pH 9.5). SDS polyacrylamide gels (20% T, 5.4% C, 0.75 x 140 x 130 mm, polymerized onto GelBond PAG film) including a 3 cm wide stacking gel (4% T, 4% C) were prepared using the discontinuous buffer system of Laemmli (1970). Electrophoresis was carried out at the same conditions as CHAPS gels. Detection of nucleoside phosphatase activity on electropherograms

After electrophoresis, the gels were removed from the glass plates and washed in several changes of HzO. Gel stripes were incubated with 2 mM substrate (AMP, ADP, ATP, CMP, CDP, CTP, IMP, IDP and ITP) or 1 mM substrate (GTP, GDP and GMP) in 0.1 M Tris/HCl (pH 7.5) containing 1.8 mM Pb(NO& and 1.4 mM Mg(NO& or Ca(NO& at 37°C for 1 hr. The gel stripes were then immersed in 2% ammoniumsulfide (to convert the Pbs(PO& precipitates into the darker PbS). Background staining was reduced under visual control with 1% nitric acid and this reaction was stopped in 5% acetic acid (Le Be1 et al., 1980). In control experiments substrate-free medium was used. cc&methylene ADP (250 PM) was directly added to the incubation mixture. p-Nitrophenylphosphate was used as substrate (2 mM) for non-specific phosphatase. Agarose gel electrophoresis

CHAPS ProSieve agarose gels were prepared as indicated in the manufacturer’s procedure and the electrophoresis of ZGM proteins carried out as for PAGE. After electrophoresis the gels were incubated with ATP or GTP 1.0 mM in 0.1 M Tris/HCl (pH 7.5) containing 1.8 mM Pb(NO& and 1.4 mM Mg(NO& at 37°C for 1 hr. After identification of the region of the gel containing the nucleoside phosphatase activity by the presence of white Pbs(PO& precipitates the band was cut and the protein recovered as indicated in the ProSieve gel system protein recovery procedure. Protein was concentrated by Spin-X UF concentrators with a 10,000 Da MW cut off. Protein detection in gels

Proteins were visualized in gels by silver staining (Morrissey, 1981).

e/ d.

Lectin Incubation

Gel stripes were preincubated overnight with 1.0 PM Con A, RCA120, SBA , GNA or 5.0 ,nM WGA before the incubation with the substrates AMP, ADP, ATP, GDP and GTP. Lectin immuno detection

After CHAPS and SDS--PAGE separation ZGM proteins were electrotransferred to PVDF membranes by Western blotting (Burnette, 198 1). Glycosyl residues were characterized by incubation of the transfer membranes with digoxigenin labelled lectins according to the instructions of the Boehringer DIG Glycan Differentiation Kit. Experiments were repeated at least three times. Inorganic phosphate assay

The method of Le Be1 et al. (1978) was used for the determination of inorganic phosphate released after incubation of ZGM proteins with di- and triphosphate nucleotides. EM-localization activity on ZGM

of

nucleoside

phosphatase

ZGM were centrifuged at 10,500g for 10 min in a Beckman Microfuge-11. For the localization of nucleoside phosphatase activity on the membranes the method of Uchiyama (1983) was modified. Membrane pellets were incubated at 37°C for 1 hr in a medium, which contained 0.1 M Tris/HCl (pH 7.5), 1.4 mM MgS04, 1.8 mM Pb(NO& and 2 mM AMP. As controls, the ZGM were incubated in substrate-free medium or in medium containing the substrate AMP and the inhibitor a$-methylene-ADP (40 mM). ZGM were prepared for electron microscopy by washing the pellets in 0.1 M Tris/HCl buffer (pH 7.5) and centrifuging at 10,500g for 10 min in a Beckman Microfuge-1 1. The pellets were fixed in 0.1 M Tris/HCl buffer (pH 7.5) containing 2% glutardialdehyde for 2 hr at 25°C and postfixed in Tris/HCl buffer containing 1% osmium tetroxide for 1 hr. The samples were then washed, dehydrated, embedded in Epon8 12, sectioned, and viewed in a Zeiss EM902 electron microscope. RESULTS

