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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↗

PubMed1996

Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma.

Coagulation is initiated by the binding of factor VIIa to tissue factor, with resultant limited factor IX and X activation and thrombin production. Owing to the feedback inhibition of the factor VIIa/tissue factor complex by tissue factor pathway inhibitor (TFPI), additional factor X activation and thrombin generation must proceed through a pathway involving factors VIII, IX, and XI. Experiments designed to elucidate the requirement for amplified factor Xa and thrombin generation in normal hemostasis show that the resistance of plasma clots to tissue plasminogen activator (tPA)- and urokinase-induced fibrinolysis is related to the extent of thrombin generation. Inhibition of fibrinolysis is mediated in part by plasma carboxypeptidase-U ([CPU] carboxypeptidase-R, procarboxypeptidase-B, thrombin-activatable fibrinolysis inhibitor), a proenzyme that is proteolytically activated by thrombin in a process enhanced dramatically by the cofactor thrombomodulin. A clot induced in factor IX-deficient plasma with limited amounts of tissue factor in the presence of urokinase (100 U/mL) lyses prematurely, and this defect is corrected by supplementation of the deficient plasma with factor IX (5 micrograms/mL) or thrombomodulin (20 ng/mL). These additions enhance the rate and extent of CPU activation: in the case of factor IX, presumably by permitting amplified generation of factor Xa and thrombin, and in the case of thrombomodulin, presumably by increasing the degree of CPU activation produced by the low levels of thrombin generated in the absence of factor IX. Pretreatment of the factor IX-deficient plasma with specific anti-CPU antibodies prevents the increased resistance to fibrinolysis produced by addition of factor IX and thrombomodulin. Likewise, when coagulation is induced by thrombin (2 U/mL) in the presence of tPA (60 U/mL), clots formed from plasmas deficient in factors VIII, IX, X, or XI lyse prematurely unless the missing factor is replaced or thrombomodulin (20 ng/mL) is added.

Animals↗

PubMedDate not supplied

[Basic carboxypeptidase activity in rats of both sexes].

The activity of carboxypeptidase (CP) H, the enzyme taking part in neuropeptide formation, and activity of recently described phenylmethylsulfonyl fluoride (PMSF)--inhibiting CP in males and females of white mongrel rats were studied. Minor differences between the CPH activities in brain regions were found in hippocampus. PMSF-inhibited activity of carboxypeptidase was significantly higher in females than in males in pituitary gland, adrenal gland, olfactory bulbus, optic and auditory bills, cerebellum, hippocampus, striatum, cerebral hemispheres and spleen. The CPH activity was 5-fold higher in ovaries than in testicles. PMSF-inhibited CP activity in testicles was 3.7-fold lower than in ovaries. Possible participation of basic CP in determination of sexual differences of some neuropeptide level and protein catabolism is studied.

Adrenal Glands↗

PubMed1978

Studies on enzyme models and on the enzyme carboxypeptidase A.

The enzyme carboxypeptidase A has been extensively studied and is thus a good candiate for chemical models and imitation. A model system was constructec which combined two of the three catalytic functional groups of the enzyme together with the appropriate substrate group, and cooperative effective catalysis was demonstrated. However, the mechanism which the model system used in enzyme-like conditions was unexpected, and this led to a study of the enzyme itself. Carboxypeptidase A was studied by examination of oxygen-18 exchange reactions and the ability of the enzyme to substitute only lytic agents, such as methanol, forthe water which is normally used. The result of these studies is proposed mechanism of action of this enzyme which accommodates the large amount of information already available, and which indicates that this enzyme uses a mechanism similar to that found in the model system. Approaches have also been made to an artificial enzyme which could hydrolyse amides. Cyclodextrin has been functionalized with two imidazole groups placed on opposite sides of the cavity. This material catalyses the hydrolysis of an amide, which binds to the cyclodextrin cavity and then is hydrolysed with the assistance of one imidazole in its basic form and the other one in its protonated form, acting together in a way reminiscent of the cooperation of some of the functional groups of hydrolytic enzymes.

Binding Sites↗

PubMed1993

Control of Saccharomyces cerevisiae carboxypeptidase S (CPS1) gene expression under nutrient limitation.

