Rat preprocarboxypeptidase H. Cloning, characterization, and sequence of the cDNA and regulation of the mRNA by corticotropin-releasing factor

Carboxypeptidase H is a putative post-translational processing enzyme which removes basic amino acid residues from intermediates during protein hormone biosynthesis. A 2.2-kilobase pair cDNA was shown to contain the complete amino acid sequence of rat carboxypeptidase H. The deduced amino acid sequence revealed that the enzyme was synthesized as preprocarboxypeptidase H, a precursor form of 476 amino acid residues. Preprocarboxypeptidase H contained a putative hydrophobic signal peptide and a short propeptide which contained 5 adjacent Arg residues at its C terminus. Northern blot analysis identified a single carboxypeptidase H mRNA of approximately 2.3 kilobases in brain, pituitary, and heart, as well as in mouse AtT20 cells. No carboxypeptidase H mRNA was detected in rat liver, spleen, kidney, lung, and mammary gland. Sequence analysis of cDNAs obtained from different rat tissues suggested that a single mRNA encodes an identical carboxypeptidase in several tissues. Treatment of AtT20 cells with dexamethasone decreased the levels of both carboxypeptidase H and preproopiomelanocortin (POMC) mRNAs by approximately 30%. Exposure of the dexamethasone-treated cells to corticotropin-releasing factor effected a 2- to 3-fold increase in the carboxypeptidase H and POMC mRNA levels relative to those of dexamethasone-treated cells exposed to control medium. This suggests that the mRNA levels of POMC and one of its putative post-translational processing enzymes, carboxypeptidase H, are co-regulated by corticotropin-releasing factor and steroid hormones.     

Effect of chloroform and ether narcosis on the activity of basic carboxypeptidases in the hypothalamic-pituitary-adrenal axis in rats

It is discovered that chloroform narcosis does not influence on carboxypeptidase H and phenylmethylsulfonyl fluoride-inhibited carboxypeptidase activity in the rats hypothalamic-pituitary-adrenal axis. Ether narcosis provokes an increase of PMSF-inhibited carboxypeptidase activity in the pituitary body approximately in 8 times and carboxypeptidase H activity in hypothalamus by 29 percents in comparison with the intact animals. It is supposed that at research neuropeptides and their metabolism enzymes and especially the answer to a stress chloroform narcosis would be the better anaesthesia method than ether narcosis.     

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.     

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.     

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.     

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.     

Distribution of Activities of Alkaline Carboxypeptidases in Tissues of Laboratory Animals of Different Species

The study deals with distribution of activities of enzymes of neuropeptide metabolism, carboxypeptidase H and phenylmethylsulfonylfluoride-inhibited carboxypeptidase (PMSF-inhibited carboxypeptidase), in tissues and organs of male cats, and breedless white rats and mice. Distribution of the carboxypeptidase H activity was very similar in different animal species, although the level of the activity differed. Essential species-specific differences in distribution of the PMSF-inhibited carboxypeptidase activity are revealed, likely due to peculiarities of metabolism of biologically active peptides and catabolism of proteins in these animal species, as well as to differences in the ratio of different isoenzymes of their PMSF-inhibited carboxypeptidase.     

The effect of prenatal stress on the carboxypeptidase H activity in the hypothalamo-pituitary-adrenal-gonadal system of the rat

Effects of dams stress on the carboxypeptidase H: the neuropeptide exchange enzyme in the hypothalamo-pituitary-adrenall-gonadal system, was studied in the litter of different age (0, 14, 28, 45, and 120 day after birth) was studied. The sex differences in dynamic of enzyme activity in intact animals are substantiated. The effect of prenatal stress on carboxypeptidase H activity dependes on age and sex of animals. Prenatal stress is altering during the age dynamics of enzyme activity. This in the pituitary gland and hypothalamus of prenatal stressed female rats was a control for the male rats. In the adrenal and gonadal gland of prenatal stressed males, this was a control for the female rats female. The role of carboxypeptidase H in pubescence and mechanisms of effect of prenatal stress on sex system functional are discussed.     

Carboxypeptidase H-like immunoreactivity in the striatum of cats and monkeys

The enzyme carboxypeptidase H was detected by immunohistochemistry in the striatum of adult cats and monkeys. Specific labelling was observed in the neuropil as well as in both medium-sized and large neuronal cell bodies. The distribution of neurons and neuropil expressing immunoreactivity to carboxypeptidase H was examined in relation to the pattern of immunoreactivity to the neuropeptides enkephalin and substance P. Carboxypeptidase H-like immunoreactivity was found both in zones rich and poor in immunostaining for the two peptides, but was usually denser in those striatal areas in which substance P-positive cell bodies are clustered (striosomes). The results further suggest a role for carboxypeptidase H in the metabolism of multiple neuropeptides in vivo.     

