Purification and properties of a peptidase acting on a synthetic substrate for collagenase from monkey kidney.

A peptidase cleaving a synthetic substrate for collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (designated as PZ-peptide) has been purified extensively (about 5200-fold) from a soluble extract of monkey kidney with a view of carrying out studies on its possible physiological role. The purified PZ-peptidase appeared essentially free of collagenase, nonspecific protease and di- and tri-peptidase activities. The properties of the purified PZ-peptidase resemble very much the granuloma enzyme. It is optimally active around pH 7.0. Its apparent Km value for PZ-peptide is 0.72 mM and V is 10.1 mumol/mg protein/min. It is reversibly inhibited by p-hydroxymercuribenzoate and HgCl2, whereas iodoactetamide does not affect the enzyme activity. N-Ethylmaleimide inhibited the enzyme partially (50%). Heavy metals like Cu-2+, Cd-2+, Ag+, Pb-2+, Ni-2+, and Zn-2+ completely inhibited the enzyme activity, while the inhibition by Co-2+ was only partial. Fe-2+ did not exert any effect on the activity. The enzyme activity was completely inhibited by EDTA and was restored almost to the original value by metal ions like Mn-2+, Mg-2+, Ca-2+ and Ba-2+. The approximate molecular weight of the purified enzyme was estimated to be 56 000.     

Purification and characteriazation of collagenase from guinea pig skin.

Guinea pig skin col-agenase, isolated from culture medium of whole skin, was separated into two enzymatically active fractions. These two fractions have been purified extensively. Peak II fraction has been purified to homogeneity as examined by polyacrylamide gel electrophoresis. Their molecular weights are approximately 130 000 (peak I) and 40 000 (peak II). Both guinea pig skin collagenase fractions are capable of degrading the native collagen fibrils and are inhibited by serum, cysteine and EDTA. They appear to be glycoproteins. Guinea pig skin (peak II) and human skin collagenase were compared. They are both glycoproteins and have similar molecular size (Mr = 40 000). Immunodiffusion assay showed that no cross-reactivity was seen between the enzymes, indicating species specificity among collagenases.     

Collagenase activity in cultures of rat prostate carcinoma.

A specific collagenase (EC 3.4.24.3) has been found and purified from serum-free culture medium of 11095 epidermoid carcinoma of rat prostate. The molecular weight of this collagenase was estimated at 71 000 and the pH optimum was approx. 7. At 26 degrees C, the collagenase cleaved collagen at a site 3/4 the length from the N-terminus. At 37 degrees C, this collagenase degraded collagen to smaller peptides. The enzyme activity was inhibited by serum, cysteine and EDTA, but not by protease inhibitors. The presence of collagenase in rat tumor tissue suggests that this enzyme might play a significant role in tissue invasion by cancer cells.     

The release of collagenase as an inactive proenzyme by bone explants in culture

1. A latent collagenase, activated only by limited proteolysis, was found in culture media of mouse bone explants. It could be activated by trypsin or, less efficiently, by chymo-trypsin. Skin explants also released latent collagenase. 2. Bone collagenase attacks native collagen at about neutral pH when it is in solution, in reconstituted fibrils or in insoluble fibres, producing two fragments representing 75 and 25% of the molecule. It requires calcium and is inhibited by EDTA, cysteine or serum. 3. Latent collagenase is not activated by trypsin-activated collagenase but by a distinct unidentified thermolabile agent present in a latent trypsin-activatable state in the culture media, or by purified liver lysosomes between pH5.5 and pH7.4. Trypsin activation decreases the molecular weight of latent collagenase from 105000 to 84000 as determined by gel filtration. 5. The latency of collagenase is unlikely to be due to an enzyme-inhibitor complex. Although some culture media contain a collagenase inhibitor, its presence is not constant and its molecular weight (at least 120000) is not compatible with the decrease in molecular weight accompanying activation; also combinations of collagenase with inhibitor are not reactivated by trypsin. Moreover, the latency remains after gel filtration, or treatment by high dilution, exposure to pH values between 2.5 and 10, or high ionic strength, urea or detergent. 6. It is proposed that latent collagenase represents an inactive precursor of the enzyme, a ;procollagenase', and that the extracellular activity of collagenase is controlled by another protease that activates procollagenase by a limited proteolysis of its molecule.     