Expression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients. Enzyme production is sensitive to nitrogen catabolite repression; i.e. the presence of ammonium ions maintains expression of the gene at a low level. Transfer of ammonium-glucose pre-grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells. Derepression of the gene by nitrogen limitation is cycloheximide-insensitive. Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions. Non-metabolizable sugar analogs (2-deoxyglucose, 6-methyl-glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy-dependent. Production of carboxypeptidase yscS also increases several-fold when ammonium-pregrown cells are transferred to media containing glucose and a non-readily metabolizable nitrogen source such as proline, leucine, valine or leucyl-glycine. Analysis of CPS1 expression in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23 degrees C) and in heat-shocked cells (38 degrees C) shows that steady-state levels of CPS1 mRNA are not controlled by a low cAMP level-signalling pathway.

Adenylyl Cyclases↗

PubMed1998

Reversed-flow affinity elution applied to the purification of carboxypeptidase Y.

In the present study we describe a novel method for obtaining highly pure carboxypeptidase Y, or derivatives thereof, in a single-step purification procedure. The method is based on affinity chromatography and the results demonstrate that an efficient method is obtained only when the affinity gel is fully saturated with enzyme. Thus, pilot experiments are required to determine the binding capacity of the resin with respect to a given enzyme. To avoid this additional experimental effort, we have developed a method utilizing reversed-flow affinity elution. The method has been successfully employed to purify hundreds of carboxypeptidase Y mutant enzymes.

Carboxypeptidases↗

PubMed1984

Nutritional studies of a carboxypeptidase inhibitor from potato tubers.

Carboxypeptidase inhibitor from potato tubers was fed to newly hatched chicks at a level equal to that present in diet containing 50% raw potato, which caused severe growth depression and 20% mortality. At this level the effects of the inhibitor on growth was small but the following effects were noted: (a) increased fecal protein (the increase mainly consisting of low molecular weight proteins); (b) poorer feed efficiency; and (c) a significant decrease in pancreatic digestive proenzyme levels, although no hypertrophy was noted. In addition, the inhibitor was not digested readily in the intestinal tract and it increased in concentration in intestinal contents as it progressed down the tract. Potato Inhibitor II, a potent trypsin inhibitor, when fed to chicks, also at the level found in a 50% raw potato diet, was severely growth depressing. It significantly increased fecal protein and caused pancreatic hypertrophy. The trypsin inhibitor may be a major growth depressing agent in raw potatoes whereas the carboxypeptidase inhibitor probably contributes little to the growth depression.

Animals↗

PubMed1994

Pea (Pisum sativum L.) seed isolectins 1 and 2 and pea root lectin result from carboxypeptidase-like processing of a single gene product.

The complete amino acid sequences of the alpha-subunits of pea (Pisum sativum L.) seed and root lectin, the C-terminal amino acids of the beta-subunits of pea seed lectin, and most of the sequence of the beta-subunit of pea root lectin were determined. In contrast to earlier reports it was shown that the beta-subunits of both seed isolectins end at Asn-181. The alpha 1 subunits end at Gln-241 (major fraction) or Lys-240 (minor fraction), whereas the alpha 2 subunits end at Ser-239, Ser-238, Ser-237 or Thr-236. psl cDNA clones from seed are identical to psl cDNA clones from root, and root PSL is identical to seed PSL2, ending at Ser-239, Ser-238 or Ser-237. It seems that the presence of Lys-240 is the sole determinant of the charge difference between pea isolectins. PSL1 can be converted into PSL2 by carboxypeptidase P from Penicillium janthinellum. These results confirm that PSL from roots is encoded by the same gene as PSL from seeds. Thus, it seems that, next to an Asn-X specific protease responsible for the processing at positions 181/182 and 187/188, a carboxypeptidase is responsible for the conversion of PSL1 and PSL2, which is probably the final processing product.

Amino Acid Sequence↗

PubMed1986

LD-carboxypeptidase activity in Escherichia coli. II. Isolation, purification and characterization of the enzyme from E. coli K 12.

A LD-carboxypeptidase from Escherichia coli K 12 was isolated by Tris-EDTA treatment and purified to electrophoretic homogeneity by DEAE-cellulose chromatography. The enzyme has a molecular weight of approximately 12,000 as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis and by Sephadex G-100 gel filtration. The studies of the substrate specificity of the enzyme revealed that UDP-MurNAc-tetrapeptide is a superior substrate, with a Km value of 1 X 10(-4) mol/l. The activity of the LD-carboxypeptidase was inhibited by D-amino acids and the beta-lactam antibiotic nocardicin A. Ki values of 0.3 and 43 mmol/l were determined for nocardicin A and D-homoserine, respectively. The properties of the purified enzyme correspond to activity I in ether treated cells.

