Isolation and Characterization of a Novel Serine Proteinase

Vol. 268, No. 1, Issue of January 5, pp. 527-533,1993 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY

0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Isolation and Characterization of a Novel Serine Proteinase Complexed with az-Macroglobulin from Porcine Gastric Mucosa* (Received for publication, June 19, 1992)

Takashi UchinoS, Yasuko SakuraiS, Masaaki Nishigail, Takayuki Takahashi$,Hideo Arakawag, Atsushi Ikais, and Kenji TakahashiSll From the $Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Bunkyo-ku, Tokyo113 and the $Laboratory of Biodynamics, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama227, Japan

stomach (16). However, none of the peptidases involved in Porcine stomach mucosa was found to contain 740a kDa proteinhavingendopeptidase activity toward the proteolytic processing of such peptides have yet been peptide 4-methylcoumaryl-7-amide substrates and low identified. molecular mass peptides. This proteinwas purified to In an attempt to elucidate the functional role of proteolytic an apparenthomogeneitybya series ofchromatoenzymes in the mammalian stomach, a high molecular weight graphic steps on DEAE-cellulose, Sepharose CL-4B, protein that preferably hydrolyzes peptide 4-methylcoumarylhydroxylapatite, and fast protein liquid chromatogra7-amide (MCA)’ substrates at theArg-MCA bond was found phy Mono Q columns. The protein was shown to be a in the homogenate. In the present study, we purified the complex of the plasma proteinase inhibitor a2-macro- protein and characterized it in detail. The results reveal that globulin and a25-kDa endopeptidase. The enzyme ac- the enzyme activity is due to an a2-macroglobulin-associated tivity was completely inhibited by diisopropyl fluorophosphate, p-amidinophenylmethanesulfonylfluoride, serine proteinase with a molecular weight of about 25,000. leupeptin, antipain, bovine pancreatic trypsin inhibi- Further investigation of the enzymatic properties including substrate specificity and effects of various inhibitors indicated tor, soybean trypsin inhibitor, and ovomucoid, indicating that the entrapped enzymeis a serine proteinase. that this proteinase is distinct from any other known serine endepeptidases thus far described. The proteinase could be released from az-macroglobulin by mild acid treatment and the released enzyme EXPERIMENTAL PROCEDURES AND RESULTS~ showed activity toward protein substrates. Substrate specificitystudies using synthetic and pepPurification of a Protein with Boc-Gln-Arg-Arg-MCA Hytide substratesindicatedthattheenzymepreferendrolyzing Actiuity from Porcine Stomach Mucosa-Table I tially hydrolyzes Arg-X bonds and, to a much lesser extent, Lys-X bonds, and is apparently distinct from summarizes the purification of a protein hydrolyzing Bocthrombin, kallikrein, plasmin, and other trypsin-like Gln-Arg-Arg-MCA from porcine stomach mucosa. The deproteinases so far reported including tryptase. Thus, tailed procedure is described in the Miniprint section. Anionexchange column chromatography (DE-52 and Mono &) althe present enzyme is thought to be a novel type of serine proteinase. The proteinase associated with aZ- waysgave rise to low recovery of activity, but inclusion of macroglobulinwas also found in porcine intestinal mu- these columns was eventually found to yield the protein in satisfactory purity asdescribed in the following section. From cosa, but not in plasma. 230 g of the mucosa, 1.4 mg of the protein was obtained with an overall yield of 2.2%. Purity and Molecular Weight-Native PAGE using a graThe stomach is a dilated segment of the digestive tract dient gelgave a single proteinband associated with the whose main function is to digest ingested food. The best activity (Fig. 5). Its apparent molecular mass was estimated studied stomach enzyme involved in the process of digestion to be approximately 730,000 by PAGE and 750,000bygel inthis organ is the aspartic proteinase pepsin (1-5). In filtration on Sepharose CL-GB (Fig. 6). Fig. 7 shows the results contrast to this extracellular proteinase, very little is known of SDS-PAGE of the sample in the presence and absence of of intracellular proteinases, although recently cathepsin E has 2-mercaptoethanol. A major protein band corresponding to been isolated and characterized (2, 6-9) anditsstructure M, = 360,000 and a very faint band corresponding to M, = analyzed (10-12). Mucosal extracts indeed contain a variety 100,000 wereobserved under nonreducing conditions, while a of peptidase activities and most of these peptidases are major protein band ( M , = 95,000) and other minor bands (M, thought to be involved in intracellular protein turnover (13- = 80,000, 56,000, and 54,000) were separated under reducing 15). In addition, there must be some peptidases that function as proteolytic processing enzymes for certain biologically imThe abbreviations used are: MCA, 4-methylcoumaryl-7-amide; portant proteinsand peptides andtheir precursors since SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophobioactive peptides such as gastrin, enkephalin, somatostatin, resis; DFP, diisopropyl fluorophosphate; TPCK, Nu-tosyl-L-phenylglucagon, and other peptides are known to be present in the alanine chloromethyl ketone; TLCK, N”-tosyl-L-lysinechloromethyl ~~