New Achromobacter collagenase and its immunological relationship with a vertebrate collagenase

Evidence is presented that Achromobacter iophagus produces two distinct collagenases. Achromobacter collagenases A and B were separated by high-performance liquid chromatography from partially purified enzyme. The main collagenase, A (EC 3.4.24.8), which has been already described, was eluted in the region of molecular mass 110-90 kDa. A minor collagenase B eluted in the region of 320 kDa, although in SDS-gel electrophoresis the apparent molecular masses of its main active forms were estimated as 55 and 110 kDa. The specificities of collagenases A and B are different. Collagenase A splits in its synthetic substrate Pz-Pro-Leu-Gly-Pro-DArg the bond Leu-Gly, collagenase B does not split this substrate. Both collagenases split bonds Gln-Gly and Leu-Gly in synthetic peptides DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-DArg-OH and DNP-Pro-Leu-Gly-Ile-Ala-Gly-DArg-NH2, respectively. Collagenase B is twice as active as A on the native collagen type I. Both enzymes are inhibited by EDTA. The antibodies raised against the human tooth collagenase specifically inhibited the collagenase B, but did not influence the activity of collagenase A. These results indicate, to our knowledge for the first time, an immunological relationship between a bacterial and a vertebrate collagenase.     

Purification and physico-chemical properties of collagenase synthesized by a bacterium of the type Acinetobacter sp.]

The Acinetobacter spec collagenase has been almost completely purified. This enzyme is a true collagenase the activity of which is high on collagen. The enzyme is active on insoluble collagen, gelatin and the synthetic Pz-peptide, but has no proteolytic activity on casein or bovine serum-albumin. The collagenase was obtained on a simple medium with gelatin and yeast extract. The enzyme was purified by (NH4)2SO4 precipitation. DEAE cellulose column chromatography, Sephadex G 200 gel-filtration. The molecular weight of the enzyme was found to be 102 000 daltons, and its isoelectric point was found to be 7,7 +/- 0,2. The optimum pH and temperature for insoluble collagen hydrolysis were 7.6 and 37 degrees C, respectively; so, this collagenase corresponds to true collagenase. Hydrolysis of Pz-peptide is activated by Ca2+ and inhibited by metal ions (Cu2+, Fe3+, Zn2+, Pb2+, Hg2+). EDTA and o-phenanthroline induced a very significant reduction in enzyme activity. Iodoacetate and p-CMB induced a slight reduction in enzyme activity only at high concentrations (10-2M). The collagenase is most stable for temperatures less than or equal to 50 degrees C.     

Purification and characterization of a streptomycete collagenase

A soil streptomycete designated as Streptomyces sp. A8 produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase' as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-a rginine into two tripeptides. The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G-150 gel filtration. The purified enzyme had an apparent molecular weight of about 75,000 by SDS-polyacrylamide gel electrophoresis. Treatment with lithium chloride did not dissociate it into subunits. A strong inhibition was observed with chelating agents such as alpha-alpha-dipyridyl and 8-hydroxyquinoline. Ethylene diamine tetraacetate completely inhibited the enzyme activity. Among the cations tested only Ca2+ and Mg2+ enhanced the collagenase activity. Heavy metal ions like Pb2+, Ag+, Cu2+ and Zn2+ strongly inhibited the enzyme. The EDTA inhibition could be reversed with Ca2+. Cysteine and reduced glutathione caused significant reduction in enzyme activity. Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase. Amino acid analysis revealed the absence of cysteine and tyrosine. Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus.     

Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence

Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [ 3H ]proline-labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly-Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin-A--Sepharose affinity chromatography and further purified by QAE-Sephadex ion-exchange chromatography and gel filtration on Sephacryl S-200. The purified enzyme had a molecular weight of approximately 105000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non-helical form, but not when in native triple-helical conformation. Dansyl-monitored end-group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly-Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly-Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non-helical as a result of collagenase cleavage.     

Different molecular forms of D-ribulose-1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides.

Ribulose-1,5-bisphosphate (Rbu-P2) carboxylase isolated from Rhodopseudomonas sphaeroides 2.4.1.Ga was separated into two different forms by DEAE-cellulose column chromatography. Both forms, designated Peak I and Peak II have been purified to homogeneity by the criterion of polyacrylamide disc-gel electrophoresis. The Peak I carboxylase has a molecular weight of 550,000, while the Peak II carboxylase is a smaller protein having a molecular weight of approximately 360,000. Sodium dodecyl sulfate electrophoresis revealed a large subunit for both enzymes which migrates similarly to the large subunit of spinach Rbu-P2 carboxylase. The Peak I enzyme also exhibited a small subunit having a molecular weight of 11,000. No evidence for a smaller polypeptide was found associated with the Peak II enzyme. Antisera prepared against the Peak I enzyme inhibited Peak I enzymatic activity, but had no effect on the activity of the Peak II enzyme. The two enzymes exhibited marked differences in catalytic properties. The Peak I enzyme exhibits optimal activity at pH 8.0 and is inhibited by low concentrations of 6-phosphogluconate, while the Peak II enzyme has a pH optimum of 7.2 and is relatively insensitive to 6-phosphogluconate.     

