Carboxypeptidase H. A regulatory peptide-processing enzyme produced by human hepatoma Hep G2 cells

Human hepatoma (Hep G2) cells have been shown to secrete nanogram quantities of carboxypeptidase N (Grimwood, B. G., Plummer, T. H., Jr., and Tarentino, A. (1988) J. Biol. Chem. 263, 14397-14401). A second carboxypeptidase with an acidic pH optimum (pH 5.5) is also secreted at levels 2-3-fold greater than carboxypeptidase N. This enzyme was partially purified from the conditioned medium and compared with pure bovine pituitary carboxypeptidase H. The two enzymes behaved in a similar fashion in DE52 ion-exchange chromatography and on gel filtration, with the Hep G2 enzyme being slightly larger than the bovine pituitary enzyme (52-54 versus 50-52 kDa). Both enzymes hydrolyzed COOH-terminal basic amino acids from typical synthetic substrates as well as from natural leuenkephalin peptides and were identical based on pH activity profiles, inhibition by EDTA or guanidinoethyl mercaptosuccinic acid, and stimulation by Co2+ ions. Inhibition of enzyme secretion from Hep G2 cells by tunicamycin indicated that the Hep G2 enzyme was glycosylated. This finding was confirmed by a parallel deglycosylation of the Hep G2 and bovine pituitary carboxypeptidase H enzymes with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Immunoblots using mouse antiserum to bovine pituitary carboxypeptidase H revealed that the Hep G2 enzyme was immunocross-reactive with the bovine enzyme but was slightly larger in size (54 versus 52 kDa). Continuous [35S]methionine labeling and purification to near homogeneity using an affinity matrix corroborated the observations that the secreted Hep G2 carboxypeptidase H was slightly larger than bovine pituitary carboxypeptidase H. The Hep G2-secreted enzyme in pulse-chase experiments was initially detected intracellularly after a 15-min pulse as a single protein of about 54 kDa and was present in the 30-min chase medium with no evidence for pre- or postsecretion proteolytic processing. The human adrenergic cell line IMR-32 continuously labeled with [35S]methionine also secreted carboxypeptidase H of the same size as the Hep G2 enzyme.     

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.     

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.     

Purification, cDNA cloning, and expression of a new human blood plasma glutamate carboxypeptidase homologous to N-acetyl-aspartyl-alpha-glutamate carboxypeptidase/prostate-specific membrane antigen

We describe the identification, cDNA cloning, and biochemical characterization of a new human blood plasma glutamate carboxypeptidase (PGCP). PGCP was co-purified from human placenta with lysosomal carboxypeptidase, cathepsin A, lysosomal endopeptidase, cathepsin D, and a gamma-interferon-inducible protein, IP-30, using an affinity chromatography on a Phe-Leu-agarose column. A PGCP cDNA was obtained as an expressed sequence tag clone and completed at 5'-end by rapid amplification of cDNA ends polymerase chain reaction. The cDNA contained a 1623-base pair open reading frame predicting a 541-amino acid protein, with five putative Asn glycosylation sites and a 21-residue signal peptide. PGCP showed significant amino acid sequence homology to several cocatalytic metallopeptidases including a glutamate carboxypeptidase II also known as N-acetyl-aspartyl-alpha-glutamate carboxypeptidase or as prostate-specific membrane antigen and expressed glutamate carboxypeptidase activity. Expression of the PGCP cDNA in COS-1 cells, followed by Western blotting and metabolic labeling showed that PGCP is synthesized as a 62-kDa precursor, which is processed to a 56-kDa mature form containing two Asn-linked oligosaccharide chains. The mature form of PGCP was secreted into the culture medium, which is consistent with its intracellular localization in secretion granules. In humans, PGCP is found principally in blood plasma, suggesting a potential role in the metabolism of secreted peptides.     

Regulation of Neuropeptide-Processing Enzymes by Nitric Oxide in Cultured Astrocytes

Abstract: Nitric oxide (NO), a recently discovered neuro-transmitter, has been shown to have a cytostatic effect on cultured glia. A NO-generating agent, S -nitroso- N -acetyl-penicillamine (SNAP), was used to treat C6 glioma and primary cortical astrocytes. The levels of a monobasic peptide-processing enzyme activity and carboxypeptidase E activity were examined. The cellular levels of these two enzymes are specifically reduced in response to treatment with SNAP. A decrease of ˜30–50% in these two'enzyme activities was seen in both primary astrocytes and C6 glioma cells. This decrease in cellular enzyme activities is not due to increased secretion because the secreted activity is also reduced in response to SNAP treatment in both the glioma cells and the primary astrocytes. Removal of SNAP treatment causes the carboxypeptidase enzyme activity to return to control levels within 3 days. Northern and western blot analyses indicate that the reduced cellular level of carboxypeptidase E is not due to reduced expression of the messenger RNA or protein, suggesting that the SNAP treatment is affecting factors that influence carboxypeptidase E activity. Taken together, these results imply that NO is involved in the regulation of peptide biosynthetic enzymes and this could lead to the antimitogenic action of SNAP on glia.     

