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At least 19 recordsLinked to original sources
PubMed1987

Product inhibition of carboxypeptidase H.

Carboxypeptidase H is one of several enzymes required for the processing of peptide hormone precursors. In this study, inhibition of carboxypeptidase H by its peptide products was investigated. Carboxypeptidase H activity in bovine adrenal medulla chromaffin granules and rat adrenal medulla homogenate was inhibited by the peptides Met- and Leu-enkephalin, vasopressin, oxytocin, luteinizing hormone-releasing hormone, substance P, and thyrotropin-releasing hormone, with oxytocin and ACTH 1-14 having the least effect, at concentrations of 2-20 mM. Inhibition by amidated peptide products (vasopressin, oxytocin, luteinizing hormone-releasing hormone, substance P, and thyrotropin-releasing hormone) show that the final products of the precursor processing pathway can regulate carboxypeptidase H. These levels of peptides are similar to known intragranular peptide concentrations indicating that product and feedback inhibition of carboxypeptidase H may play a role in the control of neuropeptide synthesis. The proenkephalin-derived peptides Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8, and Met-enkephalin-Arg6-Phe7 competitively inhibited bovine and rat carboxypeptidase H with Ki values of 12.0, 6.5, 7.0, and 5.5 mM, respectively. The significantly greater Ki for Met-enkephalin may reflect the effects of higher intragranular concentration of Met-enkephalin, since one proenkephalin molecule contains four copies of Met-enkephalin and only one copy of each of the other enkephalin peptides. Thus, the products from one multivalent precursor molecule may equivalently inhibit carboxypeptidase H activity. Product inhibition of carboxypeptidase H and perhaps other processing enzymes may serve to limit the maximum peptide concentration within the secretory vesicle.

Adrenal Medulla↗
PubMed1989

Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes.

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.

Animals↗
PubMed1992

Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules.

An investigation of the molecular properties of glycoprotein III has shown this to be a major component of molecular aggregates present in the membrane and soluble fractions of secretory vesicles from bovine adrenal medulla. These aggregates also contain components identified as glycoproteins H, J, and K which are molecular forms of Kex2-related proteases (glycoprotein H) and carboxypeptidase H (glycoprotein components J and K) and which have functions concerned with the processing of prohormones. A number of experiments indicated that these glycoproteins were associated. These components were coimmunoprecipitated from the soluble and membrane fractions of chromaffin granules. Purification of soluble glycoprotein III using wheat germ agglutinin-Sepharose resulted in the recovery of similar proportions of glycoproteins H, J, and K and gel filtration of the eluted material in combination with immunoprecipitation revealed the presence of heteroaggregates containing all of the glycoproteins. Similar results were obtained following octylglucoside solubilization of chromaffin granule membranes. Glycoprotein components III, H, J, and K were also found to have identical distributions following fractionation of chromaffin granule membranes with Triton X-114. It was concluded that the aggregates seen in the soluble fraction reflect an association of these components in the chromaffin granule membrane. This raises the possibility that these interactions are important for the targetting of these glycoproteins to secretory granules.

Adrenal Medulla↗
PubMed1989

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.

Cobalt↗
PubMed1986

Identification of two glycoproteins of chromaffin granules as the carboxypeptidase H.

An antiserum against carboxypeptidase H, an enzyme involved in the biosynthesis of neuropeptides such as the enkephalins, was used to identify the enzyme proteins in bovine chromaffin granules. Two-dimensional immuno- and lectin-blots revealed that two closely migrating glycoproteins which have been previously named J and K represented the enzyme. The same protein doublet was present in the membrane preparation and the soluble lysate of bovine chromaffin granules; however, the membrane contained significantly more enzyme protein.

Animals↗
PubMed1989

Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans.

