Purification of an acid proteinase from Aspergillus saitoi and determination of peptide bond specificity.

The specificity and mode of action of an acid proteinase (EC 3.4.23.6) from Aspergillus saitoi were investigated with oxidized B-chain of insulin, angiotensin II and bradykinin. Further purification of acid proteinase was performed with N,O-dibenzyloxycarbonyl-tyrosine hexamethylene-diamino-Sepharose 4B affinity chromatography and isoelectric focusing. The purified enzyme was free of any other proteolytic activity demonstrated in Asp. saitoi. Acid proteinase from Asp. saitoi hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu(15)-Tyr(16) bond and the Phe(24)-Phe(25) bond. Additional cleavages of the bonds His(10)-Leu(11), Ala(14)-Leu(15) and Tyr(16)-Leu(17) were also noted. Primary splitting sites at Leu(15)-Tyr(16) and Phe(24-)-Phe(25) with acid proteinase from Asp. saitoi were identical with those reported in the work of cathepsin D (EC 3.4.23.5) from human erythrocyte. Hydrolysis of angiotensin II was observed at the Tyr(4)-Ile(5) bond. In conclusion, peptide bonds which have a hydrophobic amino acid such as phenylalanine, tyrosine, leucine and isoleucine in the P'1 position (as defined by Berger and Schechter, [29]) are preferentially cleaved by the trypsinogenactivating acid proteinase from Asp. saitoi.     

Purification and characterization of goat pepsinogens and pepsins

Three type-A and two type-C pepsinogens, namely, pepsinogens A-1, A-2, A-3, C-1, and C-2, were purified from adult goat abomasum. Their relative levels in abomasal mucosa were 27, 19, 14, 25, and 15%, respectively. Amino acid compositions were quite similar between isozymogens of respective types, but different between the two types especially in the Glx/Asx and Leu/Ile ratios. NH₂-terminal amino acid sequences of pepsinogens A-3 and C-2 were SFFKIPLVKKKSLRQNLIEN- and LVKIPLKKFKSIRETM-, respectively. Pepsins A and C showed maximal hemoglobin-digestive activity at around pH 2 and 3, respectively, and specific activities of pepsins C were higher than those of pepsins A. Two subtypes of pepsin A were obvious, namely pepsin A-2/3 which maintains its activity in the weakly acidic pH region over pH 3 and pepsin A-1, which does not. Hydrolysis of oxidized insulin B chain by goat pepsins A occurred primarily at Ala14-Leu15 and Leu15-Tyr16 bonds.     

The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities

The flowers of cardoon (genus Cynara) are traditionally used in Portugal for cheese making. In this work the vegetable rennet of the species Cynara cardunculus L. was characterized in terms of enzymic composition and proteolytic specificity of its proteinases (cardosin A and cardosin B). Cardosin A was found to cleave insulin B chain at the bonds Leu15-Tyr16, Leu17-Val18 and Phe25-Tyr26. In addition to the bonds mentioned cardosin B cleaves also Glu13-Ala14, Ala14-Leu15 and Phe24-Phe25 indicating that it has a broader specificity. The kinetic parameters for the hydrolysis of the synthetic peptide Leu-Ser-Phe(NO₂)-Nle-Ala-Leu-oMe were also determined and compared to those of chymosin and pepsin. The results obtained indicate that in terms of specificity and kinetic parameters cardosin A is similar to chymosin whereas cardosin B is similar to pepsin. It appears therefore that the enzyme composition of cardoon rennet closely resembles that of calf rennet.     

Characterization of meprin, a membrane-bound metalloendopeptidase from mouse kidney.