ZG isolated from pig pancreas were free of mitochondria or other organelles as observed by electron microscopy (Fig. 1). The ftne

Nucleoside phosphatase activities on ZGM

Fig. 1. Zymogen granules from pig exocrine pancreas, purified by differential and Percoll gradient centrifugation. No other organelles or membrane fragments are present. Bar is 5 pm.

granular material between the ZG is Percoll. The purified ZG were used for the isolation of ZGM. The presence of contaminating mitochondrial membranes-the most likely contaminants-or adsorbed ZG contents was verified by measuring the activities of cytochrome c oxidase (marker for mitochondrial membranes) and a-amylase (marker for ZG content). Very low cytochrome c oxidase specific activity (in a range of lop3 U/mg of protein) was detected in the purified ZGM.

The specific amylase activity of ZGM was 11.7 +4.2 U/mg of protein, whereas pancreas homogenate and ZG had an amylase specific activity of 235.5 f 5.3 U/mg and 748.4k84.0 U/mg of protein, respectively. These values are comparable with the literature (Le Be1 and Beattie, 1984). Proteins of the ZGM were solubilized with CHAPS and subjected to non-denaturing electrophoresis in a gradient polyacrylamide gel containing 0.2% CHAPS and 0.1 M Tris/HCl

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a

b

Fig. 2. Zymogen granule membranes of pig exocrine pancreas, solubilized in the zwitterionic detergent CHAPS and separated by non-denaturing CHAPS electrophoresis in polyacrylamide gels. Protein separation on CHAPSPAGE is based on differences in molecular weight and protein charge. (a) ZGM proteins solubilized by 2% CHAPS and separated in 0.2% CHAPS-PAGE. Proteins were visualized by silver staining. Diagrammatic representation of the protein pattern is shown on the left. (b) ZGM proteins treated as in (a) and electrotransferred to PVDF membrane for glycoprotein immuno-detection by the lectin DSA digoxigenin labelled.

(pH 9.5). Three major bands, often fusing in a single broad band spread over 30% of the gel, were detected (Fig. 2a). Lectin immuno detection of carbohydrate groups after electrotransfer to PVDF membranes showed the glycoprotein nature of the major spread band. Figure 2(b) shows the positive reaction of DSA that recognizes the core disaccharide galactosefi( 1-3)N-acetylgalactosamine. We have obtained a positive reaction also in presence of SNA, MAA, WGA and PHA-L. After incubation with different nucleotide substrates (AMP, IMP, CMP, GMP, ADP, IDP, CDP, GDP, ATP, ITP, CTP and GTP) the nucleoside phosphatase activities were detected by visualization of the resulting lead phosphate precipitates in the gel, which formed in the presence of Pb(NO&. Precipitates became better visible after conver-

Fig. 3. Nucleoside phosphatase activities of CHAPS-solubilized zymogen granule membrane proteins separated by CHAPS-PAGE. (a) Incubation with GTP, Mg2+ and Pb(N03)2 at pH 7.5. (b) As in (a) but without Mg*+. (c) Incubation with GMP and Pb(NO& at pH 7.5.

sion with ammonium sulfide to brown, insoluble PbS. The nucleoside phosphatase patterns of ZGM proteins on the gels are illustrated in Fig. 3. After incubation with GTP, in presence of Mg2+ or Ca2+, we obtained a positive reaction in the same area occupied by the three major protein bands obtained in CHAPS PAGE (Fig. 3a). The activity was completely abolished in the absence of divalent cations (Fig. 3b). Identical results were obtained in the presence of ATP, ADP, GDP, ITP, IDP, CTP and CDP (data not shown). Figure 3(c) shows the presence of a sharp band with monophosphatase activity after incubation with AMP . This band hydrolysed also GMP, IMP and CMP (data not shown). The monophosphatase activity was independent of the presence of Mg2’ or Ca2+. In control studies no nucleoside phosphatase activities were detected on gel stripes incubated in substrate-free medium. q/I-Methylene ADP inhibited AMPase activity but did not interfere with the nucleoside di- and triphos-