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 7 To whom correspondence should be addressed Dept. of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 3-5689-5607;Fax: 3-5684-2394.

ketone; BOC, t-butyloxycarbonyl; Z, benzyloxycarbonyl;Bz, benzoyl; SUC,succinyl. Portions of this paper (including “Experimental Procedures,” part of “Results,” Table I, and Figs. 1-4,6,8-11, and 13-17) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are is available from included in the microfilm edition of the Journal that Waverly Press.

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sulted in a remarkableincrease in the activity toward protein substrates (Fig. 10). In addition, goat anti-human a2-macroglobulin antibody nolonger precipitated the proteinase activity when the purified protein was previously treated at pH 3 (datanot shown). Theseresults clearly indicate that the proteinase is noncovalently associated with a2-macroglobulin, and is freed from steric restraint upon the acid treatment. In order to obtain the pure enzyme from the acid-treated sample, several column chromatographic steps were tried under various conditions. The enzyme activity was completely lost on ion-exchange columns (Mono Q, Mono S, and DERelative Activity (%I FIG. 5. PAGE analysis of the purified protein. The sample 52). Poor recovery (about 20%) of the activity was also obwas separately applied to two well positions of a gradient PAGE gel served on the gel filtration over a Sephacryl S-300 column. (4-15%) without SDS. After electrophoresis at 4 “C, one lane was Thus, none of these columns was found to be suitable for the stained with Coomassie Brilliant Blue R-250 (lane2), while the other purification. It should be noted that the gel filtration gave a was sliced into pieces of 3-mm width for overnight extraction in 0.5 single activity peak eluting at a position corresponding to MI ml of the routine assay buffer. Aliquots of the extracts were assayed = 25,000 (data not shown), a value close to that obtained in for enzyme activity toward Boc-Gln-Arg-Arg-MCA and the relative activities are shown. Lane 1 shows the separation of molecular weight the [3H]DFP labeling experiment. Action on Synthetic Substrates-No significant difference marker proteins (Pharmacia LKB Biotechnology Inc.): thyroglobulin (670 kDa), apoferritin (440 kDa), lactate dehydrogenase (140 kDa), was observed when the activities toward a few MCAsubstrates and albumin (66 kDa). were compared before and after the acid treatment of the protein. This was reasonable since the substrates are small enough to freely reach the active site irrespective of whether A B the proteinase is associated with the inhibitor. Thus, the activities on synthetic substrateswere tested using the enzyme associated with a2-macroglobulin and the results are shown inTable 11. The specificities of humanthrombin ( M I = 34,000), porcine tissue kallikrein (M1=30,000),and porcine plasmin ( M I= 80,000) are also included for comparison. The enzyme was most active toward Boc-Gln-Gly-ArgMCA. Boc-Gln-Arg-Arg-MCA, Boc-Gln-Ala-Arg-MCA,Boc22Phe-Ser-Arg-MCA, and Boc-Leu-Gly-Arg-MCAwere hydroFIG. 7. SDS-PAGE analysis of the purified protein. A, the lyzed fairly wellby the enzyme and to similar extents. A protein (2 pg) was electrophoresed in 7.5% PAGE gel in SDS under common feature of the substrateshydrolyzable by the enzyme nonreducing (lane 1 ) and reducing(lane2 ) conditions. The gels were was the presence of an Arg residue at the PI position. Substained with Coomassie Brilliant Blue. B, the protein (0.87 pg) was incubated with 8 pCi of [3H]DFPin 20 mM Tris-HC1 (pH 7.5) and strates containing Lys-MCA bonds were much less susceptible subjected to SDS-PAGE using 7.5% gel under reducing conditions, to the enzyme, and substrates for chymotrypsin were not followed by fluorography. Molecular weight marker proteins used are: cleaved at all. Little orno cleavage occurred when the MCAdimer of human a*-macroglobulinsubunit (360 kDa), myosin (200 derivatives of an amino acid or a dipeptide were tested. kDa), &galactosidase (116 kDa), phosphorylase b (97 kDa), bovine Thrombin very rapidly hydrolyzed the substrates Boc-Valserum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase Pro-Arg-MCA and Boc-Gln-Ala-Arg-MCA,while the tissue (31 kDa), and soybean trypsin inhibitor (22 kDa). kallikrein most rapidly cleaved Boc-Gln-Ala-Arg-MCA, Bocr,

