Human ''creatine kinase conversion factor'' identified as a carboxypeptidase.

The effect of partially purified 'creatine kinase conversion factor' on rabbit muscle creatine kinase is shown to be that of a carboxypeptidase, removing the C-terminal lysine residue from both subunits. These changes fully explain the three-banded electrophoretic patterns of the partially and the fully modified rabbit and human enzymes. The factor also produces a similar electrophoretic pattern with haemoglobin A; comparison with the effects of carboxypeptidases A and B permits the inference that the C-terminal residues of both alpha- and beta-subunits are removed. Small synthetic peptides are poor or non-substrates. A low activity with hippuryl-L-lysine may be due to contamination of the preparation with carboxypeptidase N. The possibility has been excluded that the action of conversion factor on creatine kinase involves modification of the protein thiol groups. Mr, substrate-specificity, pH-activity profile and the effects of metal ions distinguish creatine kinase conversion factor from carboxypeptidases A, B and N. On the basis of this evidence it is proposed to give the conversion factor the provisional name of carboxypeptidase K.     

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.     

Purification and characterization of a new arginine carboxypeptidase in human serum

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37 degrees C, we named it unstable carboxypeptidase (carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl-L-arginine and hippuryl-L-lysine, but at a different relative rate than carboxypeptidase N, and has no esterase activity on hippuryl-L-argininic acid. Its activity was inhibited by o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate carboxypeptidase U from carboxypeptidase N and other known carboxypeptidases.     

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.     

Properties of the major carboxypeptidase in the larvae of the webbing clothes moth, Tineola bisselliella.

The larvae of the webbing clothes moth, Tineola bisselliella contain two carboxypeptidases (EC 3.4.12-) and one of these has been purified by preparative polyacrylamide gel electrophoresis. Its pH optimum for the hydrolysis of N-benzyloxycarbonyl-glycyl-leucine was pH 7.5-7.7 and its molecular weight as judged by gel filtration was 72 000. It is strongly inhibited by disopropylfluorophosphate, thiol reagents and some metal cations and also by 1:10 phenanthroline but not EDTA. Km and V values for the hydrolysis of 13 N-acyl dipeptides were determined. The enzyme has a strong preference for neutral aliphatic amino acid residues and does not hydrolyse C-terminal proline, arginine or lysine. It is a true carboxypeptidase, requiring an L-amino acid in the C-terminal position, with a free carboxyl group and hydrolysing peptide substrates consecutively from the C-terminal end. Dipeptides are cleaved much more slosly than tripeptides or N-acyl dipeptides.     

Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues.

Carboxypeptidases were purified from guts of larvae of corn earworm (Helicoverpa armigera), a lepidopteran crop pest, by affinity chromatography on immobilized potato carboxypeptidase inhibitor, and characterized by N-terminal sequencing. A larval gut cDNA library was screened using probes based on these protein sequences. cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymes of clan MC [Barrett, A. J., Rawlings, N. D. & Woessner, J. F. (1998) Handbook of Proteolytic Enzymes. Academic Press, London.], but with a novel predicted specificity towards C-terminal acidic residues. This carboxypeptidase was expressed as a recombinant proprotein in the yeast Pichia pastoris. The expressed protein could be activated by treatment with bovine trypsin; degradation of bound pro-region, rather than cleavage of pro-region from mature protein, was the rate-limiting step in activation. Activated HaCA42 carboxypeptidase hydrolysed a synthetic substrate for glutamate carboxypeptidases (FAEE, C-terminal Glu), but did not hydrolyse substrates for carboxypeptidase A or B (FAPP or FAAK, C-terminal Phe or Lys) or methotrexate, cleaved by clan MH glutamate carboxypeptidases. The enzyme was highly specific for C-terminal glutamate in peptide substrates, with slow hydrolysis of C-terminal aspartate also observed. Glutamate carboxypeptidase activity was present in larval gut extract from H. armigera. The HaCA42 protein is the first glutamate-specific metallocarboxypeptidase from clan MC to be identified and characterized. The genome of Drosophila melanogaster contains genes encoding enzymes with similar sequences and predicted specificity, and a cDNA encoding a similar enzyme has been isolated from gut tissue in tsetse fly. We suggest that digestive carboxypeptidases with sequence similarity to the classical mammalian enzymes, but with specificity towards C-terminal glutamate, are widely distributed in insects.     

The membrane-bound basic carboxypeptidase from hog intestinal mucosa(1).