Nucleoside phosphatase activities on ZGM

Fig. 4. Zymogen granule membranes solubilized in the zwitterionic detergent CHAPS and separated by denaturing SDS-PAGE. (a) Molecular weight markers (123, 89, 67, 50, 37, 34 kDa) for SDS-PAGE. Protein silver staining. (b) ZGM proteins solubilized by 2% CHAPS and separated in 0.2% SDS-PAGE. Protein silver staining. (c) SDS-PAGE of ZGM proteins recovered from the band with nucleoside phosphatase activity previously separated by agarose gel electrophoresis. Protein silver staining. (d) ZGM proteins treated as in (b) and electrotransferred to PVDF membrane for glycoprotein immuno-detection by the lectin WGA digoxigenin labelled.

phatase activities. No activity towards p-nitrophenylphosphate was found at pH 5.0 or 9.3. ZGM proteins solubilized with CHAPS were also separated in denaturing gradient SDS-PAGE (Fig. 4b). The electrophoretic pattern showed a major band of 92 kDa. On western blots SDS--PAGE separated ZGM proteins showed that the lectins used for immuno detection of glycoproteins on CHAPS-PAGE bound to the 92 kDa protein. As shown in Fig. 4(d) the lectin WGA, which recognizes N-acetylglucosamine or sialic acid residues, gave a positive reaction with this protein. The identification of the protein responsible for the nucleoside phosphatase activity was carried out by CHAPS agarose gel electrophoresis followed by SDS-PAGE. After native agarose electrophoresis of ZGM proteins

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nucleoside phosphatase activity was detected on gel strips in presence of ATP or GTP. The area containing the lead phosphate precipitate was excised and the proteins recovered by extraction with 50 mM Tris/HCl and 1mM EDTA at pH 8.0. SDS-PAGE of the recovered protein revealed only a single protein band of 92 kDa as detected by silver staining (Fig. 4c). The presence of glycosyl residues near the active sites of the nucleoside phosphatases was investigated by preincubating the gels after electrophoresis with different lectins (Con A, WGA, GNA, SBA and RCAIZO). The nucleoside phosphatase patterns on CHAPS-PAGE after lectins preincubation are illustrated in Fig. 5. Inhibition was qualitatively judged by visual appearance. The lectins Con A (Fig. 5b) and WGA (data not shown) completely inhibited the activity for AMP and ATP. Lectins GNA (Fig. 5c) and RCA 120 (Fig. 5d) showed a lower, but distinct inhibition towards di- and triphosphate nucleotides but no reduction of the monophosphatase activity (data not shown). The lectin SBA did not show any inhibitory effect. We have obtained the same results with ADP, GDP and GTP as substrates. Table 1 shows the effects of some ATPase inhibitors on the release of inorganic phosphate by the nucleoside phosphatase activity present on ZGM. Sodium azide, sodium fluoride, ethacrynic acid and okadaic acid, an inhibitor of phosphatases 2A and 2B (Wagner et al., 1992) showed a strong inhibition of the hydrolysing activity to ADP, ATP and GDP. The nucleoside phosphatase activity present on ZGM was insensitive to oligomycin (a mitochondrial ATPase inhibitor), to ouabain (a plasma membrane Na,KATPase inhibitor) and to levamisole (an inhibitor of alkaline phosphatases). Okadaic acid (reported to be a specific inhibitor of protein phosphatases 2A and 2B) also showed inhibition. Moreover, after incubation of the gels in the presence of different di- and triphosphate nucleotides (ATP, ADP, GTP, GDP, ITP, IDP), Mg*+, Pb(NOs)* and ATP diphosphohydrolase inhibitors (sodium fluoride, sodium azide and ethacrynic acid) reduced lead phosphate precipitates were detected as shown in Fig. 5(h)-( j). In Fig. 6 ZGM are illustrated. Figure 6(A) shows a low, and Fig. 6(B) a high magnification electron micrograph of the membrane pellet. The membrane profiles depict a poly-