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.     

Characterization of the yeast KEX1 gene product: a carboxypeptidase involved in processing secreted precursor proteins.

We have identified and partially characterized the Saccharomyces cerevisiae KEX1 gene product, Kex1p, to assess its role in processing secreted protein precursors. Anti-Kex1p antibodies identified a 113-kilodalton protein that was absent in cells in which the KEX1 gene has been disrupted and that was more abundant in cells overexpressing the KEX1 gene. Kex1p was found to be a membrane-associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide(s) was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kilodaltons. After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, alpha-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like enzyme to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. Our results provide biochemical evidence consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins.     

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.     

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.     

(Met)enkephalin and carboxypeptidase processing enzyme are co-released from chromaffin cells by cholinergic stimulation

A carboxypeptidase B-like enzyme is involved in processing of proenkephalin in adrenal medulla. Nicotine stimulated the co-release of this enzyme with (Met)enkephalin pentapeptide from bovine chromaffin cells in primary culture. The ratio of enzyme activity/immunoreactivity was determined for the released carboxypeptidase to provide an index of the level of enzyme activity per unit number of enzyme molecules. The ratio for the Co++-stimulated carboxypeptidase secreted into the cell culture medium upon nicotinic stimulation was 10.1 +/- 1.02 (pmol Met-enkephalin formed per ng carboxypeptidase immunoreactivity), while the Co++-stimulated carboxypeptidase in the soluble and membrane components of purified chromaffin granules had lower ratios of 5.46 +/- 0.70 and 1.07 +/- 0.13, respectively. Hexamethonium, a nicotinic receptor antagonist, blocked the nicotine-induced release of the carboxypeptidase processing enzyme and (Met)enkephalin. These data suggest that a pool of carboxypeptidase enzyme molecules at a high state of activation are present in functionally mature granules whose contents are released by nicotinic receptor stimulation.     

Serine Carboxypeptidase SCPEP1 and Cathepsin A Play Complementary Roles in Regulation of Vasoconstriction via Inactivation of Endothelin-1

The potent vasoconstrictor peptides, endothelin 1 (ET-1) and angiotensin II control adaptation of blood vessels to fluctuations of blood pressure. Previously we have shown that the circulating level of ET-1 is regulated through its proteolytic cleavage by secreted serine carboxypeptidase, cathepsin A (CathA). However, genetically-modified mouse expressing catalytically inactive CathA S190A mutant retained about 10–15% of the carboxypeptidase activity against ET-1 in its tissues suggesting a presence of parallel/redundant catabolic pathway(s). In the current work we provide direct evidence that the enzyme, which complements CathA action towards ET-1 is a retinoid-inducible lysosomal serine carboxypeptidase 1 (Scpep1), a CathA homolog with previously unknown biological function. We generated a mouse strain devoid of both CathA and Scpep1 activities (DD mice) and found that in response to high-salt diet and systemic injections of ET-1 these animals showed significantly increased blood pressure as compared to wild type mice or those with single deficiencies of CathA or Scpep1. We also found that the reactivity of mesenteric arteries from DD mice towards ET-1 was significantly higher than that for all other groups of mice. The DD mice had a reduced degradation rate of ET-1 in the blood whereas their cultured arterial vascular smooth muscle cells showed increased ET-1-dependent phosphorylation of myosin light chain 2. Together, our results define the biological role of mammalian serine carboxypeptidase Scpep1 and suggest that Scpep1 and CathA together participate in the control of ET-1 regulation of vascular tone and hemodynamics.     

Further characterization of the silkworm diapause hormone A

One of two diapause hormones (DH-A) was studied. DH-A was stable to acids, bases (except to 1·0 N NaOH), acylation agents and periodate oxidation. The hormonal activity was quickly lost by trypsin as well as by non-specific proteolytic enzymes but slowly or hardly at all by α-chymotrypsin and carboxypeptidase A. The hormone contains 14 kinds of amino acids and 2 kinds of amino sugars. The amino sugars appear not to be essential for the hormonal activity.     

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).     

Cultured Astrocytes and Neurons Synthesize and Secrete Carboxypeptidase E, a Neuropeptide-Processing Enzyme

Carboxypeptidase E (EC 3.4.17.10) is a carboxypeptidase B-like enzyme associated with the biosynthesis of many peptide hormones and neurotransmitters. Media collected from cultured astrocytes contain a carboxypeptidase E-like activity. Cultured astrocytes secrete approximately 73% of their cellular level of carboxypeptidase E per hour, and secretion is not substantially influenced by 35 mM KCl. In contrast, neurons secrete only 29% of their cellular carboxypeptidase E per hour, but secretion increases to 86% on stimulation with 35 mM KCl. Secretion of carboxypeptidase E activity from both neuronal and astrocyte cultures is relatively selective; neither acid phosphatase or acetylglucosaminidase is secreted in appreciable amounts. Cultured neurons and astrocytes express a carboxypeptidase E mRNA of a similar size. The levels of this mRNA differ in astrocytes cultured from different brain regions, with high levels in striatal, cortical, hippocampal, and hypothalamic astrocytes and low levels in cerebellar astrocytes. The level of carboxypeptidase E mRNA in hypothalamic astrocyte cultures is four- to fivefold higher than the level in hypothalamic neuronal cultures. These results indicate that cultured astrocytes express carboxypeptidase E mRNA and enzymatic activity and thus contain one of the enzymes required in the biosynthesis of many peptide hormones and neurotransmitters.     