Amino Acids↗

PubMed1991

Acid carboxypeptidase deficiency in galactosialidosis.

Carboxypeptidase activity with an optimal pH at 5.7 was found to be deficient in cultured lymphoblastoid cells and skin fibroblasts from 16 galactosialidosis patients of Japanese origin. The amounts of residual enzyme activities did not correlate with clinical phenotypes (early infantile and juvenile/adult). Four parents of the patients from different families showed enzyme activities at an intermediate level between the patients and normal controls. It was concluded that this enzyme deficiency is closely connected to the genetic defect of "protective protein." Further characterization with various protease inhibitors indicated that the enzyme deficient in galactosialidosis cells is a serine carboxypeptidase with histidine and cysteine residues at or near the active site.

Adolescent↗

PubMed1975

Kininase and anti-inflammatory activities of acid carboxypeptidase from Penicillium janthinellum.

The acid carboxypeptidase from Penicillium janthinellum catalyzed the rapid release of arginine, and the slow release of phenylalanine, proline, serine and glycine from the carboxy-terminal of bradykinin at pH 4.15 to 4.8. Anti-inflammatory activity of the acid carboxypeptidase seems to suggest that the enzyme hydrolyzed bradykinin in vivo.

Amino Acids↗

PubMed1988

Chemical modifications of a cysteinyl residue introduced in the binding site of carboxypeptidase Y by site-directed mutagenesis.

It is demonstrated that site-directed mutagenesis successfully can be combined with chemical modification creating enzyme derivatives with altered properties. A methionyl residue located in the S1' binding site of carboxypeptidase Y was replaced by a cysteinyl residue and the mutant enzyme was isolated and modified with various alkylating and thioalkylating reagents. Treatment of the mutant carboxypeptidase Y with bulky reagents like phenacyl bromide and benzyl methanethiolsulfonate caused a drastic reduction in the activity towards substrates with bulky leaving groups in the P1' position, i.e. -OBzl, -Val-NH2 and amino acids (except -Gly-OH), while substrates with small groups in that position, i.e. -OMe and -NH2, were hydrolysed with increased rates. The presence of a positive charge, in addition to a bulky group, had a further adverse effect on the activity towards substrates with large leaving groups, whereas the activity towards those with small leaving groups remained unaffected by such a group. The derivatives obtained by modification of the mutant enzyme with benzyl methanethiolsulfonate and methyl methanethiolsulfonate were effective in deamidations of peptide amides and peptide synthesis reactions, respectively.

Amides↗

PubMed2003

Carboxypeptidase E in rat antropyloric mucosa: distribution in progenitor and mature endocrine cell types.

Processing of most gut hormones involves cleavage between dibasic amino acids followed by carboxypeptidase-catalyzed removal of the COOH-terminal basic residue, resulting in peptides with a COOH-terminal glycine. Such peptides may subsequently be converted to amidated peptides or can be directly secreted. It is believed that carboxypeptidase E (CPE) is involved in gut hormone processing but its presence in gut endocrine cells has never been studied. We have analyzed the distribution of CPE in the antropyloric mucosa of rat stomach and report that gastrin cells and progenitor gastrin-somatostatin (G/D) cells express CPE while mature somatostatin cells and the majority of serotonin cells fail to express CPE. These data indicate that immature G/D cells are able to process gastrin to glycine-extended forms and that CPE-mediated processing is not a characteristic of mature somatostatin and serotonin cells.

Animals↗

PubMed1992

Structural determination of the essential serine and glycosylation sites of carboxypeptidase P.