Meprin is an intrinsic protein of the brush border, a specialized plasma membrane, of the mouse kidney. It is a metalloendopeptidase that contains 1 mol of zinc and 3 mol of calcium per mol of the 85,000-Mr subunit. The enzyme is isolated, and active, as a tetramer. The behaviour of the enzyme on SDS/polyacrylamide gels in the presence and absence of beta-mercaptoethanol indicates that the subunits are of the same Mr (approx. 85,000) and held together by intersubunit S--S bridges. Eight S-carboxymethyl-L-cysteine residues were detected after reduction of the enzyme with beta-mercaptoethanol and carboxymethylation with iodoacetate. The enzyme is a glycoprotein and contains approx. 18% carbohydrate. Most of the carbohydrate is removed by endoglycosidase F, indicating that the sugar residues are N-linked. The isoelectric point of the enzyme is between pH 4 and 5, and the purified protein yields a pattern of evenly spaced bands in this range on isoelectric focusing. The peptide-bond specificity of the enzyme has been determined by using the oxidized B-chain of insulin as substrate. In all, 15 peptide degradation products were separated by h.p.l.c. and analysed for their amino acid content and N-terminal amino acid residue. The prevalent peptide-bond cleavages were between Gly20 and Glu21, Phe24 and Phe25 and between Phe25 and Tyr26. Other sites of cleavage were Leu6-Cysteic acid7, Ala14-Leu15, His10-Leu11, Leu17-Val18, Gly8-Ser9, Leu15-Tyr16, His5-Leu6. These results indicate that meprin has a preference for peptide bonds that are flanked by hydrophobic or neutral amino acid residues, but hydrolysis is not limited to these bonds. The ability of meprin to hydrolyse peptide bonds between small neutral and negatively charged amino acid residues distinguishes it from several other metalloendopeptidases.     

New world monkey pepsinogens A and C, and prochymosins. Purification, characterization of enzymatic properties, cDNA cloning, and molecular evolution

Pepsinogens A and C, and prochymosin were purified from four species of adult New World monkeys, namely, common marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus oedipus), squirrel monkey (Saimiri sciureus), and capuchin monkey (Cebus apella). The occurrence of prochymosin was quite unique since this zymogen is known to be neonate-specific and, in primates, it has been thought that the prochymosin gene is not functional. No multiple form has been detected for any type of pepsinogen except that two pepsinogen-A isozymogens were identified in capuchin monkey. Pepsins A and C, and chymosin hydrolyzed hemoglobin optimally at pH 2-2.5 with maximal activities of about 20, 30, and 15 units/mg protein. Pepsins A were inhibited in the presence of an equimolar amount of pepstatin, and chymosins and pepsins C needed 5- and 100-fold molar excesses of pepstatin for complete inhibition, respectively. Hydrolysis of insulin B chain occurred first at the Leu15-Tyr16 bond in the case of pepsins A and chymosins, and at either the Leu15-Tyr16 or Tyr16-Leu17 bond in the case of pepsins C. The presence of different types of pepsins might be advantageous to New World monkeys for the efficient digestion of a variety of foods. Molecular cloning of cDNAs for three types of pepsinogens from common marmoset was achieved. A phylogenetic tree of pepsinogens based on the nucleotide sequence showed that common marmoset diverged from the ancestral primate about 40 million years ago.     

Metallo-proteinase from the seedlings of kale (Brassica oleracea L. var. sabellica):

Metallo-proteinase from 8-d-old seedlings of kale was isolated. The enzyme was extracted with 1% NaCl, concentrated by ammonium sulfate and finally purified by high-performance liquid chromatography. The isolated enzyme had a molecular weight of 22.4 kDa and showed a maximum activity at pH 9.0 using casein as a substrate. Proteolytic activity of proteinase was inhibited by chelators. The inhibition by ethylenediaminetetraacetate (EDTA) was abolished by some divalent metals ions, especially by Zn²⁺. The enzyme showed activity against the synthetic peptides Suc-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Phe-pNA, and hydrolized the following peptide bonds in the oxidized insulin B-chain: Leu6-Cya7, Leu15-Tyr16, Leu17—Val18 and Phe25-Tyr26.Abbreviations EDTA ethylenediaminotetraacetic acid - HPLC high-performance liquid chromatography - NEM N-ethylmaleimide - PCMB p-mecuribenzoic acid - PMSF phenylmethylsulfonyl fluoride This work was supported by the University Science Programme, Ministry of National Education, and Polish Academy of Science, Warsaw, Poland.     