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.     

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.     

The N alpha-acetylenkephalin carboxypeptidase activity of N-acetyltyrosine deacetylase from monkey kidney. Purification, characterization and substrate specificity.

N alpha-Acetylenkephalin carboxypeptidase was co-purified with N-acetyltyrosine deacetylase from monkey kidney. Almost 90% of the activity from the homogenate was recovered in a high-speed supernatant without the use of detergents. The crucial steps in the purification were Cibacron Blue F3GA--Sepharose chromatography (involving negative and positive binding sequentially) and metal chelate affinity chromatography. The purified enzyme showed three bands on gel electrophoresis under non-denaturing conditions. All the three bands exhibited both N-acetyltyrosine deacetylase and N-acetylenkephalin carboxypeptidase activity, indicating their co-migration, Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence and absence of 2-mercaptoethanol gave a single protein band of mol.wt. 34 000. The native enzyme was a dimer of mol.wt. 66 000 as observed on Bio-Gel P-300 gel filtration. The carboxypeptidase removed two amino acids from the C-terminal end of either N-acetyl[Met5]- or N-acetyl[Leu5]-enkephalin. Non-acetylated enkephalins were less active as substrates. Peptides with their carboxy end blocked were inactive as substrates. Models suggested for carboxypeptidase A [Hartsuck & Lipscomb (1971) Enzymes 3, 1-56] support the idea that the kidney N-acetylated aromatic amino acid deacetylase or acylase III [Endo (1978) Biochim. Biophys. Acta 523, 207-217] can act as a carboxypeptidase on peptides having hydrophobic amino acids at the C-terminal end.     

Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori

Using microarray analyses, we identified carboxypeptidase A (MF-CPA), which was induced during pupal ecdysis in the wing discs of Bombyx mori. Here, we report the functional characterization of MF-CPA. MF-CPA has amino acid sequence similarities with the proteins in the carboxypeptidase A/B subfamily, from human to nematode. The MF-CPA gene is expressed during the molting periods in the epithelial tissues. MF-CPA is detected in the molting fluid, which fills the space between the old and new cuticle during molting. By Western blot analysis, we show that MF-CPA is secreted as a zymogen and processed in the molting fluid. Recombinant MF-CPA expressed in the insect cells has carboxypeptidase A activity. We propose that MF-CPA degrades the proteins from the old cuticle during the molting periods and contributes to recycling of the amino acids.     

Inactivation of bradykinin by placental peptidases.

Normal human placental eluate (0.15 M NaCl) has very high capacity for inactivating synthetic bradykinin (its specific activity is 20--50 times higher than that of normal human serum). In chromatography on DEAE-Sephadex A 50, several fractions with bradykinin-inactivating capacity were recovered. On Cbo-Phe-Arg synthetic substrate, one fraction with kininase activity was identified as carboxypeptidase N. This is not identical with serum (pregnancy) carboxypeptidase N and does not therefore pass into the blood stream. The properties of other fractions with kininase activity were studied. Aminopeptidase activity degrading L-lysine-p-nitranilide and L-arginine-beta-naphthylamide was separate from kininase activity.     

Glu300 of rat carboxypeptidase E is essential for enzymatic activity but not substrate binding or routing to the regulated secretory pathway

Several recently discovered members of the carboxypeptidase E (CPE) gene family lack critical active site residues that are conserved in other family members. For example, three CPE-like proteins contain a Tyr in place of Glu300 (equivalent to Glu270 of carboxypeptidase A and B). To investigate the importance of this position, Glu300 of rat CPE was converted into Gln, Lys, or Tyr, and the proteins expressed in Sf9 cells using the baculovirus system. All three mutants were secreted from the cells, but the media showed no enzyme activity above background levels. Wild-type CPE and the Gln300 point mutant bound to a p-aminobenzoyl-Arg-Sepharose affinity resin, and this binding was competed by an active site-directed inhibitor, guanidinoethylmercaptosuccinic acid. The affinity purified mutant CPE protein showed no detectable enzyme activity (<0.004% of wild-type CPE) toward dansyl-Phe-Ala-Arg. Expression of the Gln300 and Lys300 mutant CPE proteins in the NIT3 mouse pancreatic beta-cell line showed that these mutants are routed into secretory vesicles and secreted via the regulated pathway. Taken together, these results indicate that Glu300 of CPE is essential for enzyme activity, but not required for substrate binding or for routing into the regulated secretory pathway.