Gut: first published as 10.1136/gut.28.10.1208 on 1 October 1987. Downloaded from

Gllt, 1987, 28, 1208- 1214 Alimentary tract and pancreas Demonstration of pepsinogen C in human pancreatic islets

P B SZECSI, LI HALGREEN, SS POULSEN, C K AXELSSON, M DAMKJ/ER-NIELSEN, T KJ/ER, AND B FOLTMANN From The Department of Clinical Chemistry, Hvidovre Hospital; Institute of Pathology, Bispebjerg Hospital; Institute of Biochemical Genzetics, In.stituite of Medical Anatomy, Departnzent B and Department of Clinical Physiology, Glostrup Hospital, University of Copenhagen. Department K of Surgery, Vejle Hospital and Department L ofSurgery, Arhu.s Municipal Hospital, University ofArhus, Denmark.

SUMMARY Pancreatic tissue from 16 post mortem kidney donors have been examined for the content of pepsinogens. A zymogen with electrophoretic mobility, isoelectric point and molecular weight equal to that of pepsinogen C of gastric origin was found in all specimens. A comparison between pepsinogen C extracted from pancreatic tissue and gastric mucosa demonstrated immunological identity. Quantitative measurements with a radioimmunoassay showed pepsinogen C concentrations in pancreatic tissue three to 80 times higher than those of blood serum. Immunohistochemical staining gave positive reaction for pepsinogen C only in the alpha cells of the pancreatic islets. http://gut.bmj.com/ Gastric belong to the superfamily of superfamily of aspartic proteases localised outside aspartic proteases (EC 3.4.23). The predominant the gastrointestinal tract have traditionally been gastric proteases of adult mammals are designated designated D or E.'" During a series of A (EC 3.4.23.1) and pepsin C (EC 3.4.23.3). investigations on the general distribution of aspartic For both proteases several isoenzymes and iso- proteases, we observed that several tissues contained zymogens may be observed. In native state the two zymogens and proteases with electrophoretic and types show no immunochemical cross reactions. immunochemical properties that were indistinguish- Within each type the isozymogens cannot be distin- able from gastric pepsinogen A and C.2 19 on September 29, 2021 by guest. Protected copyright. guished with polyclonal antisera.' This study deals with our findings of a proenzyme The proenzyme of pepsin A (pepsinogen A) is in the human endocrine pancreas that is indistin- synthesised in the chief and mucous neck cells of the guishable from gastric pepsinogen C. fundic mucosa, whereas the proenzyme of pepsin C (pepsinogen C) is found in the same cells as well as Methods in cells of cardiac, pyloric and Brunner's glands.4 Furthermore, both pepsinogen A and C have been REAGENTS shown in serum,i in gastric gland heterotopia and Agar Noble (Difco, Detroit, USA); Agarose type II metaplasia anywhere in the gastrointestinal tract3 (Sigma, St Louis, USA); Agarose HSA (Litex, and in a Meckel's diverticulum.' Pepsinogen A has Glostrup, Denmark); Aprotinin (Bayer, Lever- been demonstrated in normal urine' "''" and pepsi- kusen, FRG); Acrylamide (BioRad, Richmond, nogen C in seminal fluid,'"' the seminal vesicles2 and USA); N,N'-metylene-bis-acrylamide (Sigma, St the prostate gland."' Louis, USA); Benzamidiniumchloride (Merck, Apart from (EC 3.4.23.16) members of the Darmstadt, FRG); Human albumin (Behringwerke, Marburg, FRG); "'5I-Nal (Hoechst, Frankfurt am Main, FRG); Lactoperoxdidase (Sigma, St Louis, Addrcss for (orrcsporlndcncc 13B Szcc.s.. D)pairticrit oC(hlical (Chemistry 339. USA); Phenylmethylsulfonylfluoride (Sigma, St 11v idovrc l lospit.t. UnIvcrsity otfCopenhagcn. Kctteg.aardsa llc 23. DK-265(0 1 idowrc Dct.mrkI) Louis, USA); Sephadex G-25 (Pharmacia, Uppsala, Reccivcd tor puhlication 'S Mi.rth 1987 Sweden); Triton X-100 (Serva, Heidelberg, FRG); 1208 Gut: first published as 10.1136/gut.28.10.1208 on 1 October 1987. Downloaded from