The release of carboxypeptidase H activity from isolated rat islets was determined and compared to the secretion of immunoreactive insulin. Analysis of pancreatic islet cells sorted into beta and non-beta types indicated that approx. 80% of islet carboxypeptidase H activity is present in the beta cell. The release of both insulin and carboxypeptidase H was stimulated markedly by increasing the glucose concentration in the medium from 2.8 to 28 mM. The fractional release was in accordance with the observed cellular distribution of both proteins. The secretory response was biphasic with time, with an initial rapid transient phase of release within 5 min, followed by a more sustained response. The concentration-dependencies of glucose stimulation of release of insulin and carboxypeptidase H were similar, with a threshold for stimulation around 5.6 mM-glucose and maximal stimulatory response at 16.7-28 mM-glucose. The release of both proteins was inhibited by 20 mM-mannoheptulose, removal of Ca2+ from the medium and addition of 1 microM-noradrenaline. The combination of 10 mM-4-methyl-2-oxopentanoate and 10 mM-glutamine stimulated the release of carboxypeptidase H and insulin, as did 3-isobutyl-1-methylxanthine and 350 microM-tolbutamide in the presence of glucose. It is evident that carboxypeptidase H is released from the pancreatic beta-cell by an exocytotic process from the same intracellular compartment as insulin. The release of carboxypeptidase H by a constitutive process was at best equivalent to 0.4%/h, or less than 2% of the maximal rate of release via the regulated pathway. It is concluded that carboxypeptidase H can be used as a sensitive index of beta-cell secretion and an alternative marker to the insulin-related peptides.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Molecular heterogeneity and cellular localization of carboxypeptidase H in the islets of Langerhans.

The intracellular distribution and molecular heterogeneity of carboxypeptidase H was studied in rat insulinoma tissue and isolated islets of Langerhans by a combination of immunohistochemical, ultrastructural, subcellular fractionation, and immunoblotting analyses. Immunofluorescence microscopy of islets demonstrated the presence of carboxypeptidase H in both insulin-containing B cells and glucagon-containing A cells. Quantitative ultrastructural analyses of islet B cells indicated that the enzyme was concentrated in mature insulin secretory granules, clathrin-coated condensing granules, and to a lesser extent the Golgi apparatus. Carboxypeptidase H activity was localized principally to secretory granule subfractions of insulinoma tissue, where it was present for the major part (70%) as a form which is readily solubilizable at pH values prevailing in the granule interior (5.5). This species migrated as a diffuse band of 53-57 kilodaltons (kDa) on immunoblot analysis using antisera raised against the purified native enzyme. In contrast, the insoluble form which was associated with the granule membrane at pH 5.5, migrated as a relatively compact band of 55-57 kDa. Carboxypeptidase H activity was also present in subcellular fractions which contained Golgi membranes together with elements of the endoplasmic reticulum, and in a low density secretory granule fraction which may represent immature granules. The enzyme in these compartments, like the granule membrane species, migrated as a compact 55-57 kDa band on immunoblots. Two-dimensional electrophoretic immunoblot analysis of secretory granules suggested that both membrane and soluble forms of the enzyme were glycoproteins and that the terminal glycosylation was similar in both instances. Antiserum raised against the deduced C-terminal 11 amino acids of the cloned carboxypeptidase H sequence recognized the 55-57 kDa membrane component in granules but did not react with the 53-57 kDa soluble species. A major difference between the soluble and membrane forms therefore appears to be a structural modification or proteolytic removal of the C-terminal domain in the trans-Golgi or early secretory granule compartment. The concept that proteolysis is involved is further supported by the observation that the relative proportion of the high and low mol wt forms of the enzyme in different subcellular fractions correlated with that of proinsulin and insulin, respectively. The membrane association of the 55-57 kDa form of carboxypeptidase H is disrupted at pH values of 9 and is dependent on ionic strength. This further suggests that the C-terminus of the protein may have an important role in the sorting or concentration of the enzyme in vesicular elements of the regulated pathway of secretion.

Animals↗
PubMed1990

Arginine and lysine product inhibition of bovine adrenomedullary carboxypeptidase H, a prohormone processing enzyme.

Carboxypeptidase H (CPH) is one of the later enzymes in the cascade of proteolytic steps required for the posttranslational processing of peptide hormone precursors, including processing of proenkephalin. In this study, CPH activity in the soluble and membrane fractions of enkephalin-containing bovine chromaffin granules was competitively inhibited by its products arginine and lysine. Ki values for arginine and lysine were 4.6 +/- 1.3 and 7.6 +/- 1.9 mM, respectively, indicating that arginine was a more effective inhibitor than lysine. Other amino acids (at 10 mM) had no effect. The in vivo intragranular concentrations of lysine and arginine are similar to the measured Ki values, indicating that product inhibition of CPH by basic amino acids may occur in vivo.