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abed

ef

ghi

j

klm

Fig. 5. Sensitivity to lectins and inhibitors of nucleoside phosphatase activities of CHAPS-solubilized zymogen granule membrane proteins separated by CHAPS polyacrylamide-gel electrophoresis. (a) Incubation with ATP, Mg’” and Pb(NO& at pH 7.5 (reference). (b) As in (a) but overnight preincubated with ConA (or WGA). (c) As in (a) but preincubated with GNA. (d) As in (a) but preincubated with RCA 120. (e) Incubation with AMP and Pb(NO& at pH 7.5. (f) As in (e) but overnight preincubated with ConA. (g) Incubation with ATP, Mg2+ and Pb(NOa)z at pH 7.5 (reference), same as (a). (h) As in (a) but preincubated for 15 min with sodium azide. (i) As in (a) but preincubated with sodium fluoride. (j) As in (a) but preincubated with ethacrynic acid. (k) As in (a) but preincubated with okadaic acid. (1) As in (a) but preincubated with levamisole. (m) As in (a) but preincubated with oligomycin.

morphic pattern with stacks of membrane and vesicles. Higher magnification reveals the typical bilayer structure of the membranes. Stacks of three or more layers were observed. The hydrolytic activity towards the substrate AMP is visualized by dense lead phosphate deposits lining the ZGM (Fig. 6C and D). Only a part of the membranes contain nucleoside phosphatase activity. The enzymatic nature of the reactions was verified using controls with added inhibitor a$-methylene ADP and substrate or without substrates. No precipitates were detected in the controls.

DISCUSSION

We have used highly puritied membranes of pig pancreatic ZG. By electron microscopical

criteria the starting ZG batches were free of mitochondria (Fig. 1), other organelles or membranes. The ZGM appeared free of contamination by mitochondrial membranes as judged by the absence of cytochrome c oxidase activity. Contamination with soluble, adsorbed proteins was avoided by thoroughly washing the membranes in high ionic strength buffer. Indeed, only negligible a-amylase activity could be detected. Separation of functional ZGM proteins was possible by non-denaturing PAGE. A high pH of 9.5 was necessary for all proteins to become negatively charged. A 5-15% gradient gel is most suitable. This study demonstrates that nucleoside phosphatase activities can be detected in CHAPS-solubilized membranes subjected to

Nucleoside phosphatase activities on ZGM

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Fig. 6. Electron micrographs of purified pig pancreas zymogen granule membranes. Cytochemical detection of lead phosphate precipitates after enzymatic release of inorganic phosphate from adenosine monophosphate (AMP) in the presence of lead nitrate. (A) Representative of controls: inhibitor cQmethylene ADP plus substrate AMP, (same appearance when no substrate added). Bar is 300 nm. (B) As (A) but higher magnification. Bar is 100 nm. (C) After incubation with substrate AMP and Pb(NO&. Bar is 300 nm. (D) As (C) but higher magnification. Bar is 100 nm.

non-denaturing PAGE in gels containing CHAPS. Enzymatic activities in gels containing the non-ionic detergent Triton X-100 have been detected before (Young and Young, 1984; Van De Hoek and Zail, 1977). LeBel et al. (1980) detected ATP-diphosphohydrolase activity from pig pancreas on gels using the non-ionic detergent Triton X-100. We used the zwitterionic CHAPS, because this agent was found to be a very effective solubilizer for ZGM with a high phospholipid/protein ratio

(Freiburghaus and Schtipbach, 1991) without conferring net charge (Neugebauer, 1988). This detergent preserves the nucleoside phosphatase activities. The main hydrolytic activity of the ZGM is towards nucleoside di- and triphosphates (Fig. 3a). This activity is specific in the sense that the hydrolysis is not observed with nucleoside monophosphates or p-nitrophenylphosphate as substrate. It is also specific with respect to the number of phosphate groups.