Activity of the basic carboxypeptidases in female rat tissues at different stages of estrus cycle

It is revealed, that the activity of neuropeptide metabolism enzymes (carboxypeptidase H, phenylmethylsulfonilfluorid-inhibited carboxypeptidase) in the female rat tissues depends upon the stage of estrus cycle. The carboxypeptidase H activity in the pituitary gland is the highest in proestrus; it is almost 3 times higher in comparison with diestrus; it is a little bit higher in striatum on the stage of estrus, than in diestrus and proestrus, in adrenals on the stage of proestrus and estrus it is a little bit lower, than in diestrus; in the ovaries on the stage of proestrus it is much higher, than in estrus and diestrus. The activity of PMSF-inhibited carboxypeptidase in ovaries on the stage of proestrus and diestrus is 1.7-1.8 times higher, than at the stage of diestrus. The activity of carboxypeptidase M in adrenal tissue at the stage of proestrus is 35-40% of that at the stage of diestrus and estrus. The activity of carboxypeptidase M in the ovaries at the stage of diestrus is 45-50% of that at the stage of diestrus and estrus. The role of the investigated enzymes in cyclic changes of a level of biologically active peptides and in regulation of estrus cycle is discussed.     

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.     

Effect of chronic ethanol consumption on basic carboxypeptidase activity in regions of the rat brain

It is discovered that chronic consumption of ethanol induced decrease of carboxypeptidase H activity in striatum by 27%; increase of carboxypeptidase M activity in hippocampus by 67% and decrease in cerebral hemispheres by 34%; phenylmethylsulfonyl fluoride-inhibited carboxypeptidase activity increase in hypothalamus by 141%, in striatum by 60% and in optic and lamina quadrigemina by 34%. The role of basic carboxypeptidases in mechanisms of ethanol influence on the peptidergic systems are discussed.     

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.     

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).     

Effect of selank on the main carboxypeptidases in the rat nervous tissue

Effect of single selank administration on activity of carboxypeptidase H and of phenylmethylsulfonylfluoride-inhibited carboxypeptidase - enzymes detaching arginine and lysine from C-terminus of molecules-precursors of biologically active peptides was studied. The preparation has been shown to cause long, preserved for 24 h changes of activities of these carboxypeptidases. It is suggested that the change in activity of the studied enzymes can be one of mechanisms of regulation of level of neuropeptides at action of selank.     

Effect of sodium hydroxybutyrate on carboxypeptidase H activity and on angiotensin-converting enzyme in different regions of the rat brain

It is revealed, that analogue of the gamma-aminobutyric acid--sodium hydroxybutyrate causes decrease of activities carboxypeptidase H and angiotensin converting enzyme in pituitary gland, hypothalamus and striatum. The most expressed changes of enzyme activities were observed in pituitary gland and hypothalamus. The activity of carboxypeptidase H changes more essentially, than one of angiotensin converting enzyme. The assumption one of mechanisms of influence the hydroxybutyric acid and, possible, the gamma-aminobutyric acid, on neuropeptides level is changes of activity of enzyme of biologically active peptides exchange is expressed.     

Stabilization of the structure and activity of yeast carboxypeptidase Y by its high-mannose oligosaccharide chains

Sodium dodecyl sulfate was shown to promote both the inactivation and proteolytic degradation of the yeast glycoprotein, carboxypeptidase Y, with the former effect occurring six times faster than the latter. Although the proteolysis, as judged by polyacrylamide gel electrophoresis, was inhibited by pepstatin, which implicates the presence of proteinase A, the possibility of autodigestion could not be ruled out. A contributing role of the enzyme's carbohydrate moiety to these two processes was revealed by treating carboxypeptidase Y with endo-β-N-acetylglucosaminidase H. This treatment removes all four of the enzyme's Oligosaccharide chains in sodium dodecyl sulfate and as a consequence increases the rate of inactivation of the resulting carboxypeptidase Y by twofold and its proteolytic degradation by threefold relative to that of untreated enzyme. It thus appears that carboxypeptidase Y is a glycoprotein whose structural integrity and functional activity are influenced by its associated carbohydrate component.     

The effect of ethanol consumption by dams on the offspring locomotion in the open field test and carboxypeptidase activities in the rat brain and adrenal medulla

Consumption of dams ethanol increased the posterity locomotion activity in open field test. The increase in female rats was higher then in male ones. Differences in the carboxypeptidase H and PMSF-inhibited carboxypeptidase activities between the brain regions and adrenal medulla of prenatally exposed to ethanol and intact rats were found. The changing of enzyme activities in female rats was higher then in male ones. It is possible that dams ethanol consumption induced profound changes in locomotion mediated, at least partially, by changes in the rate of proteolytic processing of neuropeptide precursors.     

Effect of testosterone and progesterone on carboxypeptidase H activity during stress in mice

It was found that testosterone propionate (3 mg/kg of body weight) and progesterone (1 mg/kg of body weight) partially prevent an augmentation of the carboxypeptidase H activity in the mouse pituitary gland under stress caused by a single intraperitoneal administration of olive oil. In testicles, testosterone prevented augmentation of the enzyme activity within 0.5 hours but increased it through in 4 and 24 hours after treatment. Progesterone in testicles augmented the enzyme activity within 0.5 hours, but not in 4 and 24 hours. Sex steroids were not affected by stressinduced carboxypeptidase H activity in hypothalamus and adrenal medulla. It was concluded that carboxypeptidase H did not take part in integration of hypothalamus-pituitary-adrenal and hypothalamus-pituitary-gonadal axis under the stress.