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conditions. However, the sample treated with the serine proTABLEI1 teinase inhibitor DFP (3H-labeled) produced only a single Enzyme activities toward various MCA substrates band with a molecular mass of approximately 28,000 on SDSEnzyme activities were determined at pH 9.0 with 50 pM substrates PAGE with autoradiography (Fig. 7 B ) . as described under “Experimental Procedures” and are expressed as Association of a Serine Proteinase with a2-Macroglobulinpercent of activity toward Boc-Gln-Arg-Arg-MCA. The results described above suggested that the purified proPresent ThrombinKallikreinPlasmin Substrate enzyme (human) (porcine) (porcine) tein is a complex of the plasma protein inhibitor a2-macroglobulin with a serine proteinase. To substantiate thispoint, Boc-Gln-Arg-Arg-MCA 100 100 100 100 several experiments were performed. The purified protein Boc-Gln-Gly-Arg-MCA 63 10 201 354 121 3300 55 305 cross-reacted with goat anti-human a2-macroglobulin anti- Boc-Gln-Ala-Arg-MCA 97 11 105 142 body (Fig. 8). Furthermore, directelectron microscopic obser- Boc-Phe-Ser-Arg-MCA 37 5 90 304 vation of the protein revealed two similar molecular shapes Boc-Leu-Gly-Arg-MCA 48 Boc-Leu-Thr-Arg-MCA (Fig. 9). One of them looks like the letter H, while the other Boc-Gly-Arg-Arg-MCA 11 42 13 12 is a rounded structure characterized by two thick units located Boc-Val-Pro-Arg-MCA 24 217 41 4160 30 roughly in parallel with small material between them. They Boc-Leu-Lys-Arg-MCA 40 42 285 57 13 are thecharacteristic shapesof a2-macroglobulin that under- Boc-Val-Leu-Lys-MCA Boc-Glu-Lys-Lys-MCA 11 went adrastic conformational change after reactingwith 16 Z-Phe-Arg-MCA proteinase (24). Z-Arg-Arg-MCA 3 Release of a Serine Proteinase from the a2-Macroglobulin- Bz-Arg-MCA 71 0.4 proteinase Complex-The results shown in Fig. 7 suggest that Suc-Ala-Ala-Pro-Phe-MCA 0 the proteinase is dissociable from the a2-macroglobulin-pro- Suc-Leu-Leu-Val-Tyr-MCA 0 0 0 0 5 teinase complex. It was found that exposure of the purified Arg-MCA 0 protein to pH 3 for 12 h did not cause significant change in Leu-MCA ’Not tested. the activity toward Boc-Gln-Arg-Arg-MCA substrate, but re-