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Table I. ZGM proteins (0.5 mg/ml) were incubated for 1.O hr at 37°C with 2.0 mM substrate and 1.4 mM Mg(NOj )? at pH 7.5 (final volume 50 yl). The control rate of Pi spontaneously released was 0.001 W/ml. Proteins were preincubated 15 min at 37°C with inhibitors. After incubation the proteins were precipitated with 10% TCA. Supernatant containing the P, released was mixed with ammonium molybdate, Cu acetate and ELON and the blue complex read at 750 nm (Le Bel et al.. 1978). The decrease of absorbance by TCA was taken into account. KH2 PO4 was used as phosphate standard Inhibition of nucleoside phosphatase activity (%I) Substrates P, released, W/ml % Sodium azide Sodium fluoride Ethacarynic acid Ouabain Olygomycin Levamisole Okadaic acid

ATP

ADP

GTP

GDP

2.31 kO.21 (3) 100 74+ I5 (2) 73+26 (2) 50&3 (2) Ok2 (2)

1.75&0.04 (4) 100 63+ IO (4) 76k3 (3) 66k6 (3) Ok2 (3) OXI2 (3) 1 II (3) 50* I4 (3)

2.02+0.03 (3) 100 5Ok6 (2) 6759 (3) 44+8 (2) Of4 (2) 0+2 (2) 012 (2) 43+9 (2)

1.28rtO.02 (2) 100 ND ND ND ND ND ND ND

0+2 (2) 5+4 (2) 54* I (2)

Number of experiments in brackets. ND, experiment not done.

Indeed, nucleoside triphosphates reacted more strongly than the nucleoside diphosphates as confirmed by the higher release of inorganic phosphate observed in presence of ATP and GTP with respect to ADP and GDP under identical conditions (Table 1). A number of published experiments have demonstrated that a single enzyme is responsible for the hydrolysis of triphospho- and diphosphonucleosides. LeBel et al. (1980) reported that the hydrolysis of ADP and ATP occurs in a single band on the gel, following separation of pig pancreas ZGM proteins by polyacrylamide gel electrophoresis in Triton X- 100. The nucleoside phosphatase activity present on our native ZGM proteins share similarities with ATP diphosphohydrolases, such as calcium/magnesium dependence and non-sensitivity to mitochondrial and plasma membrane ATPase inhibitors (oligomycin and ouabain, respectively) and alkaline phosphatase inhibitors (levamisole) but sensitivity to sodium fluoride, sodium azide and ethacrynic acid (Table 1 and Fig. 5). Inhibition by sodium azide is unlikely to be due to an involvement of mitochondrial ATPase as the more specific inhibitor oligomycin had no effect. The significance of the strong inhibition observed in presence of okadaic acid (Table l), a reported specific inhibitor of protein phosphatases 2A and 2B remains unclear. We have demonstrated that a single glycoprotein of 92 kDa was extracted from the broad band with nucleoside phosphatase activity. This finding is compatible with the hypothesis that a single multimeric protein could be responsible for the nucleoside phosphatase activity (Fig. 4c). Homophilic protein complexes linked by noncovalent hydrophobic forces could carry the

hydrolysing activity. Freedman and Scheele (1993) have described a similar molecular structure for GP-2, the most abundant glycoprotein present on the intragranular face of the ZGM. GP-2 has been found to have a tendency for self-association into tetrameric complexes under certain conditions (Freedman and Scheele, 1991) and it has also been shown to have a phosphatidylinositol anchor in the ZGM (Le Be1 and Beattie, 1988). CHAPS solubilized ZGM proteins after denaturing separation showed a 92 kDa band (Fig. 4a) which could be identified as GP-2 by comparison with the literature (Le Be1 and Beattie, 1984). The presence of a 92 kDa glycoprotein in the dense broad band obtained by CHAPS PAGE suggests that multimeric forms of GP-2 are present. We hypothesize that GP-2 could be the carrier of the nucleoside phosphatase activity. The sharp protein band showing strong activity to nucleoside monophosphates (Fig. 3c) had no clear correlate on silver stained CHAPS gels. Probably this band is fused with the upper protein band showed in Fig. 2(a). The enzymatic nature of this activity was confirmed by inhibition by cQ-methylene ADP, a known inhibitor of AMPase activity (Rodan et al., 1977). Mannose and N-acetylglucosamine residues are probably present near the active site of AMPase and ATPase since the lectins Con A and WGA inhibited the activity to AMP and ATP (Fig. 5b). The activity towards ADP, GDP and GTP was also inhibited by these lectins. The partial inhibition of the di- and triphosphatase activity by GNA and RCA 120 (Fig. 5c and d) indicates that j?-o-galactose and terminal mannose residues are not very