Purification and characterization of bovine dental sac collagenase

1. Active type collagenase was purified as much as 140-fold from the explant medium of bovine dental sacs and showed a single band on disc gel electrophoresis. Purified collagenase cleaved native collagen at only one locus under physiological conditions, but hydrolyzed neither gelatin nor alpha-casein. The optimal pH was about 7.8. 2. The molecular weight of active type enzyme was 35,000 by gel filtration and 34,000 by gel electrophoresis. The activation of latent type of collagenase resulted in the reduction of molecular weight from 45,000 to 38,000 by gel filtration. 3. A small but detectable amount of collagenase was directly extracted from frozen and thawed bovine dental sacs. In explant media of frozen and thawed tissue and fresh tissue with actinomycin D, some activity was detected for the first 2 days, but essentially no collagenase activity was detected in the explant medium after day 3. 4. The latent type collagenase was activated by trypsin, 4-aminophenylmercuric acetate (4-APMA), thiocyanate and deoxycholate (DOC). DOC showed irreversible dissociation of latent type enzyme in similar fashion to that exerted by 4-APMA. 5. The purified collagenase was inhibited by bovine serum, EDTA, o-phenanthroline, cysteine and dithiothreitol.     

Collagenase enzymes from Clostridium: characterization of individual enzymes.

Four collagenases have been purified to apparent homogeneity from extracts of Clostridium histolyticum and partially characterized. The four purified enzymes are devoid of hydrolytic activity against casein and the synthetic substrate, benzolyarginine naphthylamide, but all retain activity against native collagen. The enzymes are initially spearated by isoelectric focusing where three of the enzymes show distinct isoelectric points: collagenase I = 5.50, collagenase II = 5.65, and collagenases IIIa and IIIb = 5.90-6.00. Collagenases IIIa and IIIb can be subsequently separated on diethylaminoethylcellulose. The four purified enzymes show single bands upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Calibration of the molecular weights on the basis of migration distance shows a marked dependence on gel porosity. At high acrylamide concentration, collagenases I, II, and IIIa appear to converge to a limiting molecular weight congruent to 81 000, while collagenase IIIb has a distinctly lower value congruent to 72 000. The similarity between these molecular weight values and those derived from the sedimentation and diffusion coefficients of the native enzyme indicates that each collagenase is a single polypeptide chain. All of the collagenases have comparable catalytic activities against a series of natural and synthetic substrates and are immunologically cross-reactive. Although all four enzymes are evident upon initial electrofocusing of the crude extract, it is possible that the multiplicity of forms is, at least in part, a consequence of lysis following initial secretion from the cell.     

Purification and characterization of a murine basement membrane collagen-degrading enzyme secreted by metastatic tumor cells

A type IV collagen-degrading enzyme activity secreted by a highly metastatic mouse tumor was purified by concanavalin A- and type IV collagen-agarose affinity chromatographies followed by gel filtration on Bio-Gel A-0.5 m. The apparent molecular weight of the enzyme was 160,000 but about 70,000 when Triton X-100 was added to the column buffer. The purified enzyme protein was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptide chains of about 68,000 and 62,000 daltons. The enzyme activity could be increased by preincubation with trypsin and it is possible that the two chains represent latent and active enzyme forms. The enzyme activity was not reduced in the presence of dithiothreitol, it had a pH optimum of 7.6 and was inhibited by EDTA but not N-ethylmaleimide, phenylmethylsulfonyl fluoride, or Trasylol. The inhibition with EDTA was reversible. The pro-alpha 1(IV) and pro-alpha 2(IV) chains of the type IV procollagen substrate were both degraded at a similar rate to form two pairs of degradation fragments corresponding in molecular weights to about 70 and 30% of the original size chains. The presence of Triton X-100 increased slightly the activity of the enzyme and diminished the reduction of its activity upon freezing, indicating that the enzyme is a hydrophobic protein.     

Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen

A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices.     