a2-Macroglobulin-complexed Proteinase Serine Val-Leu-Lys-MCA, and Boc-Val-Pro-Arg-MCA. Plasmin cleaved Boc-Gln-Arg-Arg-MCA efficiently, but hydrolyzed Boc-Gln-Gly-Arg-MCA,a good substrate for the present enzyme, rather slowly. Theseresults clearly show that the specificity of the currentproteinase is different from those of thrombin, tissue kallikrein, and plasmin. Action on Peptide Substrates-Four peptide substrates were tested for hydrolysis by the proteinase, and theresults of high performance liquid chromatography analysis of the digests are shown in Fig. 11. As summarized in Fig. 12, cleavages occurred only on the COOH-terminal side of Arg or Lys residues. The specificity is thus consistent with that found with MCA substrates. Effects of Inhibitors-The effects of various inhibitors on the activity were examined, and the results are shown in Table 111. DFP, p-amidinophenylmethanesulfonyl fluoride, antipain, leupeptin, and bovine pancreatic trypsin inhibitor inhibited strongly the activity of the az-macroglobulin-associated proteinase. Strong inhibition by soybean trypsin inhibitor and ovomucoidwas observed only after the purified protein was treated at pH 3, indicating again the release of the proteinase from az-macroglobulin by thistreatment. These results clearly indicate that the enzyme is a trypsinlike serine proteinase. Presence of the az-Macroglobulin-Proteinase Complex in Porcine Intestinal Mucosa-In order to examine whether or

529

not thesame a2-macroglobulin-proteinasecomplex is present in the mucosa of the porcine intestine, a partial purification experiment was conducted as described inthe Miniprint section. Electrophoretic analysis of the fractions obtained by chromatography on Sepharose CL-GB revealed that wmacroglobulin was eluted in fractions 45-50, which corresponded to one of the peaks with proteinase activity (Fig. 14). Association of the proteinase with cuz-macroglobulin was confirmed by assaying the activity in the gel slice extracts as described under “ExperimentalProcedures.” The activities toward various MCA substrates were assayed for the proteinase in this peak. The relative activities were essentially the same as those of the purified stomach enzyme, suggesting that the same serine proteinase is present in the intestinal mucosa in the az-macroglobulin-associatedform. The specific enzyme activity toward Boc-Gln-Arg-Arg-MCA was estimated to be 23.9 milliunits/mg a2-macroglobulin (the amount of the inhibitor was densitometrically determined in PAGE of the above Sepharose CL-GB fraction). The value is about half the specific activity (42.1 milliunits/mg protein) of the az-macroglobulin-protein complex purified from stomach. As shown in Fig.15, porcine plasma did notcontaina significant amount of the inhibitor-proteinase complex. DISCUSSION