1053

Nucleoside phosphatase activities on ZGM

distant from the active site(s) of the enzyme. Inhibition of AMPase and ATPase activity by lectin binding can be explained by steric hindrance (interference with the neighbouring active site) or allosteric alteration of the substrate-binding affinity (change of steric conformation). Electron microscopical preparations confirmed the localization of the AMPase activity on the ZGM. Epon-embedded ZGM preincubated with AMP and lead nitrate revealed distinct lead deposits along membranes. Controls with added inhibitor a,/?-methylene ADP or without substrate did not show any precipitates. Beaudoin et al. (1980) similarly observed lead precipitates on the apical plasma membranes and on the inner surface of ZGM at the outset of fusion in the presence of Ca2+ as activator and ATP, ADP and IDP as substrates. This and experiments by Uchiyama (1983) indicate that the enzymatic activity is located on the inner side of the ZGM. AMPase activity was cytochemically detected in rat pancreatic exocrine cells after infiltrating lightly fixed pieces of intact tissue. Activity was found on the Golgi cisternae, lysosomal bodies and condensing vacuoles, but not on ZG because the nucleotides do not diffuse into the ZG (Uchiyama, 1983). In our cytochemical experiments the lead deposits were not present on all membranes. The enzyme activities were clustered on some membrane sheets but not present on others (Fig. 6C and D). This could be explained by a tendency of these membrane proteins to aggregate in the two dimensional plane of the isolated membrane sheets. As the phospholipid/ protein ratio is high (Freiburghaus and Schiipbach, 1991) large areas of protein-free membrane sheets can be expected. The phosphatidylinositol anchor of S-nucleotidase (Low, 1987) would allow the protein to diffuse freely in the two dimensional plane of the membrane. Also, true heterogeneity of ZG, as has been suggested for contents (Beaudoin et al., 1988; Mroz and Lechene, 1986) and membranes (Beaudoin et al., 1988; Kan and Bendayan, 1989) could explain our finding. In conclusion, we have (1) separated native proteins of the ZGM of pig pancreas by nondenaturing (CHAPS-) polyacrylamideand agarose gel electrophoresis, (2) identified a calcium/magnesium dependent di- and triphosphate nucleotidase activity and a monophosphatase activity, (3) isolated a 92

kDa glycoprotein responsible for this activity, (4) detected and characterized glycosyl residues near the active site of these nucleoside phosphatases, and (5) cytochemically localized the monophosphatase activity on the ZGM. The role of these phosphatases during exocytosis remains to be elucidated. Our results suggest that GP-2 carries the nucleoside diand triphosphatase activity. Acknowledgement-This

Amelie-Waring-Foundation,

work was supported Zurich, Switzerland.

by the

REFERENCES

Azzi A., Bill K., Broger C., Casey R. P. and Riess V. (1981) Purification of cytochrome c oxidase by affinity chromatography. In Membrane Proteins (A Laboratory Manual) (Edited by Azzi A., Brodbeck U. and Zahler P.), pp. 97-100. Springer, Berlin. Baron M. D., Pope B. and Luzio J. P. (1986) The membrane topography of ecto-5’-nucleotidase in rat hepatocytes. Biochem. J. 236, 495-502. BayerdiirtTer E., Haase W. and Schulz I. (1985) Na/Ca countertransport in plasma membrane of rat pancreatic acinar cells. J. Membr. Bioi. 87, 107-I 19. Beaudoin A. R., Gilbert L., St Jean P., Grondin P. and Cabana C. (1988) Heterogeneity of the zymogen granule membranes in rat pancreas. Eur. J. cell Biol. 47, 233240. Beaudoin A. R., Laliberte J. F., Le Be1 D., Lord A., Grondin G., Phaneuf S. and St.Jean P. (1980) Localization and physiological role of the ATP-diphosphohydrolase in the pancreatic acinar cell. In INSERM Symposium E.xocrine

