PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “CARBOXYPEPTIDASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 631 records · Page 35Linked to original sources
PubMed1988

pK values for active site residues of carboxypeptidase A.

The phenolic group of active site residue Tyr-248 in carboxypeptidase A has a pKa value of 10.06, as determined from the pH dependence of its rate of nitration by tetranitromethane. The decrease in enzyme activity (kcat/Km) in alkaline solution, characterized by a pKa value of approximately 9.0 (for cobalt carboxypeptidase A), is associated with the protonation state of an imidazole ligand of the active-site metal ion, as indicated by a selective pH dependence of the 1H NMR spectrum of the enzyme. Inhibition of the cobalt-substituted enzyme by 2-(1-carboxy-2-phenylethyl)phenol and its 4,6-dichloro- and 4-phenylazo-derivatives confirms that the decrease in enzyme activity (kcat/Km) in acidic solution, characterized by a pKa value of 5.8, is due to the protonation state of a water molecule bound to the active-site metal ion in the absence of substrate. Changes in the coordination number of the active-site metal ion are seen in its visible absorption spectrum as a consequence of binding of the phenolic inhibitors. Conventional concepts regarding the mechanisms of the enzyme are brought into question.

Binding Sites↗
PubMed1986

Colorimetric determination of carboxypeptidase A activity in serum.

This simple, reproducible colorimetric method for determining the activity of carboxypeptidase A (EC 3.4.17.1) is based on measuring the absorbance at 505 nm of a quinoneimine dye produced from the action of this enzyme on the new substrate p-hydroxybenzoyl-glycyl-L-phenylalanine. The enzyme acts on the substrate to produce p-hydroxybenzoyl-glycine and L-phenylalanine. The former is then hydrolyzed by hippuricase (EC 3.5.1.14) to produce p-hydroxybenzoic acid and glycine. Finally, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by sodium periodate forms a quinoneimine dye. The Km for the reaction with this substrate is 3.6 mmol/L; the optimum pH is 7.8. Our within-run and between-run CVs are 4.3% and 6.6%, respectively. The activity of carboxypeptidase A in serum correlates well with that of lipase (r = 0.96) and immunoreactive elastase-1 (r = 0.76).

Amidohydrolases↗
PubMedDate not supplied

Use of circular dichroism to study the interaction of carboxypeptidase A with substrates and inhibitors.

The phenylalanyl circular dichroism (CD) bands of peptides were used to assay peptidase activity of carboxypeptidase A (EC.3.4.12.2.). Hippuryl-L-phenylalanine has a sharp, negative CD band at 254 nm whilst L-phenylalanine (the optically active product) has positive CD. Thus the hydrolysis of this substrate as well as the inhibition effect of dipeptides, may be measured from the CD change at 254 nm. The addition of the dipeptide GLy-Tyr to carboxypeptidase A makes the CD spectrum more positive in the region from 270-295 nm. This alteration can result from the tyrosyl and tryptophanyl CD bands of the protein as well as from the tyrosyl CD band of the inhibitor.

Carboxypeptidases↗
PubMed1985

Assay of carboxypeptidase N activity in serum by liquid-chromatographic determination of hippuric acid.

We describe conditions for determining carboxypeptidase N (EC 3.4.17.3) activity by liquid chromatography. Serum (10 microL) is mixed with the artificial substrates hippuryl-L-arginine (30 mmol/L) and hippuryl-L-lysine (100 mmol/L) in 50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution at pH 8.2 and 7.8, respectively. The hippuric acid product is separated from the substrate in less than 2 min by reversed-phase "high-performance" liquid chromatography and measured spectrophotometrically. o-Methyl hippuric acid is used as internal standard. By this method, optimized for activity and sensitivity of detection, carboxypeptidase N activities are 60-fold greater than those by another procedure (J Chromatogr 266:173-177, 1983). The mean value for 80 normal control subjects was 74.8 (SD 10.3) nmol of hippuric acid released per milliliter of serum per minute for hippuryl-L-arginine substrate, 378 (SD 55) for hippuryl-L-lysine substrate. The sensitivity and precision of the method make it suitable both for routine clinical determinations and as a reference procedure.

Adolescent↗
PubMed1985

2.8-A Structure of penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase from Streptomyces R61 and complexes with beta-lactams.