ADRENAL MEDULLARY CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.—REGULATORY PROPERTIES OF THREE ENZYME ACTIVITIES

Abstract— Cyclic nucleotide phosphodiesterase from bovine adrenal medulla was fractionated into multiple activities by two different procedures, sucrose gradient centrifugation and gel filtration. Extracts of frozen and thawed adrenal medulla homogenates gave two phosphodiesterase activity peaks following density gradient centrifugation. The higher molecular weight activity hydrolyzed both cyclic AMP and cyclic GMP; ethylene glycol-bis(aminoethyl ether)- N,N' -tetraacetic acid (EGTA) inhibited only the hydrolysis of cyclic GMP. The lower molecular weight activity hydrolyzed only cyclic AMP and was not inhibited by EGTA. The two activities were not interconverted by recentrifugation.
Gel filtration of cyclic nucleotide phosphodiesterase activity extracted from frozen and thawed adrenal medulla on Ultrogel AcA 34 resolved the enzyme into three distinct peaks of enzyme activity with molecular weights of 350,000 (Peak I), 229,000 (Peak II) and 162,000 (Peak III). The enzyme from fresh tissue was resolved into peak I and II and only a small fraction of Peak III. Peak I hydrolyzed both cyclic nucleotides, while peak II was a cyclic GMP-specific enzyme and peak III was specific for cyclic AMP. The hydrolysis of cyclic AMP by the activity in Peak I was markedly stimulated by cyclic GMP; the hydrolysis of cyclic GMP by peak II was inhibited by EGTA and stimulated by calcium and CDR (calcium-dependent regulator protein). Peak III, which appears to be particulate, is not activated by either cyclic GMP or calcium and CDR.     

Purification and properties of the extracellular metallo-proteinases of Chromobacterium lividum (NCIB 10926).

Four extracellular proteolytic enzymes (I-IV) (EC 3.4.22.-) were identified in static cultures of Chromobacterium lividum (NCIB 10926) by agar gel electrophoresis and isoelectric focusing. Proteinases I-III were freed of non-enzymic protein by chromatography on TEAE-cellulose and CM-cellulose. The enzyme mixture was then fractionated in a pH gradient by isoelectric focusing. All three enzymes were shown to be heat-labile metallo-enzymes. Optimal activity occurred at pH 5.6 for enzyme I and at pH 6.2 for enzymes II and III. Remazolbrilliant Blue-hide powder was a sensitive substrate for these enzymes. Proteinase I was also shown to degrade haemoglobin and casein effectively, but not myoglobin, ovalbumin or bovine serum albumin. Proteinases I-III exhibited molecular weight values of 75 000, 72 000 and 67 000 by exclusion chromatography and 71 000 and 66 000 by sodium dodecyl sulphate-poly-acrylamide-gel electrophoresis for enzyme I and II, respectively. The amino acid compositions of enzymes I and II were somewhat similar. Proteinase I was inhibited by EDTA, 1,2-di(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic activity. Mg2+ could substitute for Ca2+ or Mn2+ for Co2+. The interrelationship of proteinases I-III is discussed.     

Purification and properties of rat brain dipeptidyl aminopeptidase

Dipeptidyl aminopeptidase, which hydrolyzes the 7-(Gly-Pro)-4-methylcoumarinamide, has been purified from the brains of 3 week-old rats. It was purified about 2,600-fold by column chromatography on CM-cellulose, hydroxyapatite and Gly-Pro AH-Sepharose. This enzyme hydrolyzed Lys-Ala-beta-naphthylamide well with an optimum pH of 5.5. It was inhibited by diisopropyl fluorophosphate, phenyl-methanesulfonyl fluoride, some cations, and puromycin, but was not inhibited by p-chloromercuribenzoate, N-ethylmaleimide, dithiothreitol, EDTA, iodoacetic acid, and bacitracin, indicating that rat brain dipeptidyl aminopeptidase is a serine protease. This enzyme showed a molecular weight of 220,000 by gel filtration and of 51,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The properties of purified rat brain dipeptidyl aminopeptidase were similar to those of bovine pituitary dipeptidyl peptidase II, but the molecular weight and substrate specificity of these enzymes were different.     

Isolation and properties of cathepsins A from chicken liver]

Four highly purified enzyme preparations, which belongs to the cathepsine A group in their substrate specificity, are isolated from the extract of a hen liver acetone powder. The preparations are designated as A1, A2, A3 and A4 according to their electrophoretic mobility. The A1 component is a protein with molecular weight of about 200 000, it degrades a synthetic substrate and, to a small degree, hemoglobin. This protein is suggested to be the fragment of a liver enzyme complex. The A2 component has molecular weight of about 70 000 and the highest activity. The A3 component has molecular weight 100 000 and the lowest activity. The A2 and A3 components are similar to cathepsine A from other tissues. THe A4 component is characterized by a high electrophoretic mobility, a resistance in neutral and weakly alkaline media and a low molecular weight (of about 60 000). Cu2+, Ag+ and diisopropylphosphofluoridate completely inhibited the activity of all the enzyme preparation studied. Tosyl-alpha-amino-phenylethyl-chloremethylketone inhibited the enzymes activity only by 50-70%.     

