Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.     

CHARACTERIZATION OF A RAT BRAIN CATHEPTIC CARBOXYPEPTIDASE (CATHEPSIN A) INACTIVATING ANGIOTENSIN-II

—Catheptic carboxypeptidase (cathepsin A) is present in lysosomal-enriched fractions of rat brain at levels approximately 5-fold that of cathepsin B1 and of the classical carboxypeptidase A but lower than that of cathepsin C and carboxypeptidase B. Cathepsin A was purified 40-fold by extraction of calf brain with an acetate buffer containing 0.5% desoxycholate followed by heat treatment, salt precipitation and chromatography on DEAE-Sephadex. Purification revealed the presence of two distinct isoenzymatic forms of high mol. wt that were very stable when frozen in the presence of sucrose and KCl. Among N-protected dipeptides used as substrates the highest activity was given by Z-Glu-Tyr and Z-Phe-Tyr at a pH optimum of 5.5, K_m1.1 mm and V_max 0.5 μmol (Tyr)/mg protein per min. Brain cathepsin A was completely inhibited by low concentrations of DFP and PCMB but unaffected by thiols, EDTA and specific inhibitors of other cathepsins (pepstatin and chymostatin). The carboxypeptidase A-like specificity of cathepsin A was confirmed by breakdown of Ile⁵-angiotensin with release of C-terminal Phe. Cathepsin A may play a role in the turnover of selected hormonal peptides containing C-terminal neutral amino acids and in the sequential breakdown of proteins associated with degenerative conditions such as demyelination.     

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing.

A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.     

Enzyme preparation from extract of wheat bran koji by alcohol precipitation

A method for the preparation method of enzymes for shoyu making was studied. When enzyme proteins were extracted with water from a column of wheat bran koji culture of Aspergillus oryzae 460, the tailing phenomenon resulted in low recovery. However, a better yield was obtained by the use of the solution passed at the latter stage of extraction as the extraction liquid for the succeeding extraction. Thus, in case of leucine aminopeptidase (Leu-Gly-Gly as substrate) the extraction yield reached 89%. Considering the extraction yield, and the amount of alcohol required to precipitate enzyme proteins, an appropriate volume of extract was found to be 1.5 to 2 times the amount of wheat bran koji. Moreover, less sporulation of koji culture due to a shortening of the culture time to 48 h, or 38 h by the use of germinated spores as seed culture, resulted in less water repellence, and a higher yield of extraction of enzymes than in the old culture (58.5 h). The temperature of the mixture of extract and alcohol should be kept at 5°C to increase the yield of leucine aminopeptidase and acid carboxypeptidase. By the concentration of enzyme proteins through ultrafiltration, the use of alcohol could be reduced, and an enzyme preparation with high specific activity and recovery could be obtained.     

Crayfish carboxypeptidase. Affinity chromatography, characterization and amino-terminal sequence.

In an effort to trace the evolutionary history of the pancreatic metalloexopeptidases, carboxypeptidase has been isolated from the cardia of the crayfish Astacus fluviatilis. The isolation procedure included affinity chromatography on a column of potato carboxypeptidase inhibitor covalently linked to Sepharose. Approximately 25 mg of pure enzyme can be obtained by the present procedure from 50 ml of cardia fluid. The pure enzyme resembles bovine carboxypeptidase B in specificity and is inhibited both by 3-phenyllactate and by 6-aminohexanoate. The pH optimum of activity is about pH 6.5, and the isoelectric point,pH 4.0. Inhibition by typical metal chelating agents (i.e. ethylenediamine tetraacetate and 1,10-phenanthroline) and neutron activation analysis indicate that, like the mammalian enzyme, crayfish carboxypepetidase is a zinc metalloenzyme. The purified enzyme migrates as a single band in cellulose acetate, disc gel and sodium dodecylsulfate gel electrophoresis. The amino acid composition is similar to that of pancreatic carboxypeptidases except for a higher content of acidic amino acid residues. The amino acid sequence of the first 19 amino-terminal residues reveals significant homology to that of pancreatic carboxypeptidases A and B.     