The carboxypeptidase activity occurring in hog intestinal mucosa is apparently due to two distinct enzymes which may be responsible for the release of basic COOH-terminal amino acids from short peptides. The plasma membrane-bound carboxypeptidase activity which occurs at neutral optimum pH levels was found to be enhanced by CoCl(2) and inhibited by guanidinoethylmercaptosuccinic acid, o-phenanthroline, ethylenediamine tetraacetic acid and cadmium acetate; whereas the soluble carboxypeptidase activity which occurs at an optimum pH level of 5.0 was not activated by CoCl(2) and only slightly inhibited by o-phenanthroline, ethylenediamine tetraacetic acid, NiCl(2) and CdCl(2). The latter activity was presumably due to lysosomal cathepsin B, which is known to be present in the soluble fraction of hog intestinal mucosa. Although the membrane-bound enzyme was evenly distributed along the small intestine, it was not anchored in the phospholipidic bilayer via a glycosyl-phosphatidylinositol moiety, as carboxypeptidase M from human placenta is. The enzyme was not solubilized by phosphatidylinositol-specific phospholipase C, but was solubilized to practically the same extent by several detergents. The purified trypsin-solubilized form is a glycoprotein with a molecular mass of 200 kDa, as determined by performing SDS-PAGE and gel filtration, which differs considerably from the molecular mass of human placental carboxypeptidase M (62 kDa). It was found to cleave lysyl bonds more rapidly than arginyl bonds, which is not so in the case of carboxypeptidase M, and immunoblotting analysis provided further evidence that hog intestinal and human placental membrane-bound carboxypeptidases do not bear much resemblance to each other. Since the latter enzyme has been called carboxypeptidase M, it is suggested that the former might be carboxypeptidase D, the recently described new member of the carboxypeptide B-type family.     

Electrochemiluminescence assay for basic carboxypeptidases: inhibition of basic carboxypeptidases and activation of thrombin-activatable fibrinolysis inhibitor

Carboxypeptidases catalyze the removal of the C-terminal amino acid residues in peptides and proteins and exert important biological functions. Assays for carboxypeptidase activity that rely on change of absorbance generally suffer from low sensitivity and are difficult to adapt to high-throughput screening. We have developed a sensitive, robust assay for basic carboxypeptidase activity that makes use of electrochemiluminescent (ECL) detection of reaction product. In this assay, a peptide substrate contains the epitope for antibody (G2-10) binding which is masked by a C-terminal arginine. Carboxypeptidase activity exposes the epitope, allowing the binding of ruthenylated G2-10 which is then detected using ECL. High sensitivity allowed detection limits of 1-2 pM enzyme for carboxypeptidase B and activated thrombin-activatable fibrinolysis inhibitor (TAFIa). The inhibition of several basic carboxypeptidases by commercially available inhibitors was studied. This antibody-based method can be extended to other sensitive detection techniques such as amplified luminescent proximity homogeneous assay. The high sensitivity of the assay allowed the determination of the activatable levels of TAFI in human and other animal plasma in the presence of epsilon -aminocaproic acid, an active-site inhibitor that stabilizes TAFIa. A method to isolate in situ activated TAFIa from human serum in the presence of epsilon -aminocaproic acid was also developed.     

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.     

Prodynorphin processing by rat CNS fractions and purified enzymes: Formation of Dynorphin A 1–8 by sulfhydryl-activated carboxypeptidase and peptidyl dipeptidase

The conversion of selected prodynorphin fragments to form the octapeptide Dynorphin A 1–8 was studied in rat brain or spinal cord fractions, and the results compared to the action of purified carboxypeptidases and angiotension converting enzyme. The particulates were shown to convert Dynorphin A or 1–13 to the octapeptide as measured by radioimmunoassay, and by reverse phase high performance liquid chromatography. Detergent extracts of these particulates contained and enzyme converting 1–13 to 1–12 with release of C-terminal lysine, and active over a wide pH range of 4.8–7.6. Purification of these extracts by affinity chromatography (p-amino-benzoyl-arginine-Sepharose-6B) using Bz-Ala-Arg as the substrate led to isolation of a carboxypeptidase converting 1–13 to 1–12 active over the same pH range. Since Dynorphin 1–13 was converted to 1–8 by the consecutive use of purified carboxypeptidase B and angiotensin converting enzyme, the possibility exists that this mechanism might account for some octapeptide production in situ.

The properties and substrate specificity of the carboxypeptidase B were compared to a carboxypeptidase A active optimally at pH 5.5 and assayed with Z-Glu-Tyr. The carboxypeptidase B acted only on prodynorphins with C-terminal basic residues as contrasted to a nonspecific action by the carboxypeptidase A. The carboxypeptidase B was characterized by a strong activation by -SH agents and Zn²⁺, and thus could be differentiated from other opioid converting enzymes. The enzyme was inhibited by guanidinoethyl succinic acid (GEMSA), and p-chloromercuriphenyl-sulphonic acid (PCMS) but not by benzylsuccinic acid, a potent inhibitor of carboxypeptidase A.     