The crystallographic structure of the penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase from Streptomyces R61 has been solved to 2.8-A resolution. The 38,000-dalton serine peptidase has two regions of secondary structure, an alpha/beta cluster, and a region which contains five helical segments. The beta sheet is composed of five beta strands. The tertiary structure has no homology with the classic serine proteases or with the zinc carboxypeptidases. The binding at a common site of three types of beta-lactam (a penicillin, a cephalosporin, a monocyclic beta-lactam) and a desazacyclobutanone has been observed in Fourier difference maps. The binding site sequence is Val-Gly-Ser-Val-Thr-Lys. The beta-lactam ring lies near the enzyme's catalytic serine at position 37, and the C3 substituent of a cephalosporin falls near lysine 40.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Interaction of immobilized carboxypeptidase with a natural inhibitor from human blood plasma].

Carboxypeptidase N was bound covalently to CNBr-activated Sepharose 4B without decrease in the enzymatic activity. Interaction of the immobilized enzyme with native low molecular inhibitor from human blood plasma was accompanied by formation of the stable Co2+-dependent complex, which is partially dissociated in presence of 2 M NaCl. The data obtained suggest that the inhibitor studied possesses the specific effect on the carboxypeptidase N activity.

Carboxypeptidases↗
PubMed1982

Hydrolysis of esters by carboxypeptidase A requires a penta-coordinate metal ion.

The catalytic role of the metal ion in bovine carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) is investigated by application of cryoenzymologic and electron paramagnetic resonance methods with use of the Co2+-reconstituted enzyme. Incorporation of 17O into oxygen-donor ligands induces a substantial change in the spin-lattice relaxation probability of the paramagnetic ion. While a change in spin-lattice relaxation is observed for the free Co2+-enzyme in 17O-enriched water, no change is observed for the enzyme complexed to glycyl-L-tyrosine. These results are consistent with x-ray crystallographic studies showing that the metal-bound water molecule in the active site is displaced upon binding of the peptide inhibitor. A change in spin-lattice relaxation of the Co2+ ion in the mixed anhydride, acyl-enzyme intermediate formed with the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate is observed when 17O is enriched either into water or into the carbonyl oxygen position of the scissile bond of the substrate. Since the protein supplies three amino acid side chains as ligands to the metal ion, these results indicate that the metal ion is altered from a tetra-coordinate species in the free enzyme to a penta-coordinate species in the acyl-enzyme reaction intermediate. In addition, the results provide structural support for our assignment of ionization of a metal-bound water molecule in rate-limiting deacylation (Makinen, M. W., Kuo, L. C., Dymowski, J. J., and Jaffer, S. (1979) J. Biol. Chem. 254, 356-366) and affirm that the metal-hydroxide species is the nucleophile responsible for the breakdown of the mixed anhydride reaction intermediate of carboxypeptidase A.

Animals↗
PubMed1983

Purification and characterization of enkephalin convertase, an enkephalin-synthesizing carboxypeptidase.

Enkephalin convertase, an enkephalin-synthesizing carboxypeptidase present in adrenal medulla chromaffin granules, has also been detected in brain and pituitary. To determine whether these three carboxypeptidase activities represent the same enzyme, we purified and characterized enkephalin convertase from adrenal medulla, whole brain, and whole pituitary. Enzyme from all three tissues co-purifies on DEAE-cellulose, gel filtration, concanavalin A, and L-arginine affinity columns, resulting in a 135,000-fold, 110,000-fold, and 2,800-fold purification for bovine adrenal medulla, brain, and pituitary, respectively. Purified enkephalin convertase appears homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showing a single band with an apparent molecular weight of 50,000 for enzyme isolated from all three tissues. Adrenal, brain, and pituitary enkephalin convertase are similarly inhibited by hexapeptide enkephalin precursors and active site-directed inhibitors. Both [Met]-and [Leu]enkephalin-Arg6 inhibit enkephalin convertase with Ki values between 50 and 80 microM, while [Met]-and [Leu]enkephalin-Lys6 are 3-fold less potent. Two active site-directed inhibitors, guanidinopropylsuccinic acid and guanidinoethylmercaptosuccinic acid, are potent inhibitors of all three enzymes with Ki values of 8-9 nM. A series of dansylated di-, tri-, and tetrapeptide substrates are hydrolyzed by enkephalin convertase with similar kinetic properties (Km, Vmax, and Kcat/Km) for the three enzymes. This evidence suggests that enkephalin convertase activity represents the same enzyme in adrenal medulla, brain, and pituitary. Enkephalin convertase may be involved in the production of other peptide neurotransmitters and hormones besides enkephalin.