Carboxypeptidase T--intracellular carboxypeptidase of Thermoactinomycetes--a distant analog of animal carboxypeptidase

Carboxypeptidase T, an extracellular carboxypeptidase from Thermoactinomyces sp. was isolated and purified by affinity chromatography on bacitracin adsorbents. The enzyme homogeneity was established by SDS electrophoresis (Mr = 38 000) and isoelectrofocusing in PAAG (pI 5.3). Carboxypeptidase T reveals a mixed specificity in comparison with pancreatic carboxypeptidases A and B and cleaves with nearly the same efficiency the peptide bonds formed by the C-terminal residues of basic and neutral hydrophobic amino acids. The enzyme is insensitive to serine and thiol proteinase inhibitors but is completely inhibited by EDTA and o-phenanthroline. The maximal enzyme activity is observed at pH 7-8. With an increase of temperature from 20 to 70 degrees C the enzyme activity is enhanced approximately 10-fold. In the presence of 1 mM Ca2+ the enzyme thermostability is also increased. In terms of some properties, e.g. substrate specificity carboxypeptidase T is similar to metallocarboxypeptidase secreted by Streptomyces griseus. The N-terminal sequence of carboxypeptidase T: Asp-Phe-Pro-Ser-Tyr-Asp-Ser-Gly- Tyr-His-Asn-Tyr-Asn-Glu-Met-Val-Asn-Lys-Ile-Asn-Thr-Val-Ala-Ser-Asn-Tyr- Pro-Asn - Ile-Val-Lys-Thr-Phe-Ser-Ile-Gly-Lys-Val-Tyr-Glu-Gly-Xaa-Gly-Leu- coincides by 21% with that of pancreatic carboxypeptidases A and B. Thus, it may be concluded that these enzymes originate from a common precursor.     

A brain protein (P30) that immunoreacts with a polyclonal anti-pancreatic carboxypeptidase A antibody shows properties that are shared with tubulin carboxypeptidase

A preparation of tubulin carboxypeptidase partially purified from bovine brain was found to contain a protein of molecular mass 30 kDa (P30) as determined by SDS-PAGE, that is recognized by a polyclonal anti-bovine pancreatic carboxypeptidase A. However, this protein is different from pancreatic carboxypeptidase A as judged by the isoelectric point and the pattern of peptides produced by trypsin digestion. The isoelectric point of P30 was similar to that found for tubulin carboxypeptidase (9 ± 0.2). When the tubulin carboxypeptidase preparation was subjected to gel filtration chromatography under low salt concentration, P30 behaved as a protein of molecular mass 38 kDa whereas tubulin carboxypeptidase eluted at a position of 75 kDa molecular mass. However, when the chromatography was performed at relatively high salt concentration they behaved as proteins of 49 and 56 kDa, respectively. We considered that P30 may be an inactive monomeric form of the dimeric tubulin carboxypeptidase. However we can not rule out the possibility that it represents another carboxypeptidase not yet described.     

Characterization of proteins from Ascaris lumbricoides which bind specifically to carboxypeptidase.

Inhibitors of the peptidase and esterase activities of carboxypeptidases A and B have been isolated from extracts of Ascaris lumbricoides var suis. These proteins were obtained by treatment of the aqueous extracts at low pH, precipitation with ammonium sulfate, molecular sieving on Bio-Gel P-4, and chromatography on DEAE-cellulose. The inhibitors were resolved into three homogeneous peaks on CM-cellulose. These components, CM-A, CM-B, and CM-C, have constant specific activity and were recovered in a 41% yield. They moved as single bands when subjected to electrophoresis at high or low pH on polyacrylamide gels and they have similar amino acid compositions. Methionine, tyrosine, and cysteine are absent from each of the inhibitors. The 65 residues of CM-B suggest a minimum molecular weight of 7530, in close agreement to the value of 7600 +/- 200 determined on a Bio-Gel P-100 column. Each of the proteins has the same NH2-terminal residues, NH2-Asx-Glx-Val-Glx- and the same COOH-terminal residue, leucine. A plot of per cent acrylamide versus log relative mobility suggests that the three proteins are charge isomers. CM-B appears to be stable to high NaCl concentrations, extremes of pH, high temperatures, and digestion by intestinal proteases. Carboxypeptidase C, carboxypeptidase N, and yeast protease C are not inhibited by CM-B. However, the exopeptidase and esterase activities of human carboxypeptidase A are inhibited. The inhibitors appear to bind to bovine carboxypeptidase A with an atypical stoichiometry. Two moles of CM-B inhibitor bind to 1 mol of enzyme. The evidence is: (a) a demonstrated purity of bovine carboxypeptidase A, (b) minimal and maximal inhibitor molecular weights by different methods, of 7600 and 8300, and (c) a maximum specific activity of apparently homogeneous inhibitors which is 50% of that predicted for unit stoichiometry.     