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.     

A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein".

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.     

Purification and some properties of camel carboxypeptidase B

A carboxypeptidase B-like enzyme was purified 116-fold with a recovery of activity of 29% from a crude extract of camel pancreas by a four-step procedure consisting of two anion exchange chromatographies in succession, gel filtration and hydrophobic interaction chromatography. The enzyme was homogeneous on SDS and non-denaturing gel electrophoresis and on gel isoelectric focusing. Its molecular mass was found to be 31.5 kDa and its isoelectric point was estimated as 6.1. It was active towards a number of substrates that are cleaved by carboxypeptidases B from other species and was also susceptible to inhibition by inhibitors of such enzymes. The camel enzyme showed a pH optimum of 8.0 and it was seen to be a relatively potent kinase in vitro. The enzyme purified in this study was very similar to carboxypeptidases B isolated from other species in size, charge, substrate specificity and susceptibility to inhibition and thus it can be identified as camel carboxypeptidase B.     

Purification of carboxypeptidase B from human pancreas.

Carboxypeptidase B of the human pancreas was purified by chromatography on DEAE-cellulose and CM-cellulose columns. Two forms of the enzyme, named carboxypeptidase B1 and B2, were separated. They have similar mol.wts. (34250 +/- 590) as established by polyacrylamide-gel disc electrophoresis and by gel filtration. Carboxypeptidase B2 migrates further towards the anode in disc electrophoresis. When the amino acid content of the enzymes was analysed, carboxypeptidase B2 had four more glycine and three more aspartic acid residues than had form B1. The amino acid sequence of the human carboxypeptidase B1 differs from that of the bovine enzyme only in two places in the N-terminal 20-amino-acid sequence. The N-terminal amino acid in carboxypeptidase B1 and B2 is alanine. The peptide 'map' of the tryptic digest of carboxypeptidase B1 contained more peptides than did that of form B2. The Km, the Vmax. and the pH optimum of the cleavage of the peptide substrate hippurylarginine and the ester substrate hippurylargininic acid were similar for both enzymes. CoCl2 accelerated the peptidase activity, and cadmium acetate enhanced the esterase activity, of human carboxypeptidases B1 and B2. Urea and sodium dodecyl sulphate inhibited the enzymes.     

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.     

Carboxypeptidase from <Emphasis Type="Italic">Streptomyces bikiniensis</Emphasis>: Primary structure,isolation, and properties

A metallocarboxypeptidase produced by Streptomyces bikiniensis 27 strain (VKPM Ac-1783) (CPSb) was purified and characterized. The enzyme cleaves both basic and hydrophobic C-terminal amino acid residues from synthetic peptides, that is, it possesses specificity of mammalian carboxypeptidases A and B. The enzyme also hydrolyzes peptides bearing glutamic acid at the C-end. CPSb exhibits its maximal activity at pH 7.0–7.6 and 55°C. The nucleotide sequence encoding the mature CPSb in S. bikiniensis 27 (VKPM Ac-1783) genome (Accession No. GU362077) was determined. It is shown that the primary structure of the mature enzyme has a moderate degree of identity with orthologs from Streptomyces griseus (79% identity) and Streptomyces avermitilis (85% identity).     

Molecular cloning and sequencing of the cDNA for human membrane-bound carboxypeptidase M. Comparison with carboxypeptidases A, B, H, and N

Carboxypeptidase M, a widely distributed membrane-bound carboxypeptidase that can regulate peptide hormone activity, was purified to homogeneity from human placenta (Skidgel, R. A., Davis, R. M., and Tan, F. (1989) J. Biol. Chem. 264, 2236-2241). The NH2-terminal 31 amino acids were sequenced, and two complementary oligonucleotide probes were synthesized and used to isolate a carboxypeptidase M clone from a human placental cDNA library. Sequencing of the cDNA insert (2009 base pairs) revealed an open reading frame of 1317 base pairs coding for a protein of 439 residues. The NH2-terminal protein sequence matched the deduced amino acid sequence starting with residue 14. Hydropathic analysis revealed hydrophobic regions at the NH2 and COOH termini. The NH2-terminal 13 amino acids probably represent part of the signal peptide, and the COOH-terminal hydrophobic region may act either as a transmembrane anchor or as a signal for attachment to a phosphatidylinositol glycan moiety. The carboxypeptidase M sequence contains six potential Asn-linked glycosylation sites, consistent with its glycoprotein nature. The sequence of carboxypeptidase M was 41% identical with that of the active subunit of human plasma carboxypeptidase N, 41% identical with bovine carboxypeptidase H (carboxypeptidase E, enkephalin convertase), and 15% with either bovine pancreatic carboxypeptidase A or B. Many of the active site residues identified in carboxypeptidases A and B, including all of the zinc-binding residues (2 histidines and a glutamic acid), are conserved in carboxypeptidase M. These data indicate that all of the metallocarboxypeptidases are related, but the nondigestive carboxypeptidases with more specialized functions, present in cell membranes, blood plasma, or secretory granules (i.e., carboxypeptidase M, carboxypeptidase N and carboxypeptidase H), are more closely related to each other (41-49% identity) than they are to carboxypeptidase A or B (15-20% identity).     