Adrenal Medulla↗
PubMed1994

Identification of carboxypeptidase H transcripts by antisense RNA.

Carboxypeptidase H is a metallopeptidase involved in the processing of neuropeptide precursors and prohormones. In this study, northern analysis with antisense CPH RNA as probe detected the presence of three CPH mRNA transcripts of 2.2, 2.6, and 5.9 kb that are expressed in a tissue-specific manner in several species. Specifically, mouse brain and the mouse AtT-20 corticotroph cell line possess all three transcripts, but mouse pituitary and adrenal possess the 2.2 kb CPH mRNA as the main form. In bovine, the 5.9 kb CPH mRNA was detected in adrenal medulla, but not in pituitary; whereas, both of these bovine tissues possess the 2.2 kb form. Also, in rat brain, adrenal, heart, and pituitary, the 5.9 m,kb form was detected mainly in brain, and the 2.6 kb form was most abundant in pituitary; all rat tissues examined possessed the 2.2 kb form. Additional evidence for the existence of the largest CPH transcript was provided from a bovine adrenal medulla cDNA library by in vitro transcription of total library phage DNA, followed by analysis of resultant RNAs by Northern blot. It will be important in future studies to define the structural similarities and differences among these multiple CPH mRNA transcripts that are expressed in a tissue-specific manner.

Adrenal Medulla↗
PubMed1990

Two soluble forms of bovine carboxypeptidase H have different NH2-terminal sequences.

Carboxypeptidase H is an important enzyme in the biosynthesis of many peptide hormones. Development of a rapid isolation procedure led to the purification of two soluble forms from acidic extracts of bovine pituitary glands. These two forms differed in apparent molecular size (56 and 53 kDa). Both forms were found in the anterior lobe while only the 53-kDa form was found in posterior lobe. Digestion with N-glycosidase F demonstrated that these two forms are not due to alternative glycosylation of a common polypeptide core. Both forms bind antibodies raised against a COOH-terminal peptide of the full-length protein showing that the difference between them is not due to proteolysis at the COOH terminus. These results also argue against the idea that proteolysis of COOH-terminal domains converts the membrane-associated form of this protein into a soluble form. NH2-terminal sequence analysis demonstrated different NH2 termini. The NH2-terminal sequence of the 56-kDa form begins at the site predicted for signal peptide cleavage. Ion-exchange chromatography resolved the 56-kDa form from the 53-kDa form. The two forms were catalytically active with very similar properties. These results show that bovine carboxypeptidase H can be posttranslationally processed at alternative sites and provide evidence against the idea of a prosequence that must be removed before enzyme activity can be expressed.

Amino Acid Sequence↗
PubMed1987

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.

Adenoma, Islet Cell↗
PubMed1989

Catalysis of slow C-terminal processing reactions by carboxypeptidase H.

A hypothesis was examined that carboxypeptidase H (CpAse H), which is known to catalyse the release of lysine and arginine from the C-terminus of peptides, can also release histidine, tyrosine, and phenylalanine. Synthetic peptides terminating in -His-Lys or -Tyr-Lys were used as model substrates for the enzyme and amino acid analysis was employed to detect release of the terminal amino acids. With N-acetyl-beta-Ala-Asn-Ala-His-Lys and N-acetyl-beta-Ala-Asn-Ala-Tyr-Lys, which correspond to intermediates in the processing of porcine and human beta-endorphin, lysine was removed rapidly and quantitatively but no release of histidine or tyrosine could be detected. To allow more sensitive analysis, radiolabelled substrates were employed and the amounts of the products formed on incubation with CpAse H were determined after separation by ion-exchange chromatography. With 125I-D-Tyr-Ala-His-Lys-Lys as substrate at pH 5.7, very small amounts of D-Tyr-Ala were released; the main product was D-Tyr-Ala-His. At pH 5.0 the release of histidine from 125I-D-Tyr-Ala-His took place 6,000 times more slowly than the release of lysine from 125I-D-Tyr-Ala-Lys. When the tripeptides were incubated at pH 5 with porcine pituitary secretory granules, the lysine was released rapidly but no release of histidine could be detected. The results demonstrate that CpAse H catalyses the release of C-terminal histidine with great difficulty.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Distribution of carboxypeptidase H messenger RNA in rat brain using in situ hybridization histochemistry: implications for neuropeptide biosynthesis.