Purification and characterization of cathepsin D from herring muscle (Clupea harengus)

Cathepsin D was purified and concentrated 469-fold from a homogenate of Clupea harengus muscle. The purified enzyme is a monomer with a molecular weight of 38000-39000. It is inhibited by pepstatin and has optimal activity at pH 2.5 with hemoglobin as the substrate. The isoelectric point is at pH 6.8. Glycosidase treatment and binding to Concanavalin A indicated that the enzyme contains one N-linked carbohydrate moiety of the high-mannose type per molecule. The first 21 amino acid residues of the N-terminal showed high similarity to cathepsin D from antarctic icefish liver (Chionodraco hamatus) and trout ovary (Oncorhynchus mykiss). Digestion of the beta-chain of oxidized insulin resulted in preferential cleavage at Leu(15)-Tyr(16), (47%), Tyr(16)-Leu(17) (34%) and Ala(14)-Leu(15) (18%). Incubation with myofibrils from herring muscle at pH 4.23 showed that the enzyme mainly degraded myosin, actin and tropomyosin.     

The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B.

The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu13-Ala14, Leu17-Val18 and Phe23-Tyr26; minor cleavage sites are the bonds Asn3-Gln4, Ser9-His10 and Leu15-Tyr16. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers smaller neutral amino acid residues in the subsites S2 and S3, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P2 and P3 positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH2F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Phe-CH3 and Z-Phe-Lys(Z)-CH3 inhibit competitively both cathepsin S and cathepsin L in the micromolar range.     

Isolation and characterization of a proteinase from the sarcocarp of melon fruit.

A proteinase from the sarcocarp of melon (Cucumis Melo L. var. Prince) was purified by a three-step procedure involving batch-wise treatment with CM-cellulose fibers, column chromatography on CM-cellulose powder and gel filtration on Sephadex G-75. The final enzyme preparation was homogeneous on acrylamide gel electrophoresis. Its molecular weight was estimated by two different methods to be about 50,000. Anlayses indicated tha presence of 475 amino acid residues and at least 7 moles of hexose. The maximum activity was found in the alkaline pH region against casein as a substrate. The optimum temperature against casein was 70 degrees at pH 7.1. The enzyme was strongly inhibited by diisopropyl fluorophosphate, partly inhibited by HgCl2 and not inhibited by EDTA, p-chloromercuribenzoic acid, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor. The reduced and carboxymethylated insulin B-chain was cleaved at the peptide bonds of Asn3-Gln4, Cm-Cys7-Gly8, Glu13-Ala14, Leu15-Tyr16, Cm-Cys19-Gly20, Phe25-Tyr26, Pro28-Lys29, and Lys29-Ala30 by the enzyme.     

Purification and some properties of two proteinases from Crotalus adamanteus venom that inactivate human alpha 1-proteinase inhibitor.

Two proteinases (proteinases I and II) have been purified from Crotalus adamanteus venom to the stage of electrophoretic homogeneity and proteinase II has been crystallized. The proteinase differ slightly in molecular weight and amino acid composition. Both are metalloenzymes requiring Zn2+ or Ca2+, or both; neither requires thiol compounds for activation. The proteinases are free of esterolytic activity against benzoly-L-arginine ethyl ester and benzoyl--tyrosine ethyl ester. Proteinase II cleaves the oxidized B chain of insulin at the bonds Phe1-Val2, His5-Leu6, His10-Leu11, Ala14-Leu15, Leu15-Tyr16, and Tyr-16-Leu17. Digestion of polylsine and polyarginine by proteinase II liberates products ranging from dodecapeptides to hexapeptides. Proteinases I and II catalytically inactive human plasma alpha 1-proteinase inhibitor (54,000 daltons). Electrophoretic analysis of the reaction of proteinase II with alpha 1-proteinase inhibitor reveals that an inactivated inhibitor species of 50,000 daltons is formed, and a peptide of 4,000 daltons is released. The gradual disappearance of the native inhibitor results in the corresponding loss of inhibitory activity against trypsin and chymotrypsin.     

Exogenous adenosine 3': 5'-monophosphate can release yeast from catabolite repression.