Amino acid sequence of the N-terminus and selected tryptic peptides of the active subunit of human plasma carboxypeptidase N: comparison with other carboxypeptidases

Human plasma carboxypeptidase N was purified to homogeneity and its active and inactive subunits were separated. By introducing a novel technique, both forms of the active subunit (Mr = 55,000 and Mr = 48,000) were isolated. N-terminal sequencing of the active subunit of human carboxypeptidase N revealed significant homology with the N-terminal sequence of bovine carboxypeptidase H (43% identity) and to a lesser extent with carboxypeptidase A (29% identity) or carboxypeptidase B (18% identity). The active subunit of carboxypeptidase N was hydrolyzed with trypsin and 4 of the tryptic peptides were isolated by HPLC and sequenced. The sequences of the four peptides were homologous (39-64% identity) with regions of carboxypeptidase H corresponding to the middle (residues 148-175) and C-terminal portion (residues 321-408). These regions had essentially no homology with carboxypeptidase A or B. These data indicate that carboxypeptidase H and the active subunit of carboxypeptidase N may have diverged from a common ancestral gene.     

Isolation and molecular cloning of mast cell carboxypeptidase A. A novel member of the carboxypeptidase gene family

Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells (CTMC) and from a mouse Kirsten sarcoma virus-immortalized mast cell line (KiSV-MC), and a cDNA that encodes this exopeptidase has been cloned from a KiSV-MC-derived cDNA library. KiSV-MC-derived mast cell carboxypeptidase A was purified with a potato-derived carboxypeptidase-inhibitor affinity column and was found by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a Mr 36,000 protein. Secretory granule proteins from KiSV-MC and from mouse peritoneal CTMC were then resolved by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to polyvinylidine difluoride membranes. Identical aminoterminal amino acid sequences were obtained for the prominent Mr 36,000 protein present in the granules of both cell types. Based on the amino-terminal sequence, an oligonucleotide probe was synthesized and used to isolate a 1,470-base pair cDNA that encodes this mouse exopeptidase. The deduced amino acid sequence revealed that, after cleavage of a 15-amino acid hydrophobic signal peptide and a 94-amino acid activation peptide from a 417-amino acid preproenzyme, the mature mast cell carboxypeptidase A protein core has a predicted Mr of 35,780 and a high positive charge [Lys + Arg) - (Asp + Glu) = 17) at neutral pH. Although critical zinc-binding amino acids (His67, Glu70, His195), substrate-binding amino acids (Arg69, Asn142, Arg143, Tyr197, Asp255, Phe278), and cysteine residues that participate in intrachain disulfide bonds (Cys64-Cys77, Cys136-Cys159) of pancreatic carboxypeptidases were also present in mast cell carboxypeptidase A, the overall amino acid sequence identities for mouse mast cell carboxypeptidase A relative to rat pancreatic carboxypeptidases A1, A2, and B were only 43, 41, and 53%, respectively. RNA and DNA blot analyses revealed that mouse peritoneal CTMC, KiSV-MC, and bone marrow-derived mast cells all express a prominent 1.5-kilobase mast cell carboxypeptidase A mRNA which is transcribed from a single gene. We conclude that mouse mast cell carboxypeptidase A is a prominent secretory granule enzyme of mast cells of the CTMC subclass and represents a novel addition to the carboxypeptidase gene family.     

Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B

Carboxypeptidase activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different carboxypeptidases in lepidopteran protein digestion, the effect of potato carboxypeptidase inhibitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of Soybean Kunitz Trypsin Inhibitor. Analysis of carboxypeptidase activity in the guts showed that ingested PCI remained active in the gut, and completely inhibited the activity of carboxypeptidases A and O. Interestingly, carboxypeptidase B activity was not affected by PCI. All previously described enzymes from the same family, both from insect or mammalian origin, have been found to be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of carboxypeptidase B activity resistant to PCI in most of them. The H. zea carboxypeptidase B enzyme (CPBHz) was purified from gut content by affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recombinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The substrate specificity of recombinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to be highly selective for C-terminal lysine residues. Inhibition by PCI appeared to be pH-dependent.