Carboxypeptidase H is an exopeptidase which is highly specific for C-terminal basic amino acids and thought to play a role in neuropeptide biosynthesis. The distribution of carboxypeptidase H mRNA was examined in adult rat brain using in situ hybridization histochemistry. Enzyme transcripts were detected in all major brain areas. Very high levels of carboxypeptidase H mRNA were found in the hippocampus associated with pyramidal cells. Other brain regions showed varied levels of labelling, ranging from high levels in the magnocellular cells of hypothalamic nuclei to very low levels in the striatum. The appearance of enzyme transcripts throughout brain supports a role for this enzyme in the biosynthesis of many neuropeptides. The expression of transcripts in certain ventricular ependymal cells identified them as a new potential peptidergic cell type. The variations in levels of expression of carboxypeptidase H mRNA may reflect differences in peptidergic activity in different neuronal systems.

Animals↗
PubMedDate not supplied

[Effect of testosterone on carboxypeptidase H activity in the murine hypothalamus and hypophysis].

Effects of single intraperitoneal administration of testosterone propionate (3 or 30 mg/kg body weight) on activity of carboxypeptidase H in pituitary body and hypothalamus of female white mouse were studied. It was found that testosterone propionate treatment (3 and 30 mg/kg body weight) increased the carboxypeptidase H activity in pituitary through 0.5 hour and it decreased one in 24 hours after treatment. The carboxypeptidase H activity in hypothalamus was lower as compared with control animals in 24 hours after testosterone propionate treatment in the dose 3 mg/kg body weight. However, the carboxypeptidase H activity in hypothalamus was lower in 0.5 and 24 hours and it was higher in 4 h after testosterone propionate treatment in the dose 30 mg/kg body weight as compared with the control. These data suggest that testosterone affects the carboxypeptidase H activity by changing the level of enzyme gene expression.

Animals↗
PubMedDate not supplied

[Effect of glucocorticoids on activity of soluble and membrane-bound forms of carboxypeptidase H in vivo].

Inhibition of soluble and membrane-bound carboxypeptidase H in rat hypothalamus, striatum and pituitary gland by hydrocortisone and dexamethasone treatment in vivo has been established. Differences in the dynamics of change in the activity were determined by the action of hydrocortisone and dexamethasone. Differences of the dynamics of change in soluble and membrane-bound carboxypeptidase H activity were also observed. Possible causes of these differences are discussed.

Amino Acid Sequence↗
PubMed1988

Regulation of carboxypeptidase H by inhibitory and stimulatory mechanisms during neuropeptide precursor processing.

1. Carboxypeptidase H is one of several proteolytic processing enzymes required for conversion of large neuropeptide precursors into the small peptide neurotransmitters and hormones. 2. Because of the importance of posttranslational processing as a regulatory step for the production of active peptides, recent studies investigating control mechanisms for carboxypeptidase H (CPH) are reviewed. 3. Evidence is discussed which illustrates how CPH can be inhibited and activated. These findings suggest that a processing enzyme can play a role in the control of neuropeptide production. 4. It will be important in further studies to understand how the multiple processing enzymes--endopeptidase(s) and aminopeptidase, along with CPH--are coordinately regulated for the synthesis of active peptides.

Adrenal Medulla↗
PubMedDate not supplied

[Effect of ethanol on carboxypeptidase H in the rat brain].

It is established that under a single-dose introduction of ethanol activity of carboxypeptidase H increases. During chronic consumption of ethanol changes of enzyme activity was less than during a single-dose introduction of ethanol. Most considerable changes in carboxypeptidase H activity are observed in the pituitary gland, hypothalamus and striatum. Ethanol effect on the activity of membrane-bound and soluble forms of enzyme was distinguished. It is supposed that carboxypeptidase H is involved into the formation of response to ethanol.

Animals↗
PubMedDate not supplied

[Effect of stress factors on carboxypeptidase H activity in areas of the rat brain].

It is established that carboxypeptidase H activity increases as affected by various stress factors in the rat brain departments. The increase of the enzyme activity because of the emotional-pain stress is of continuous character. Possible role of carboxypeptidase H in the development of the stress response is discussed.

Animals↗