Demtionistration ojpepsinogen C in hlumlan pancreatic islets 129()

3-3' diaminobenzidine tetrahydrochloride (Sigma, St E LECTROPHORESES Louis, USA); HAWP nitrocellulose (Millipore, Agar gel zone electrophoresis was carried out in l1(o Molsheim, France); Sodium dodecylsulfate (Merck, (w/v) agar gels in 0(05 M sodium phosphate pl I 6() Schuchardt, FRG); Donkey antirabbit antibody for two hours at a gradient of 15 V/cm at constant coated cellulose (Wellcome, Dartford, UK). voltage as described previously."' Crossed tiindeiii immunoelectrophoresis was carried out in 1 %O (w/v) SAMP1 ES OF TISSUES agarose gels." Pancreatic tissue was obtained from post mortem Sodium dodecylsulphate (SDS) polyacrylamide kidney donors. The donors were victims of accidents gel electrophoresis was carried out in vertical slab and without systemic disease. The time of ischaemia gels (T=15%, C=0-5%).- The proteases were de- was kept as short as possible (15-120 min). The tected by indirect peroxidase staining after trans- pancreas were dissected free from fat and connective ferring of proteins to nitrocellulose." tissue, washed with ice cold 0-05 M sodium phos- Isoelectric focusing was performed in I mmin thick phate p1i 6-0 and cut into pieces of 0-5 cm.' polyacrylamide gels (T=7.5%0, C=3(o ) coiitdiiniflg Extraction of 1 g wet weight tissue took place in a 500 (v/v) Pharmalyte (pH 2-5-5) for 3 h at 1500 V Potter-Elvehjem homogeniser with 3 ml of 0-05 M (constant voltage) after one hour of prefocuising. sodium phosphate pH 6-0 made 5 mM with phenyl- methylsulfonylflouride, 20 mM with benzamidine DETECTION OF ACTIVITY AFTER and 40 FtM with aprotinin. Triton X-100 was then El ECTROPHORESIS added to the homogenate at a final concentration of After electrophoresis the containing zonles 2% (v/v) and sonication took place for three times 1Os were visualised as caseograms - that is, overlaying a in an ice bath. Cell debris were removed by centri- skim milk containing gel on top of the electrophoresis fugation at 20000 g at 5°C for 30 min. The super- gel and observation of clotted casein. natant was stored at -20°C until assayed. Extraction of samples of liver and spleen tissues for QUANTIFICATION OF PtEPSINOGFNS control experiments took place as described for Pepsinogen C was measured by a double antihody pancreatic tissues. Fixation for immunohistochemis- solid phase radioimmunoassay (RIA) with stalndards try took place in Bouins fixative for six hours at 4°C. purified from human gastric mucosa.-`