A new simple fast and reproducible purification procedure for the proteinase from rat liver mitochondria has been worked out. The specificity of cleavage of peptide bonds in glucagon, oxidized A and B chains of insulin and yeast proteinase B inhibitor by the proteinase of the inner mitochondrial membrane has been studied. The proteinase hydrolyzed three peptide bonds in glucagon, Tyr (13) - Leu (14), Trp (25) - Leu (26) and Phe (22) - Val (23) (minor cleavage site); none in the insulin A chain; one in the B chain of insulin, Tyr (16) - Leu (17); and three in the yeast proteinase B inhibitor, Phe (4) - Ile (5), Phe (20) - Leu (21) and Tyr (41) - Thr (42) (minor cleavage site).Thus, the mitochondrial proteinase cleaves peptide bonds at the carboxyl site of an aromatic amino acid and the amino site of a leucine, isoleucine, threonine or valine. The comparison with chymotrypsin A shows that the mitochondrial proteinase cleaves peptide bonds in a more restricted manner.     

Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action.

A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto). Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al. and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA. The cleavage sites in the oxidized B-chain of insulin by the proteinase were CySO3H7-Gly8, Val12-Glu13, Try16-Leu17, and Phe25-Tyr26. The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or alpha 2-macroglubulin.     

Specificity of elastases: degradation of the oxidized beta-chain of insulin by porcine pancreatic elastase II and dog leucocyte elastase.

Porcine elastase II (EC 3.4.21.-), a pancreatic proteinase with elastolytic activity, hydrolyses the oxidized beta-chain of insulin with major cleavages occurring at Leu17-Val18, Phe24-Phe25, Phe25-Tyr26 and Tyr26-Thr27. Canine leucocytic elastase splits the same substrate with major sites at Val12-Glu13 and Val18-Cys19 O3H. This indicates similarity of elastase II to chymotrypsins (EC 3.4.21.1 or 3.4.21.2) and of dog leucocyte enzyme to human granulocyte elastase and porcine pancreatic elastase I (EC 3.4.21.11).     

Purification of a New Extracellular 90–kDa Serine Proteinase with Isoelectric Point of 3.9 from Bacillus subtilis (natto) and Elucidation of Its Distinct Mode of Action

A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto). Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al. and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA. The cleavage sites in the oxidized B-chain of insulin by the proteinase were CyS0₃H7-Gly8, Val12-Glu13, Tyr16-Leu17, and Phe25-Tyr26. The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or α₂-macroglubulin.     

Pancreatic proteolytic enzymes of ostrich purified on immobilized protein inhibitors. Characterization of a new form of chymotrypsin (Chtr1)

Four forms of chymotrypsin (Chtr1, Chtr2, Chtr3, Chtr4), one form of trypsin and one form of elastase were purified from a slightly alkaline extract of ostrich (Struthio camelus) pancreas. The zymogens in the crude extract were activated with immobilized trypsin and then separated by affinity chromatography using immobilized inhibitors and ion exchange chromatography. One of the purified forms of chymotrypsin (Chtr1) exhibited an unusual interaction with the highly selective protein trypsin inhibitor from Cucurbita maxima (CMTI). Interactions with other protein trypsin inhibitors such as basic pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor (STI), trypsin inhibitors from Cyclanthera pedata (CyPTI), Cucurbita pepo (CPTI), Cucurbita pepo var. giramontia (CPGTI) and Linum usitatissimum (LUTI) were also investigated. This study demonstrated the affinity of Chtr1 to inhibitors containing Arg at P1 position. Studies of substrate specificity of Chtr1 using oxidized B-chain of insulin revealed four susceptible bonds: Tyr15-Leu16, Phe24-Phe25, Phe25-Tyr26 and, surprisingly, Arg22-Gly23. The amino acid composition, as well as the first 13 residues of the N-terminal amino acid sequence, was determined. Studies of ostrich elastase showed that it can interact with immobilized CMTI in the presence of 5 M NaCl. This unusual characteristic is reported for the first time and suggests that elastase specificity depends on ionic strength. The kinetic constants K(M), k(cat) and k(cat)/K(M) for purified ostrich trypsin, chymotrypsin 4 and elastase were also determined.     

Isolation and properties of a metalloproteinase from buckwheat (Fagopyrum esculentum) seeds.