No. IS: Pancreatic

Biology Cells

of Normal

and

Cancerous

(Edited by Ribet A., Pradayrol L. and Susini C.), pp. 273-280. Elsevier/ North-Holland, Amsterdam. Burnette W. N. (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analyt. Biochem. 112, 195-203. Carraway K. L., Doss R. C., Huggins J. W., Chesnut R. W. and Carraway C. A. (1979) Effects of cytoskeletal perturbant drugs on ecto 5’-nucleotidase, a concanavalin A receptor. J. cell Biol. 83, 529-543. Davis B. J. (1964) Disc electrophoresis. II. Method and application to human serum proteins. Ann. NY Acad. Sri.

121, 404427.

De Lisle R. C., Schulz I., Tyrakowski T., Haase W. and Hopfer U. (1984) Isolation of stable pancreatic zymogen granules. Am. J. Physiol. 246, G411418. Drummond G. 1. and Yamamoto M. (1971) Nucleoside cyclic phosphate diesterases. In The Enqw~es (Edited by Boyer D.), Vol. 4, pp. 337-354. Academic Press, New York.

Freedman S. D. and shows pH and ion merit complexes. J. Freedman S. D. and induced homophilic

Scheele G. A. (1991) Globular GP-2 dependent self-association into tetracell Biol. 115,

197a.

Scheele G.A. (1993) Reversible pHbinding of GP-2, a glycosyl-phos-

1054

Marco

Sorlam

phatidyl-anchored protein in pancreatic zymogen granule membranes. Elrr. J. (,(,I/. Biol. 61, 229-238. Freiburghaus, A. U. and Schiipbach. J. (1991) A universal method for solubilizing membrane proteins for 2DPAGE with isolelectric focussing on immobilized pHgradients. In 2D-PAGE ‘91 Proceeditrgs of the Irlterrlufiord

Meeting

017

TUY)

Dimen.sioncrl

London, July 16-l 8, 1991 (Edited by J.). pp. 50-51). Department of Cardiothoracic NHL Institute (UK), London. J., Reddy M. K. and Reddy J. K. (1983) of secretory protein and membrane composecretory granules isolated from normal and pancreatic acinar cells of rats. Proc,. nuts.

Electrophoresi.s

Dunn M. Surgery, Hansen L. Comparison sition of neoplastic Acud.

Sci.

U.S.A.

80, 43794383.

Harper F., Lamy F. and Calvert R. (1978) Some properties of a Ca”+- and (or) Mg’+-requiring nucleoside diand triphosphatase(s) associated with the membranes of rat pancreatic zymogen granules. Can. J. Biochem. 56, 565-576. Kan F. W. and Bendayan M. (1989) Topographical and planar distribution of Helix pomatia lectin-binding glycoconjugates in secretory granules and plasma membrane of pancreatic exocrine acinar cells of the rat: demonstration of membrane heterogeneity. Am. J. Anatom. 185, 165-176. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature

227, 680-685.

Le Be1 D., Poirier G. J. F. and Beaudoin purification of a drolase from pig 1233. LeBel D. and Beattie eral proteins of

G., Phaneuf S., St. Jean P., Lalibertt A. R. (1980) Characterization and calcium-sensitive ATP diphosphohypancreas. J. Biol. Chem. 255, 1227-