Low molecular weight angiotensin I converting enzyme from rat lung.

A low molecular weight angiotensin I converting enzyme (light angiotensin enzyme) was isolated from a homogenate of rat lung subjected to dialysis against sodium acetate at pH 4.8. This enzyme has a molecular weight of 84 000 on Sephadex G-200 and a molecular weight of 91 000 on SDS-poly-acrylamide gel as compared with a molecular weight of 139 000 for angiotensin I converting enzyme on SDS-polyacrylamide. Light angiotensin enzyme was activated by NaCl and inhibited by EDTA, angiotensin II, and bradykinin potentiating factor nonapeptide. Light angiotensin enzyme cross-reacted with antibody prepared against angiotensin I converting enzyme and stained with periodic acid-Schiff reagent as a glycoprotein. The evidence suggests that light angiotensin enzyme is a fragment of the higher molecular weight enzyme.     

Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes.

1. The respiratory nitrate reductase of Klebsiella aerogenes was solubilized from the bacterial membranes by deoxycholate and purified further by means of gel chromatography in the presence of deoxycholate, and anion-exchange chromatography. 2. Dependent on the isolation procedure two different homogeneous forms of the enzyme, having different subunit compositions, can be obtained. These forms are designated nitrate reductase I and nitrate reductase II. Both enzyme preparations are isolated as tetramers having sedimentation constants (s20,w) of 22.1 S and 21.7 S for nitrate reductase I and II, respectively. The nitrate reductase I tetramer has a molecular weight of about 106. 3. In the presence of deoxycholate both enzyme preparations dissociate reversibly into their respective monomeric forms. The monomeric form of nitrate reductase I has a molecular weight of about 260 000 and a sedimentation constant of 9.8 S. For nitrate reductase II these values are 180 000 and 8.5 S, respectively. 4. Nitrate reductase I consists of three different subunits, having molecular weights of 117 000; 57 000 and 52 000, which are present in a 1:1:2 molar ratio, respectively. Nitrate reductase II contains only the subunits with a molecular weight of 117 000 and 57 000 in a equimolar ratio. 5. Treatment at pH 9.5 in the presence of deoxycholate and 0.05 M NaCl or ageing removes the 52 000 Mr subunit from nitrate reductase I. This smallest subunit, in contrast to the other subunits, is a basic protein. 6. The 52 000 Mr subunit has no catalytic function in the intramolecular electron transfer from reduced benzylviologen to nitrate. However, it appears to have a structural function since nitrate reductase II, which lacks this subunit, is much more labile than nitrate reductase I. Inactivation of nitrate reductase II can be prevented by the presence of deoxycholate. 7. The spectrum of the enzyme resembles that of iron-sulfur proteins. No cytochromes or contaminating enzyme activities are present in the purified enzyme. Only reduced benzylviologen was found to be capable of acting as an electron donor. 8. p-Chlormercuribenzoate enhances the enzymatic activity at concentrations of 0.1 mM and lower. At higher p-chlormercuribenzoate concentrations the enzymatic activity is inhibited non-competitively with either nitrate or benzylviologen as a substrate. The inhibition is not counteracted by cysteine.     

Purification and properties of three intracellular proteinases from Candida albicans

Three intracellular proteinases termed A, B and C were purified to homogeneity from the unicellular form of the yeast Candida albicans. Enzyme A is an aspartic proteinase that acts on a variety of proteins. Its optimal pH is around 5 and it is displaced to 6.5 by KSCN. It is not significantly inhibited by PMSF, TLCK (Tos-Lys-CHCl₂) or soybean trypsin inhibitor but it is inhibited by pepstatin. Its molecular weight is 60 000. Enzyme B is a dipeptidase that acts on esters or on dipeptides without blocks in either the carboxyl or amino ends. Its pH optimum is around 7.5 and the molecular weight is 57 000. It is inhibited by PMSF, TLCK and DANME (N₂Ac-Nle-OMe). Proteinase C is an aminopeptidase with an optimum pH around 8. Its molecular weight was 67 000 when determined by SDS gel electrophoresis and 243 000 when determined by gel filtration. It is active towards dipeptides in which at least one amino acid is apolar and is not active when the N-terminal amino acid is blocked. It is inhibited by EDTA or o-phenanthroline and activated by several divalent cations.