In the present study we have isolated from porcine stomach mucosa a high molecular weight protein (approximately M,= 83 3 740,000) with Boc-Gln-Arg-Arg-MCAhydrolyzing activity. 1a-Neoendorphin YGSLFdW% Detailed characterization of the protein revealed that it is a complex of a major plasma protein, a2-macroglobulin,and an 5211 Tv enzyme. This enzyme, which is responsible for the activity, BAM-1PP YGGFMRRVGFIPE was demonstrated to be a serine peptidase with a molecular 7 9 11 mass of approximately 25,000 Da. v r I VIP HSDAVFTDNMRLRKQMAVKK~SI~-NHz Cleavage specificity studies using syntheticand peptide substrates indicated that theenzyme preferentially hydrolyzes 36 Arg-X bonds and, to a much lesser extent, Lys-X bonds. In T Neurotensin pELYUJKPRRPYIL addition, the enzyme appears to contain multiple amino acid FIG. 12. Sites and extents of cleavage by the proteinase in side chain binding sites in the active site (25,26).As suggested peptide substrates.Arrowheads show the sites of cleavage, and the by the finding that tripeptide MCA substrates such as Bocnumber above each arrowhead indicates the estimated extent of Gln-Arg-Arg-MCA and Boc-Gln-Gly-Arg-MCA,but not dicleavage in percent of the peptide bond. Large and small arrowheads show major and minor cleavage sites, respectively. The amino acid peptide MCA substrates such as 2-Arg-Arg-MCAand Z-Phesequences of peptides are shown in one-lettercodes. p E , pyroglutamic Arg-MCA, are good substrates, filling at least four binding acid residue, -NH2,amide. sites (SS, Sz, S1,and Sl’) seems to be a prerequisite for hydrolysis. Although further systematic kinetic studies are required to clarify the specificity of each binding site, the SI TABLE I11 subsite evidently favors basic (Arg, Lys) side chains. The Effects of inhibitors on the proteinase activity enzyme has only endopeptidase activity, and its association Inhibitor Concentration Inhibition with the plasma proteinase inhibitora*-macroglobulinis conmM % sistent with the well-known fact that the inhibitor interacts DFP 2 98 only with enzymes having endopeptidase activity (27). p-Amidinophenylmethanesulfonylfluoride 0.5 97 The mechanism of inhibition of proteinase activity by azIodoacetic acid 1 9 macroglobulin is thought to be physical trapping of proteinp-Chloromercuribenzoic acid 0.4 4 EDTA 1 23 ases by the inhibitor (27, 28). We suspected that the enzyme 1 3 o-Phenanthroline in the present study might be one of the well-characterized TLCK 0.2 34 proteinases. Several authors have reported that isolated azTPCK 0.25 4 macroglobulin fractions have low kallikrein-like (29, 30) and Benzamidine 1 69 trypsin-like (31) activities. More recently, thrombin associBestatin 0.065 9 Amastatin 0.042 0 ated with a2-macroglobulin was isolated from mammalian Antipain 0.1 98 cells cultured in thepresence of the small proteinase inhibitor Leupeptin 0.1 98 leupeptin (32,331. In this context, itis particularly interesting Elastatinal 0.2 11 to compare the current proteinase with kallikrein (34) and Pepstatin 0.01 a thrombin (35) since these enzymes appear to have overlapping Bovine pancreatic trypsin inhibitor 0.1 mg/ml 98 (95)” Soybean trypsin inhibitor 0.1 mg/ml 44 (100) enzymatic properties such as molecular size (Mr = 25,0000.1 mg/ml 0 (98) Ovomucoid 35,000) and endopeptidase nature with trypsin-like cleavage specificity. However, the results shown in Table I1 indicate a Values in parentheses are the results obtained with the sample treated at pH3. that the at-macroglobulin-associated enzyme has a substrate