(1984) The integral and periphzymogen granule membrane. Biochim. hiophys. Acta 769, 61 l-621. LeBel D. and Beattie M. (1988) The major protein of zymogen granule membranes (GP-2) is anchored via covalent bonds to phosphatidylinositol. Eiochem. hiophys. Res. Commun. 154, 818-823. LeBel D., Poirier G. G. and Beaudoin A. R. (1978) A convenient method for the ATPase assay. Analyr. Biochem. 85, 9&97. Low M. G. (1987) Biochemistry of the glycosyl-phosphatidylinositol membrane protein anchors. Biochem. J. 244, l-13. Meldolesi J., Jamieson J. D. and Palade G. E. (1971) Composition of cellular membranes in the pancreas of the guinea pig. 3. Enzymatic activities. J. cell. Eiol. 49, 15&158. Montero J. M. and Fes J. B. (1982) Purification and characterization of bovine brain 5’-nucleotidase. J. Neurochem.

M. the

39,982-989.

Morrissey J. H. (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analyt. Biochem. 117, 307-310.

(‘t (I/

Mroz E. A. and Lechine granules differ markedly 232, 871 873. Neugebauer J. ( 1988) In U.se.s of’ Detergents

it,

C‘. (1986) in protein A Guide Biology

Pancreatic composition. to the

cd

zymogen

Propertie\

Biochemi.s/r~,

Scren~

tmtl

Vol.

81X3-10-89, p. 22. Calbiochem Corp., La Jolla, CA. Newby A. C.. Luzio J. P. and Hales C. N. (1975) The properties and extracellular location of 5’-nucleotidase of the rat fat-cell plasma membrane. Biochem. J. 146, 625-633. Palade G. (1975) Intracellular aspects of the process of protein synthesis. Science 189, 347-358. Picher M., Beliveau R., Potier M., Savaria D.. Rousseau E. and Beaudoin A.R. (1994) Demonstration of an ectoATP-diphosphohydrolase in non vascular smooth muscles of the bovine trachea. Biochim. hiophys. Artu 1200, 167Tl74. Rodan G. A., Bourret L. A. and Cutler L. S. (1977) Membrane changes during cartilage maturation. Increase in 5’-nucleotidase and decrease in adenosine inhibition of adenylate cyclase. J. ceil. Biol. 72, 493--501. Sinjorgo K. M. C.. Steinebach 0. M., Dekker H. L. and Muijsers A. 0. (1986) The effects of pH and ionic strength on cytochrome c oxidase steady-state kinetics reveal a catalytic and a non-catalytic interaction domain for cytochrome c. Biochim. hiophys. Acta 850, 108-l 15. Spaans M. C., Tobler M., Ammann R. W. and Freiburghaus A. U. (1994) Separation and analysis of pig pancreatic zymogen granules with free flow electrophoresis and lectins. Electrophoresi.~ 15, 572-576. Traverso-Cori A., Chaimovich H. and Cori 0. (1965) Kinetic studies and properties of potato apyrase. Arch. Biochem. Biophys. 109, 173-l 74. Uchiyama Y. (1983) A histochemical study of variations in the localization of 5’-nucleotidase activity in the acinar cell of the rat exocrine pancreas over the twentyfour hour period. CeN Tissue Res. 230, 411420. Van Den Hoek A. and Zail S. S. (1977) Polyacrylamide gel electrophoresis of human erythrocyte membrane enzymes solubilized with Triton X-100. Clin. chim. Acts 79, 7-14. Wagner A. C., Wishart M. J., Yule D. I. and Williams J. A. (1992) Effects of okadaic acid indicate a role for dephosphorylation in pancreatic stimulus-secretion coupling. Am. J. Physiol. 263, C1172-Cl180. Wilcox D. K., Kitson R. P. and Widnell C. C. (1982) Inhibition of pinocytosis in rat embryo fibroblasts treated with monensin. J. ceil. Biol. 92, 859-864. Young T. M. and Young J. D. (1984) Protein-mediated intermembrane contact facilitates fusion of lipid vesicles with planar bilayers. Biochim. hiophy.7. Acta 775, 44th 445. Zekri M., Harb J., Bernard S. and Meflah K. (1988) Purification of bovine liver cytosolic 5’-nucleotidase. Kinetic and structural studies as compared to the membrane isoenzyme. Eur. J. Biochem. 172, 93-99.