The tissues were kept in 70% (v/v) alcohol until final Pepsinogen C was labelled with ''2I by a lacto- http://gut.bmj.com/ embedding in paraffin. peroxidase method.4 In brief, 1-63 ug pepsinogen C dissolved in 115 Rl 0-5 M sodium phosphate pH1 7-5 AND HORMONES was mixed with 18X5 MBq "'lI in 100 RI water, 5 [tg Pepsinogen A was prepared from human gastric lactoperoxidase in 10 1t 0.5 M sodium phosphate p1i mucosal or human urine"'; pepsinogen C was pre- 7-4 and 10 RI 352 RM solution of hydrogen peroxide. pared from human gastric mucosa"; human renin lodination was terminated after 30 min by addition of was a gift from Dr I Rubin, Copenhagen, Denmark; I ml water. '2I-pepsinogen C was obtained by insulin and glucagon was from NOVO, Bagsvxrd, gelfiltration on Sephadex G-25 eluted with ()0I M on September 29, 2021 by guest. Protected copyright. Denmark; somatostatin was from Cambridge Re- sodium phosphate pH 7-4. Hluman albumin (200 itl search Biochemicals, Harston, UK. 2% (v/v)) was added to 6 ml of tracer and aliquots of 500 tl were stored frozen. ANTISERA Immediately before use the tracer was diluted with Antisera against pepsinogens were raised in rabbits'' the RIA buffer: 0-04 M sodium phosphate pl1 7-4 at Statens Seruminstitut, Copenhagen, Denmark by with 0-15 M sodium chloride, 0-01 M EDTA, 5 g/l the courtesy of Dr N H Axelsen. The immuno- human albumin and 5 mg/l phenylmercuric acetate. globulin fractions of the antisera were purified by salt With freshly prepared tracer the dilution factor was precipitation and ion exchange chromatography.' determined allowing a radioactive concentration of The following antibodies were obtained from 1.1 MBq/l of diluted tracer solution. The radio- Dakopatts, Copenhagen, Denmark: horseradish immunoassay procedure was as follows: 50 Itl pepsi- peroxidase conjugated porcine antirabbit IgG nogen C standard (2-6-191 [g/l) or test substance (P 217), porcine antirabbit IgG (Z 196), peroxidase were mixed with 200()l "'"I-pepsinogen C solution, antiperoxidase complex (Z 113). Rabbit antiinsulin 200 itl rabbit antipepsinogen C (1:30000) and 300 [dl and rabbit antiglucagon was a gift from Dr J J Hoist, RIA-buffer. The mixture was incubated for 48 h at Copenhagen, Denmark. Rabbit antisomatostatin room temperature. Counting of bound pepsinogen C was from Cambridge Research Biochemicals, was performed after precipitating with 50 Fil of at Harston, UK. Rabbit antirenin was a gift from Dr K suspension of donkey antirabbit aintibody coated Poulsen, Copenhagen, Denmark. cellulose. Detection limit was 2-6 Ftg/l. The intra- Gut: first published as 10.1136/gut.28.10.1208 on 1 October 1987. Downloaded from

121() Szecsi, Halgreen, Poulsen, Axelsson, Damkjwr-Nielsen, Kjwr, and Foltmann

Pg Pn

A3 -

A3- C-

A4. A5 C1l C2 - C3 - ,..,u,,,.,.;/!i!;-_ &is«,;;'. : g C ii ':. ..:..'::.;. .1-' 4..~~~..:.;.t ... .o Fig. 2 Tandet-em crossed immunoelectrophoresis of' panc reatic extract C. The saimples appi ainidpepsinogeni followinig http://gut.bmj.com/ were applied: P well (extended), 25 Fl of'humatn panicreatic extrtrct; G well, 5 [tl chromatographically puirified gastric pepsinogen C (09 gll). First dintiensioni electrop)horesis (anode to the right) was runl (it 10 V/cmfor 60 mimi at 12°C in "' agarose gel (10%) with TrislBarbital bufferpH 8-6. Se-otnd Fig. I (aseogramn (ijier aigair gel electrophoresis in 0-05 M dimenisioni electrophoresis (aniode (it the top) was run at .sodlii,n Phosl)lphtae, pH 60(andtl 15 V/cm ftor two houirs. Lanle 2 Vlcm at 120C overnight, witli the sane agarose gel anid M. p)artly aclitvile(l extract ofgastric l nucos(i; lante P: extract buffer as in thefirst-dimension, blut with acntibodlies against oJftlinal panicreas. Applicalion voliine 2O[lperslit. pepsinogent C (4 tllcm '). Stainied with Coomnasie on September 29, 2021 by guest. Protected copyright. Pg =,)epsiniogel, Pn=pepsin. AppIl.=applic(itionpoillt. Brilliacnt Anode (it top). Blue R 250. assay and the interassay precision were 6-&l o and antipepsinogen C preabsorbed with pepsinogen C, 10%, respectively. Cross reaction with pepsinogen A pepsinogen A, glucagon, somatostatin or human was less than 0-3%. renin; (d) sections of human gastric mucosa served as positive control for the pepsinogen C staining. IMMUNOHISTOCHEMISTRY Series of paraffin sections (approx 6 [tm) from Results pancreas were immunostained by the peroxidase antiperoxidase method."' The primary antibodies By agar gel zone electrophoresis and detection of were diluted in 0-005 M TRIS buffer pH 7-6 contain- proteolytic activity by the caseogram technique,"' a ing 0-15 M sodium chloride, 0-3% (v/v) Triton X-100 zymogen with electrophoretic mobility correspond- and 5% (v/v) normal porcine serum: antipepsinogen ing to that of gastric pepsinogen C was observed in all C 1:400; anti-insulin 1:3200; antiglucagon 1:1600; of 16 examined extracts of human pancreas. The antisomatostatin 1:1600 and antirenin 1:1600. Sec- results from agar gel electrophoresis are exemplified tions were incubated overnight at 4°C. The following in Figure 1, which shows a reference of partly controls were undertaken: (a) omitment of incuba- activated mucosal extract run in parallel with a tion with primary antibody; (b) incubation with pancreatic extract. The zymogen present in pan- homologous non-immune serum; (c) incubation with creatic extract has the mobility of pepsinogen Cl. Gut: first published as 10.1136/gut.28.10.1208 on 1 October 1987. Downloaded from