530

az-Macroglobulin-complexed Proteinase Serine

specificity clearly distinct from those of tissue kallikrein and and localization of the a2-macroglobulin-proteinase complex thrombin. in the tissues, are necessary. However, the present finding It should be noted that the substrate specificity of the that the proteinase associated with a2-macroglobulin hydroenzyme is somewhat similar to that of rat lung tryptase; the lyzes peptide substratesat theArg-X and Lys-X bonds tempts relative activities of the tryptase toward Boc-Phe-Ser-Argus to speculate its possible involvement in the proteolytic MCA,Boc-Val-Pro-Arg-MCA, and Boc-Val-Leu-Lys-MCA processing or degradation of some bioactive peptides known are 100, 50.6, and 6.1 (36), respectively, while those of the to be present in the gastrointestinal tract. Sucha possibility present enzyme are 100, 39, and 12.9, respectively (Table 11). is now under examination. In contrast, the behavior of the two enzymes toward polypepAcknowledgments-We thank Dr. Takaaki Aoyage (Institute of tide proteinase inhibitors such as bovine pancreatic trypsin Microbial Chemistry, Tokyo, Japan) for the generous gift of leupepinhibitor and soybean trypsin inhibitor is different. The pro- tin, antipain, elastatinal, bestatin, and pepstatin. We also thank Dr. teinase dissociatedfrom az-macroglobulinhas beencomSenarath B. P. Athauda for his valuable suggestion. pletely inhibitedby the above inhibitors, but the tryptase was REFERENCES reported to be inhibited t o a limited extent even at a higher 1. Fruton, J. S. (1971) The Enzymes 3 , 119-164 inhibitor concentration (36). Furthermore, native tryptase is 2. Samloff, I. M. (1969) Gastroenterology 67,659-669 3. Kageyama, T., and Takahashi, K. (1983) J. Biochem. (Tokyo) 9 3 , 743-754 known to be a tetramer (Mr = 144,000) consisting of two 4. Sogawa,K., FuJn-Kuriyama, Y., Mizukami, Y., Ichihara, Y., and Takahashi, = 30,900 and 31,600) (37, 38). Smith species of subunits (M, K. (1983) J. Biol. Chem. 258,5306-5311 5. Hayano, T.,Sogawa, K., Ichihara, Y., Fujii-Kuriyama, Y., and Takahashi, et al. (37) reported the failure of pure a2-macroglobulin to K 11988) J- . R I A C- .h..m 283. ..... - - , 1382-1385 . -- - . . inhibit human lung tryptase, suggesting that thisenzyme may 6. Kageyama, T., and Takahashi,K. (1980) J. Biochem. (Tokyo) 87,725-735 7. Matsuzaki, O., and Takahashi, K. (1988) Biomed. Res. 9,515-523 be toolarge to be trapped by a2-macroglobulin (28). Additional 8. Samloff, I. M., Taggart, R. T., Shiraishi, T., Branch, T., Reid, W. A., Heath, differences between the two enzymes are also observed in p H R. W., Valler, M. J., and Kay, J. (1987) Gostroenteroloty9 3 , 77-84 profiles for stability and activity. From these considerations, 9. Athauda, S. B. P., Takahashi, T., Inoue, H., Ichinose, M., and Takahashi, K. (1991) FEBS Lett. 292,53-56 the enzymefound inthecurrentstudy is thoughttobe 10. Azuma, T., Pals, G., Mohandas, T. K., Couvreur, J. M., and Taggart, R. T. (1989) J. Biol. Chem. 2 6 4 , 16748-16753 different from tryptase. Thus,we tentatively suggest that the 11. Athauda, S. B: P., Matsuzaki, O., Kageyama, T., and Takahashi, K. (1990) proteinase is a new protein which has not beendescribed Biochem. Bw hysResCommun. 166,878-885 12. Athauda, S. B. 6., Takahashi, T., Kageyama, T., and Takahashi, K. (1991) before. Biochem. BW hysResCommun. 176,152-158 We have considered the possibility that the a2-macroglob- 13. Woolley, D. E.,&ucker, J: S., Green, G.,and Evanson, J. M. (1976)Biochem. 163, 119-126 ulin-proteinase complex is artificially produced during puri- 14. deJ.Bruin, P. A. F., Griffioen, G., and Verspaget, H. W. (1987) Cancer Res. 47,4654-4657 fication. Since methylamine treatment is known to rapidly A. J. (1977) in Proteinases in Mammalian Cells and Tissues inactivate a2-macroglobulin (39),addition of the reagentcould 15. Barrett, (Barrett, A. J., ed) pp. 209-248, North-Holland Publishmg Co., Amsterdam prevent the proteinase trappingby inhibitor if the enzyme is 16. Fujita, T., Kanno, T., and Kohayashi, S. (1988) in The Paraneuron, pp. originally present in a free form in the tissue homogenate. 