Through a series of kinetic studies involving the inactivation effects of diisopropylfluorophosphate, an affinity label that modifies the active site serine residue involved in the mechanism of action, it has been firmly established that carboxypeptidase P (CPP) requires a serine residue for catalytic activity. The essential kinetic parameters were determined to be 1.33 mM for the apparent dissociation constant with a limiting half-life of inactivation of 20.1 min. Structural elucidation of the primary amino acid sequence surrounding the essential serine, and comparing that with the reactive site of carboxypeptidase Y (CPY), revealed a significant degree of homology at the active site between these two enzymes. These regions, however, were quite divergent from other known serine proteases, leading to the speculation that these serine exopeptidases may comprise a unique family in the overall classification of serine proteases. It was established that CPY could be inactivated with either of the classic histidine affinity labels tosylphenylalanylchloromethyl ketone (TPCK) or carbobenzoxyphenylalanylchloromethyl ketone (ZPCK) with Ki's of 1.2 and 12.8 microM, respectively. This is in marked contrast to CPP, which was unaffected by saturating levels of the known histidine affinity labels, TPCK, tosyllysylchloromethyl ketone, or ZPCK. This point may be a significant element in differentiating specificity among these two serine proteases. Further investigation into the structural nature of CPP revealed that it is a glycoprotein with a single site of carbohydrate attachment. In addition, the carbohydrate moiety itself appears to contribute 1217 Da to the overall molecular weight and it is characterized as an asparagine linked high mannose type. This is significantly different from CPY with its four sites of carbohydrate attachment contributing approximately 17% to its molecular weight.

Amino Acid Sequence↗

PubMed1975

Human pancreatic carboxypeptidase B. I. Isolation, purification, and characterization of fraction II.

Human carboxypeptidase B fraction II has been purified from pancreatic juice by DEAE-'Sephadex' chromatography, isoelectric focusing, and 'Sephadex' G-100 gel filtration. The enzyme has been characterized by analytical polyacrylamide disc-gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis Km determination, molecular weight determination on 'Sephadex' G-100, zinc analysis, and inhibition by metal chelating agents. Human carboxypeptidase B fraction II appeared homogeneous in analytical polyacrylamide disc-gel electrophoresis, but showed two components of 23,500 and 9,200 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Zinc analysis revealed 0.96 gram atoms of zinc per mole of enzyme, and a Km of 65 +/- 3 muM was determined for hydrolysis of hippuryl-L-arginine.

Amino Acids↗

PubMed1976

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.

Animals↗

PubMed1977

Studies on the enzymatic hydrolysis of polyglutamyl folates by chicken liver folyl poly-gamma-glutamyl carboxypeptidase. II. Structural studies.

Further studies on the purified chicken hepatic folyl poly-gamma-glutamyl carboxypeptidase (peptidyl-L-glutamate hydrolase, EC 3.4.12.10) have elucidated some of the structural characteristics of the enzyme. Various analytical studies described reveal 424 amino acid residues in the isolated native enzyme with molecular weight of around 57 900. beta-Mercaptoethanol (14.3 mM) activated the enzyme 2.2-fold and induced reductive cleavage of an interchain disulfide linkage resulting in the splitting of the native enzyme into two active polypeptides (molecular weights 43 000 and 18 000). The constituent polypeptides have identical NH2-terminal residues (valine) and exhibit a high degree of sequence homology as revealed by finger print analyses of their tryptic digests. The 10-fold greater sensitivity of the reductively cleaved enzyme to p-chloromercuribenzoate would imply that active site related sulfhydryl groups are not readily accessible in the native enzyme. Ionic strength effects in the presence of Mn2+ and Na+ and the presence of low urea concentration (0.55 M) result in a further up to 5-fold stimulation of reductively cleaved native enzyme. Citrate inhibited and phosphate induced autolytic degradation of the enzyme. The physiological role of gamma-glutamyl carboxypeptidase has been discussed.

Amino Acids↗

PubMed1981

Isolation, purification and some chemical properties of an acid carboxypeptidase from Aspergillus niger var. Macrosporus.

Acid carboxypeptidase (peptidyl-L-amino-acid hydrolase, EC 3.4.16.1) was purified to a homogeneous state from the water extracts of Koji cultures of Aspergillus niger var. macrosporus. The molecular weight of the enzyme was determined to e 136 000 by sedimentation equilibrium method. The denatured specimen of the enzyme exhibited a molecular weight of 60 000 in the sedimentation equilibrium in 6 M guanidinium chloride, suggesting that the native enzyme is composed of two identical subunits. However, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the enzyme showed an anomalous Ferguson plot, which may account for the inconsistent values of apparent molecular weights obtained by this method. The acid carboxypeptidase was found to be an acidic glycoprotein (pI, 4.1), composed of 955 amino acid, 140 mannose, 14 galactose and 30 glucosamine residues/molecule.

Amino Acids↗