Detnionistrationt ofpepsiniogen C in human pancreatic islets 1211

pI

4.4- Mr

4Q000-

4'1 -

3.5- http://gut.bmj.com/

Fig. 3 Ilmmtiunlobloto(DSDSpolyar(rviltamide gel Fig. 4 Caseograiniofinoel('(ti( flliii /tuittli i(Ac electroplioresis (T= 15%Y, C=0=5(,). Detectiomi indirect (T= 75',, ( ) 5'Y, bY polyatcry'latuuinldegels cotaining on September 29, 2021 by guest. Protected copyright. peroxidase sitiininlg. 1: ilicidbtitioll Witli raibbit antipepsinogen PhairntlVte (pH12 5-). The .samln/)1e5s were(t(tiva(ted il'ne C (1:1000)ft)r 16 hourS at oonti tcnp/neraiti1re. 2: incutbation hloalr iitldi equal iolilne of 0) / M [1I( /)prior to (a/pp)lic(atioi: Wit/i porciel anitirabbit lg(g conijugated wit/i hlOrWsera(dish Itiil', P, 15 Itl extracti ofhllna/ill palt/i(reati( lissue; 1(m/i (, It/tl p)erOxidahsa (1: 1000)0) ft'Orli) hours (it roofli temn/perature. cliro/nito,gr(i/liicalll /)i,urified gastric pepsin ( (containing 3: /peroxidasest(ingilatigwd 3-3 (diamfliniObelinzildin 11(01g). tetrah idro()cllori(dc. Slamples: lane P. 15 ttl (1:2) e tract of /lii(tlpan(tlcre(itic tissue; liie C,, I(10'ilcl(lOgr(t/)hiicallv of the pancreatic zymogen with pepsi- urified g(astric pepsiiogeI (' (contailiang 90 ig). olt/i Comparison p) nogen C gastric origin, showed immunological s(am/)le.s was re(duce(d wit/i 5 %, dit/hioerYtritol for 5 mini (it of 1000C. identity when tested by crossed tandem immuno- electrophoresis; the precipitates fused without spurs (Fig. 2). This isozymogen is only a minor component in gastric Determined by SDS-polyacrylamide gel electro- mucosa but is the predominant isozymogen of the phoresis the molecular weight of the pancreatic seminal pepsinogen C. The isozymogens are not well zymogen was equal to that of gastric pepsinogen C characterised, but upon activation the three isozy- (Mr 40000) (Fig. 3). mogens give rise to a pepsin C with the same mobility By isoelectric focusing of activated gaistric pepsi- (data not shown). It should be noted that at pH 6-), nogen C, pl is 3-5 and activated pancreatic extract pepsin C and pepsinogen A3 have about the saime gave three proteolytic bands pl 4-4, 4 1 aind 3-5 electrophoretic mobilities. Zymogens corresponding (Fig. 4) of which only the laist had immunological to pepsinogen A was present in seven of the extracts. homology with pepsinogen C. Gut: first published as 10.1136/gut.28.10.1208 on 1 October 1987. Downloaded from