A homogeneous preparation of metalloproteinase, purified 1000-fold, was obtained from buckwheat (Fagopyrum esculentum) seeds. The Mr of the enzyme, determined by SDS/PAGE, was 34,000 (it was 39,000 by gel chromatography). Its pH optimum was 8.0-8.2 with 13 S globulin, from buckwheat seeds, as substrate. Atomic-absorption spectroscopy revealed the presence of one Zn2+ ion per enzyme molecule. The enzyme was completely inhibited by EDTA (1 mM), zincone (1 mM) and 1, 10-phenanthroline (1 mM). The metalloproteinase performed limited proteolysis of the following seed storage proteins: 13 S globulin from buckwheat seeds and 11 S globulin from soybean (Glycine max) seeds. It hydrolysed three peptide bonds formed by the amino groups of Leu15, Tyr16 and Phe25 in the oxidized B-chain of insulin. In its main properties the enzyme is similar to metalloproteinases of animal and bacterial origin.     

Modulating calcitonin fibrillogenesis: an antiparallel alpha-helical dimer inhibits fibrillation of salmon calcitonin

We have investigated the prefibrillar state of salmon (s) and human (h) calcitonin (CT). Size exclusion chromatography at pH 3.3 and 7.4 indicates that sCT is present in solution as a dimer, whereas hCT elutes as a monomer at pH 3.3 and as monomer-dimer at pH 7.4. Guanidine hydrochloride unfolding experiments show that dimerization is stabilized by hydrophobic interactions. We investigated the dimeric structure by multidimensional nuclear magnetic resonance spectroscopy and calculations by using an sCT mutant (LAsCT) in which Pro23 and Arg24 were substituted for Leu23 and Ala24. As indicated by the Leu9-Tyr27 and Leu12-Leu19 contacts, the mutated hormone forms a head-to-tail dimer whose basic unit is an alpha-helix in the region Leu12-Tyr22. The solution behavior of LAsCT is identical to that of sCT, so the dimeric structure can safely be extended to sCT: we believe that such a structure inhibits fibril maturation in sCT. No stable dimer was observed for hCT, which we attributed to the absence of a defined helical structure. However, we suggest that intermolecular collisions of short ordered regions (for example, a sequence of turns) in hCT favors intermolecular contacts, and specific orientation can be obtained through hydrogen bond formation involving Tyr12, Phe16, and Phe19, with the aromatic ring acting as an acceptor. Taken together, our results indicate that hCT fibrillation can be reduced by favoring a helical dimer, obtainable by replacing the three aromatic amino acids with leucines.     

Characterization of Ac3-proteinase from the venom of Agkistrodon acutus (hundred-pace snake)

Ac3-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. Ac3-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, dimethylcaseinolytic and hide powder azure hydrolytic activities. These activities were inhibited when Ac3-Proteinase was incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), tetraethylenepentamine (TEP), 1,10-phenanthroline, phosphoramidon or beta-mercaptoethanol. The toxin also hydrolyzed the oxidized A and B chains of both insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25). The A alpha chain of fibrinogen was digested first followed by hydrolysis of the B beta chain. Toxicological and biochemical properties of Ac3-Proteinase were investigated further and are reported in this paper.     

Substrate specificity of Bacillus subtilis intracellular serine protease. Hydrolysis of insulin beta-chain, native ribonuclease A and p-nitroanilide peptide substrates

Intracellular serine protease, termed ISP-103, was isolated from Bacillus subtilis, strain 103. The substrate specificity of the enzyme was compared to that of secretory subtilisins. Similar to subtilisins, ISP-103 cleaves a single peptide bond Ala20-Ser21 within the native pancreatic ribonuclease A, which results in the accumulation of trypsin-sensitive ribonuclease S, consisting of a non-covalently bound S-peptide (20 amino acid residues) and S-protein (104 amino acid residues). The enzyme hydrolyzes a single peptide bond Leu15-Tyr16 of the B-chain of oxidized bovine insulin, in contrast to the subtilisins cleaving four additional bonds. ISP prefers Leu rather than Phe in the P1 binding site of the rho-nitroanilide peptide substrates and shows a more strict dependence of the activity on the presence of the hydrophobic residues in the P2 and P3 sites. The data obtained indicate that the substrate specificity of ISP, being within the borders of subtilisin specificity, is nevertheless much more restricted.     

Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.