Isolation and characterization of a basic carboxypeptidase from human seminal plasma

A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent Mr 98,000 and after purification with an Mr 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at Mr 53,000 was seen which was converted to two smaller bands (Mr 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (67.8 mumol/min/mg) with the ester substrate benzoyl (Bz)-Gly-argininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 microM, kcat = 43 min-1), Arg6-Met5-enkephalin (Km = 103 microM, kcat = 438 min-1), and Lys6-Met5-enkephalin (Km = 848 microM, kcat = 449 min-1). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction.     

Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli

Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively high level in the cytoplasm (4 mg/L culture medium). Although the recombinant protein essentially accumulated as inclusion bodies, as much as 30% of the fusion protein was recovered in a soluble form at low growth temperature and could therefore be purified to homogeneity in a single-step procedure by metal-affinity chromatography. The recombinant precursor form of bovine carboxypeptidase A was recognized by a monoclonal antibody directed against purified bovine pancreatic carboxypeptidase A. Moreover, upon tryptic activation it gave rise to an enzyme, the N-terminal sequence, molecular size,and specific activity of which were comparable to those of the enzyme derived from the native precursor purified from bovine pancreas.     

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of proteolytic enzyme activities in different tissues of germinating corn (Zea mays L.)

Profiles of pH dependence and activities of live proteolytic enzymes, amino- and carboxypeptidase and endopeptidases active at pH 3.8, 5.4 and 7.5, with casein as substrate, were determined in crude extracts from the various organs of corn seedlings during germination and early development (30°C, dark, 8 d). With respect to the endopeptidases, caseolytic activities at pH 3.8, 5.4 and 7.5 in extracts from endosperm increased concurrently with loss of endosperm N during germination; however, the relative amounts of the pH 7.5 activity were very small. In scutellum extracts, caseolytic activities at both pH 5.4 and 7.5 increased during the initial stages of development but only the increase at pH 5.4 was concurrent with loss of scutellar N. In shoot extracts, caseolytic activities at pH 5.4 and 7.5 were very low and remained relatively constant. There was a progressive increase in shoot N with time. In root extracts, caseolytic activities at pH 5.4 and 7.5 were higher (3-fold) than in shoot extracts. The activity at pH 5.4 remained constant while the activity at pH 7.5 increased during germination. The rate of accumulation of N by the root was low after day 5. The pattern and ratio but not the amounts of the pH 5.4 and 7.5 caseolytic activities of the root were similar to those observed in senescing leaves of field-grown corn. Addition of mercaptoethanol increased (several-fold) the caseolytic activities at pH 3.8 and 5.4, especially the latter, but not the pH 7.5 activity in endosperm extracts and increased the pH 5.4 activity in extracts from scutellum (30%) and roots (30%) while the effect in shoot extracts was negligible. Carboxypeptidase activity was relatively low in young tissue (root tip, 3-d-old shoots) and increased with development of the various organs except the roots (whole) where the activity remained relatively constant. The increases in carboxypeptidase activities were concurrent with decreases in N from endosperm and scutellum; this result indicates that this enzyme in these tissues may be involved (cooperatively with endopeptidases) in the mobilization of reserve protein.Of all the enzymes tested, only carboxypeptidase activity was markedly (in excess of 50%) inhibited by phenylmethylsulfonylfluoride. Only aminopeptidase activity was found in appreciable amounts in endosperm and scutellum of dry kernels. Aminopeptidase activity was highest in organs with high metabolic activity (scutella, shoot, root tips) and decreased in plant parts undergoing rapid loss of nitrogen (endosperm, senescing leaves).Abbreviations AP aminopeptidase - CA caseolytic activity - CP carboxypeptidase - ME mercaptoethanol     

Human carboxypeptidase M. Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones

A membrane-bound neutral carboxypeptidase B-like enzyme was solubilized from human placental microvilli with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and purified to homogeneity by ion-exchange chromatography and affinity chromatography on arginine-Sepharose. It gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 62,000 with or without reduction. The enzyme is a glycoprotein as shown by its high affinity for concanavalin A-Sepharose and reduction in mass to 47,600 daltons after chemical deglycosylation. It has a neutral pH optimum, is activated by CoCl2, and inhibited by o-phenanthroline, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, or cadmium acetate, indicating it is a metallopeptidase. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides (e.g. Bz-Gly-Arg, Bz-Gly-Lys, Bz-Ala-Lys, dansyl-Ala-Arg, where Bz is benzoyl and dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) as well as from several biologically active substrates: dynorphin A(1-13), Met5-Arg6-enkephalin (Km = 46 microM, kcat = 934 min-1), bradykinin (Km = 16 microM, kcat = 147 min-1), Met5-Lys6-enkephalin (Km = 375 microM, kcat = 663 min-1), and Leu5-Arg6-enkephalin (Km = 63 microM, kcat = 106 min-1). Although the enzyme shares some properties with other carboxypeptidase B-like enzymes, it is structurally, catalytically, and immunologically distinct from pancreatic carboxypeptidase A or B, human plasma carboxypeptidase N, and carboxypeptidase H ("enkephalin convertase"). To denote that the enzyme is membrane-bound, and to distinguish it from other known carboxypeptidases, we propose the name "carboxypeptidase M." Because of its localization on the plasma membrane and optimal activity at neutral pH, carboxypeptidase M could inactivate or modulate the activity of peptide hormones either before or after their interaction with plasma membrane receptors.     

Proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Purification and characterization of the carboxypeptidase.

We described earlier the facilitated purifications of the trypsin and aminopeptidase components present in Pronase (Vosbeck, K. D., Chow, K. -F., and Awad, W. M., Jr. (1973) J. Biol. Chem. 248, 6029-6034). A partially resolved protein mixture left over after one of the steps in that procedure was passed through a Sephadex G-75 column. By this means, a component with carboxypeptidase activity was separated from associated serine endopeptidases. Further purification of this exopeptidase to apparent homogeneity was acheived by refiltration through the same Sephadex column and by CM-cellulose chromatography. A single protein band was observed after acrylamide gel electrophoresis; analysis by sedimentation equilibrium using the meniscus depletion method gave a molecular weight of 30,300. This enzyme demonstrates activity against Nalpha-benzyloxycarbonylglycyl-L-leucine and hippuryl-D,L-phenyllactate; no activity was found against Nalpha-acetyl-L-tyrosine ethyl ester, Nalpha-benzoyl-D,L-arginine-p-nitroanilide, or L-leuckne-p-nitroanilide. The maximum activity lies between pH values of 7 and 8; the enzyme is stable between pH values of 6 and 10. At room temperature 1,10-phenanthroline inactivates the enzyme completely whereas EDTA has no effect. Of the many cations tested, only Co2+, Ni2+, or Zn2+ restores activity to the 1,10-phenanthroline-treated enzyme; Co2+ provided 3 times the native activity. The metal in the native protein was found to be zinc. These findings are similar to those recorded with bovine pancreatic carboxypeptidase A, and suggest the possibility that the present enzyme may ge genetically related to the mammalian protein, as in previously noted examples of homology of three Pronase endopeptidases to pancreatic serine enzymes.     

Kinetic studies of wheat carboxypeptidase-catalyzed reaction: differences in pressure and temperature dependence of peptidase and esterase activities

A kinetic study of hydrolytic catalysis by wheat bran carboxypeptidase (carboxypeptidase W) was carried out using 3-(2-furyl)acryloyl-acylated (Fua-) synthetic substrates. This enzyme showed high esterase activity in addition to the intrinsic carboxypeptidase activity. The optimum pH for the peptidase activity (kcat/Km) was at pH 3.3 and the kcat/Km value decreased with increasing pH with an apparent pKa of 4.50, while the esterase activity increased with pH up to pH 8 with an apparent pKa of 6.04. Optimum pH's for kcat for the peptidase and esterase reactions were also very different and their apparent pKa values were 3.80 and 6.15, respectively. From a measurement of the pressure dependences of kcat and Km, the activation volumes (delta V not equal to) and reaction volumes (delta V), respectively, were determined. delta V not equal to for kcat was -7 to -8 ml/mol for peptidase and -2 to -3 ml/mol for esterase. These results lead us to propose that the peptidase and esterase activities of carboxypeptidase W are different not in the rate-determining steps in a common reaction pathway, but in the binding modes and/or catalytic site(s).     