12 12 .Szecsi, Halgreen, Poulsen, Axelsson, Damkjer-Nielsen, Kjar, and Foltmann

- ~. t. , http://gut.bmj.com/

- g ;~~~ktt4N,'~~~~~~~~A.A'' 1 .V.l ' w on September 29, 2021 by guest. Protected copyright.

Fig. 5 Serial seclion7s ofJ hiimoal pancreas stainie( with (A) antipepsinogen C (1:400) and (B) antipepsinogen C (1:400) pre- inculated with pep.sinOogen C (fina(il concentration 0(25 mg/l) for two houirs at room temperatuire before staining. Serial sections of llioii(iii/paincreals.stained wit/i (C) antipepsinogen ( (1:400) ant1d (D) antiglucagon (1:1600). Counterstained with Meyers hiaenl(itoxvlil. A rroivs idzlaic i/cidtiicalac/lls.

Quantitative determination of pepsinogen C in 16 centration range of pepsinogen C in serum of normal extracts from different pancreatic glands showed that human controls is 6-37 ng/ml (median 12 ng/ml). the concentration range of pepsinogen C was 32-816 In sections of pancreatic tissue from 11 individuals, ng (median 297) per gram wet weight of tissue. An staining for pepsinogen C was positive in all. Positive additional pancreas was cut into 11 sections from staining was observed in cells predominantly in the caput to cauda. This experiment showed that the peripheral parts of the pancreatic islets (Fig. 5A). middle part of the pancreatic body contained most Staining of serial sections for glucagon showed pepsinogen C: 1078-2004 ng per gram tissue, the positive reactions in the same cells as those stained caput contained 368-512 ng per gram tissue and the with antipepsinogen C (Fig. 5C and 5D). The cauda 176-311 ng per gram tissue. Extracts of spleen pepsinogen C positive staining was not inhibited by and liver from four individuals contained pepsinogen prior incubation with pepsinogen A (10 ,uM), C in concentrations not exceeding 5 ng/g weight of glucagon (10 ,uM), somatostatin (10 FM) or renin tissue. Determined by the present method, the con- (38000 Goldblatt units/l). By contrast, absorbtion Gut: first published as 10.1136/gut.28.10.1208 on 1 October 1987. Downloaded from