165-184, Springer-Verlag, Tokyo Barrett, A. J. (1980) Biochem. J. 187,909-912 However, purification experiments with or without methyla- 17. Barrett, A. J., and Kirschke H. (1981) Methods Enzymol. 80, 535-561 18. mine produced no significantdifference in theyield of the a2- 19. Sogawa, K., and Takahashi,'K. (1978) J. Biochem. (Tokyo) 84,763-770 Laemmli, U. K. (1970) Nature 227,680-685 macroglobulin-proteinase complex. Furthermore, we could 20. 21. Smith, P. K., Krohn, R. I., Hermanson, G . T., Mallia, A. K., Gartner, F. not find thecorresponding free proteinase in the homogenate H., Provenzano, M. D., FuJlmoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D.C. (1985) Anal. Biochem. 160,76-85 of the stomachmucosa and theyield of the a2-macroglobulin- 22. Towbin. H.. Staehelin. T.. and Gordon.. J . (1979) Proc. Natl. Acad.Sci. . U. S. A. 76,4350-4354 proteinase complex did not change significantly whether it T., Sasaki, T., and Ikai, A. (1988) J. Biochem. (Tokyo) 1 0 3 , 212was purified from fresh mucosa immediately after killing or 23. Osada, 217 after storage of the mucosa at -20 "C for months. Moreover, 24. Nishigai, M.,Osada, T., and Ikai, A. (1985) Biochim. Biophys. Acta 8 3 1 , 236-241 inclusion of trypsininthe homogenizationbuffer neither 25. Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 2 7 , 157-162 resulted in recovery of the a2-macroglobulin-trypsin complex 26. Schechter, I., and Berger, A. (1968) Biochem. Biophys. Res. Commun. 3 2 , nor affected the activity in fractions containing the az-mac898-902 Starkey, P. M., and Barrett,A. J. (1977) in Proteinases in Mammalion Cells 27. roglobulin complex of thepresent enzyme. Theseresults and Tissues (Barrett, A. J., ed) pp. 663-696, North-Holland Publishing strongly suggest that the proteinase-az-macroglobulincomCo., Amsterdam Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724 plex does not form during storage or after homogenization 28. 29. Vogt. W., and Dugal, B. (1976) Naungu-Schmiederbmg's Arch. Pharmacol. 294,75-84 and that the enzyme is only present in a form associated with 30. McConnell, D.J. (1972) J. Clin. Invest. 61,1611-1623 a2-macroglobulin in uiuo. The same complex is also present 31. Laurell, C.-B., and Jeppsson,J.-0. (1975) in The P h m a Proteins (Putnum, F. W., ed) 229-264, Academic Press, NewYork in the intestinalmucosa, but not in the plasma. 32. Tsuji, A., ant%urachi, K. (1989) J . Biol. Chem. 264,16093-16099 At present, thephysiological significance of the complex in 33. Tsuii. A.. Arai. T.. Furcinitti, P. S., Langmore, J. P., and Kurachi, K. (1991) B"i&him. B b hys Acta 1078,85-93the digestive organs is not clear. The enzyme is very stable 34. Fiedler. F.. Fin[. E.: Tschesche., H... and Fritz. H. (1981) Methods Enzymol. when complexed with az-macroglobulin, but appears to be80,493L532 35. Lundblad, R. L., Kingdon, H. S., and Mann,G . M. (1976)Methods Enzymol. comeratherunstableafter releasefroma2-macroglobulin. 46,156-176 Therefore, a2-macroglobulin may be important in stabilizing 36. Kido, H., Fukusen, N., and Katsunuma, N. (1985) Arch. Biochem. Biophys. 239,436-443 the enzyme and/or restricting the enzyme action only tolow 37. Smith. T.J.. Houdand. M. W.. and Johnson, D. A. (1984) J. Biol. Chem. 2 6 9 , 11046-11651 molecular weight peptides. To clarify its physiological roles, 38. Cromlish, J. A., Seidah, N. G.,Marcinkiewicz, M., Hamelin, J., Johnson, furtherstudies, including detailedcharacterization of the D A,, and Chretien, M. (1987) J . Biol. Chem. 262,1363-1373 trapped enzyme, identification of cells producing theenzyme, 39. Travis, J., and Salvesen, G . S. (1983) Annu. Rev. Biochem. 62,655-709 >"",

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cr2-Macroglobulin-complexedSerine Proteinase Supplemental Materials

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ISOLATION AND CHARACTERIZATION OF A NOVELSERINEPROTEINASE CXX4F"F'D WITH arMACROGLOBULlN FROMPORCINEGASTRICMUCOSA

by Takashi Uchino. YasukoSakurai.Masaaki Nishigai. Takayuki Takahashi. Hidco Arskawa.Atsushi Ikai. and K e j i Takahashi

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