Characterization and cDNA cloning of midgut carboxypeptidases from Trichoplusia ni

Carboxypeptidase A and carboxypeptidase B activities from the midgut of Trichoplusia ni larvae were characterized. In the T. ni larval midgut, the primary digestive carboxypeptidase activity was attributed to carboxypeptidase A, which was eight times more active than carboxypeptidase B. Both the midgut carboxypeptidase A and carboxypeptidase B exhibited maximal activities at pH 8.0-8.5 and were similarly susceptible to inhibition by potato carboxypeptidase inhibitor and phenanthroline. The midgut carboxypeptidase activities were analyzed in T. ni larvae fed on various diet sources and the results indicated that midgut carboxypeptidase activities per milligram of gut were similar regardless of the amount of dietary proteins or amino acids. However, midgut carboxypeptidase A activity was significantly higher in larvae exposed to soybean trypsin inhibitor and was significantly lower in larvae fed on broccoli foliage. From the T. ni larval midgut, five putative carboxypeptidase cDNAs were cloned, demonstrating that midgut carboxypeptidase activities are composed of multiple carboxypeptidase types. Sequence analysis indicated that the midgut carboxypeptidases were produced as secreted proenzymes which could be activated after removal of an N-terminal activation fragment by a trypsin. Two cloned cDNAs are predicted to code for carboxypeptidase A and one cDNA is predicted to code for a putative carboxypeptidase B. The other two cDNAs are highly similar to carboxypeptidase A and carboxypeptidase B in sequences, but their activity was not predictable.     

Interaction of zinc ions with arsanilazotyrosine-248 carboxypeptidase A

The interaction between arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]) and excess zinc ions has been studied by stopped-flow and spectrophotometric methods at pH 8.2 and 7.7, I = 0.5 M (NaCl), and 25 degrees C. When excess zinc ions bind to arsanilazotyrosine-248 carboxypeptidase A, the characteristic red color, which arises from the intramolecular complex of the arsanilazotyrosine-248 residue with the active site zinc of the enzyme, changes to yellow with the inhibition of peptidase activity of the enzyme. Excess zinc ions have two binding sites for arsanilazotyrosine-248 carboxypeptidase A, and the binding constants of the first site (3.9 X 10(5) M-1 at pH 8.2; 7.1 X 10(4) M-1 at pH 7.7) are much larger than those of the second site (1.8 X 10(3) M-1 at pH 8.2; 7 X 10(2) M-1 at pH 7.7). The binding of excess zinc ions to the first site is completely correlated with the inhibition of the enzyme peptidase activity and the color change of the enzyme. The results can be understood in terms of zinc ions reacting with only one of three conformational states of arsanilazotyrosine-248 carboxypeptidase A [Harrison, L. W., Auld, D. S., & Vallee, B. L. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4356].(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris.

The crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the three-dimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed beta sheet. The active site is located at the C-edge of the central (parallel) part of the beta sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.     

Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase).

Carboxypeptidases H and M differ in their distribution and other properties, but both are activated by Co2+ and inhibited by guanidinoethylmercaptosuccinic acid. The higher degree of activation or inhibition of carboxypeptidase H by these agents at acid pH has been employed to identify this enzyme in tissues. We found that the activation or inhibition of both purified and plasma-membrane-bound human carboxy-peptidase M depends on the pH of the medium. CoCl2 activated over 6-fold at pH 5.5, but less than 2-fold at pH 7.5. Guanidinoethylmercaptosuccinic acid inhibited the membrane-bound carboxypeptidase M more effectively than the purified enzyme, and the IC50 was about 25-30 times lower at pH 5.5. As purified human plasma carboxypeptidase N and pancreatic carboxypeptidase B were also activated more at pH 5.5, we conclude that the increased activation by CoCl2 is due to the enhanced dissociation of Zn2+ below the pKa of the ligands that co-ordinate the cofactor in the protein. Thus increased activation or inhibition at acid pH would not differentiate basic carboxypeptidases.     

Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.