Demonistration ofpepsinogen C in hwnman panc reatic islets 1213 experiments with pepsinogen C (10 [tM) resulted in serve in regulatory functions. Whether the general complete abolition of staining (Fig. 5B). The same and unspecific proteolytic activity is involved in antipepsinogen C stained the chief and mucous neck degenerative diseases of the pancreatic gland cells of gastric mucosa. remains to be investigated. An zymogeti have also benci Discussion isolated from monkey lungs." This wast called procathepsin D-ll, but in all analysed propcr- Pepsinogens have until now been localised only in ties it was indistinguishable froni pepsinogen C. cells of exocrine glands. The major source of pepsi- These results support the view thalt the occurrence of nogens is the gastric mucosa, even though extra- extra gastric pepsinogens is more widespread than gastric pepsinogens have been observed. The pepsi- previously thought. nogens in serum and urine are assumed mainly to arise from the gastric glands due to reabsorbtion Mrs Charlotte Fagerlund, Henny Falsing, and Jette during cell turnover.' In spite of use of inhibitors of Schousboe are gratefully acknowledged for their the pancreatic serine proteases small amount of non- excellent technical assistance, Miss Irmelin Krabbe pepsin proteases in Figure 1 are interpreted as serine for iodinating pepsinogen C. We thank Dr J Hoiriis, proteases. In nearly all extracts small amounts of Hagedorn Research Laboratories, Denmark for active were present. supply of human pancreatic tissue and Dr S Thorsen In all examined extracts zymogens with the same for discussion and encouragement. Part of this work properties as gastric pepsinogen C were observed. was supported by grants from Nordic Insulin The concentration of pepsinogen C is three to 80 Foundation, Novo Foundation and The Danish times higher in pancreatic tissue than in serum. Diabetes Association. Furthermore, organs rich in blood like the spleen and the liver did not show higher pepsinogen C levels References it is than that of serum. From this we conclude that I Foltmann B. Gastric proteinascs - structurc lunction. not trapped serum pepsinogen C that is observed. cvolution and mechanism of iaction. Ls.vvs Bioche('m As regards to the specificity of the immuno- 1981; 17: 52-84. chemical reactions one has to consider the possibility 2 Foltmann B. Tarasova NI. Szccsi PB. Mcthods Ior that the staining for pepsinogen C might be because detection of' proteinaises. Elcctrophorctic and immun1.l0o- of antigenic determinants that are common to other logical comparisoon of aspartic protci nascs of diffcre it http://gut.bmj.com/ aspartic proteases. Dreyer et al6 have recently found origins. In: Koskia V. ed. A.parhitc 1proleilla.ses a111( Ih('ir that, although the primary structure of protease A inhibitors. Berlin-New York: Walter dle Gruyter. 1985: from baker's yeast has only 37% of identity with calf 491-508. A 3 Varis K. Peptic cells. In: KreuningiJ SamnlolffIM Rotter , protease may be detected with anticalf JI Eriksson AW. eds. Peps.sinogens in manl: (liical (1a1(1 chymosin by immunoblotting. In spite of such un- geieticadv'ances. New York: Alaln R l1iss. 1985: 177-84. certainty we must emphasise that the presence of 4 Samloff M, Liehmaln WM. Cellular localization ol the pepsinogen C was demonstrated in pancreatic ex- group 11 pepsinogens in human stomrach antld dtuodenuiii on September 29, 2021 by guest. Protected copyright. tracts with several techniques and that in the by immunofluorescence. GasIroenherolog£, 1973; 65: immunohistochemical experiments only the alpha 36-42. cells gave positive reaction for pepsinogen C. 5 Axelsson CK. Nielsen MD. Kappeigaard AM. Solid- As mentioned above, pepsinogen A was present in phase double antibody radioimmunoaL10assay of pcpsi- about half of the pancreatic specimens that were nogen f in serum. Cli/i (Chil Acta 1982: 121: 309-19. 6 Ichinose M. Miki K, Furihata C. et al. Radioimmun10- examined. These investigations are still in progress. assay ot group 11 pepsinogen in huLIMI seruL11. (Clil (hill The significance of the pancreatic pepsinogen C in Acml 1982; 122: 61-9. endocrine cells is unknown. An acid environment is 7 Samloff IM. Pepsinogens I and 11. Purilication f'rom necessary for pepsinogen activation and for its pro- giastric mucosa aind radioirnmunoassay in serum11. (,Isl)o- teolytic activity. In intracellular compartments, how- eiierologx' 1982: 82: 26-33. ever, pH could be low enough for activation of 8 Samloff IM. Liebman WM. Radioimiunoassay of pepsinogens and for pepsins to exert their pro- group I pepsinogens in serLl1l. (Gasvroenmmerology 1'974: teolytic activity.27 These enzymes are generally 66: 494-502. regarded as rather unspecific in sense that general 9 Liebman WM. Bujalnover Y. (iroups I and 11 pepsi- nogens in Meckels's diverticuLumLI. J IlisioC/e11 (Cia- and clear cut rules for their specificities have not been chemn 1978; 26: 867-8. found. But these enzymes have an extended binding 10 Axelsson CK, Axelsen Nil. Svendsen PJ. Purification o cleft that may accommodate a substrate peptide pcpsinogen I from human urinc by nicans ol' DEAE- chain with at least eight amino acid residues.' Thus chromatograiphy and isotaichophorcsis. Elecirophoresis pepsin C may have a potential specificity that may 1980; 1: 164-7. Gut: first published as 10.1136/gut.28.10.1208 on 1 October 1987. Downloaded from

1214 Szecsi, Halgreeen, Polulsen, Axelsson, Damkjwtr-Nielsen, Kjwvr, an(I Foltmnann

11 Pals G, Biemond I, Delize J, et al. In: Kreuning J, determination of the aimino acid scquenice of the pro- Samloff IM, Rotter JI, Eriksson AW, eds. Pepsinogen in part. Elo J Bioclem 1982; 128: 63-70. macn: clinical andt genetic adivances. New York: Alan R 21 Axelsen NH, Kroll J. Weeke B. eds. A mainuial ot Liss, 1985: 91-1()(). quantitative immIuLinociectrophorcsis. Scand J litiIIiol 12 Pals G. Humani epsinogens. Amsterdam Free Univer- 1973; 2: suppl. 1. sity: Thesis, 1986: 110-19. 22 Laemmnli UK. Cleavage of structure proteins during the 13 Lundquist F, Seedorf HH. Pepsinogen in human assembly of the heiad of bacteriophage T4. Natlir-e seminal fluid. Nature 1952; 170: 1115-6. (Loaitl) 1970(; 227: 680-5. 14 Samloff IM, Liebman WM. Purification and immuno- 23 Bjerrum OJ, Larsen KP, Wilken M. In: Tschescke H ed. chemical characterisation of group II pepsinogens in Modern methods in protein chemistry. Berlin: Walter seminal fluid. Clin Exp Immunol 1972; 11: 405-14. de Gruyter, 1983: 79-124. 15 Ruenwongsa P, Chulavatnatol M. Acidic protease from 24 Dermody WC, Levy AG, Daivis PE, Plowmain JK. human seminal plasma. Purification and some proper- Heterogeneity ot chlorarmine T- and lactoperoxidase- ties of active enzyme and proenzyme. J Biol C(hem 1975; radioiodinated humran calcitonin. Clin Clieisi 1979; 25: 250: 7574-8. 989-95. 16 Chiang L, Contreras L, Chiang J, Ward PH. Human 25 Sternberger LA, Hardy PH, Cuculis JJ. Meyer HG. The prostatic gastricsinogen: The precursor of seminal unlabelled antibody-enzyme method of immunohisto- fluid acid proteinase. Arch Biochem Biophvs 1981; 210: chemistry. Preparation and properties of soluble 14-20. antigen-antibody complex (horseradish peroxidase- 17 Reid WA, Vongsorasak L, Svasti J, Valler MJ, Kay J. antihorseradish peroxidase) aind its use in identifi- Identification of acid proteinase in human seminal fluid cation of spirochetes. J Hi.tochen Cwviochlin 1970:, as a gastricsin originating in the prostate. Cell Tissue Res 18: 315-33. 1984; 236: 597-600. 26 Dreyer T, Halkier B, Svendsen J, Ottesen M. Primary 18 Barrett AJ, ed. Proteinases in mnammcaliacn cells anid structure of aspairtic proteinatse A from saccharomyces tissues. Amsterdam: North-Holland, 1977. cervisiae. Cairlsbetrg Res Comin 1986; 51: 27-41. 19 Foltmann B, Szecsi PB, Tarasova NI. Detection of 27 Anderson RGW, Pathak RK. Vesicles and cistcrnac in proteases by clotting of casein after gelelectrophoresis. the trans Golgi apparatus of human fibroblasts are acidic Anal Biochem 1985; 146: 353-6(1. compartments. Cell 19855; 40: 635-43. 20 Foltmann B, Jensen AL. Human progastricsin. Analysis 28 Moriyama A. Kageyama T, Takiahashi K. Monkey lung of intermediates during activation into gastricsin and procathepsin D-1I. Elar J Biochemn 1983; 132: 687-97. http://gut.bmj.com/ on September 29, 2021 by guest. Protected copyright.