Adrenal Glands↗
PubMed1984

Role of metal ions in goat carboxypeptidase A-catalysed hydrolysis of acyl peptides.

The carboxypeptidase A purified from goat pancreas has been found to have a molecular weight of 34,600 +/- 300. The enzyme is a zinc-protein and the molar ratio of zinc to enzyme protein is 1:1. Removal of zinc yields an inactive apocarboxypeptidase A. The loss of activity of the native enzyme and restoration of the activity of the apoenzyme run parallel with the zinc content of the protein, thus showing the essentiality of zinc for the enzymatic activity. The exact role of zinc in the enzyme catalysed hydrolysis of the acylpeptides has been investigated after preparing metallo proteins by substituting the zinc of carboxypeptidase A with Co2+, Mn2+, Ni2+, Fe2+, Cd2+, Hg2+, and Cu2+ and determining the kinetic parameters of such metalloproteins. These studies indicate that the metal ion is involved in both binding the substrate and polarising the peptide bond.

Animals↗
PubMed1983

Serum carboxypeptidase B levels during acute and mild Babesia divergens infections of adult cattle.

Serum carboxypeptidase B levels were measured in adult cows suffering from both acute and mild babesiosis caused by Babesia divergens. In severe infections serum carboxypeptidase B levels started to fall three days after infection and were significantly lower than both preinfection levels and those of mildly affected cows for a period of 10 days starting at the time of maximum parasitaemia. Normal levels were regained on the 23rd day after infection.

Animals↗
PubMed1983

Potentiation of the anaphylatoxins in vivo using an inhibitor of serum carboxypeptidase N (SCPN). I. Lethality and pathologic effects on pulmonary tissue.

Carboxypeptidase N (EC 3.4.12.7) (SCPN) is a plasma enzyme that efficiently inactivates the anaphylatoxins C3a and C4a and significantly reduces C5a spasmogenic activity by removing the C-terminal arginyl residue from each of these factors. The arginine analog DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (SCPN-INH) is a potent competitive inhibitor of SCPN with a Ki for this carboxypeptidase in serum of 2 x 10(-9) M. Therefore, we have used the SCPN inhibitor to potentiate biologic activity of the anaphylatoxins in vivo. Infusion via the carotid artery of about 40 mg of SCPN-INH into each of 8 adult guinea pigs inactivated the SCPN for at least 3 hours and caused no measurable toxic effects. When cobra venom factor (CVF) is infused into guinea pigs, it activates the alternative pathway of complement, thereby generating the anaphylatoxins C3a and C5a. Ordinarily, infusion of CVF is nonlethal, because the generated anaphylatoxins are rapidly converted to C3a des Arg and C5a des Arg by SCPN. However, CVF (200 micrograms) plus SCPN-INH delivered intravenously in 5 animals induced a lethal reaction in less than 5 minutes. The authors conclude that the lethal effect is due largely to the anaphylatoxins. Histologic sections of the lungs from treated animals show dramatic structural changes consistent with peripheral small airway constriction, bronchial constriction, and vasoconstriction of small muscular arteries. Also, cell aggregates are present in blood vessels. Other histologic changes include severe congestion, pulmonary edema, and an interstitial infiltrate of mononuclear cells. Large doses of chlorpheniramine prevent this lethal reaction. Lethality is apparently attributable to asphyxia and is dependent on the level of CVF administered: eg, 100 micrograms CVF was not lethal in 4 animals given SCPN inhibitor, although signs of respiratory distress were observed. On histologic examination of lungs from guinea pigs given CVF and SCPN-INH, the features are similar to those described when anaphylatoxins are instilled into guinea pig lungs. Intravenous application of purified C3a plus SCPN-INH also proved lethal in 3 of the 6 animals challenged. This is the first evidence that the C3a anaphylatoxin can elicit a lethal response.

Anaphylatoxins↗
PubMedDate not supplied

[Theoretical conformational analysis of a substrate component of tetrahedral intermediates and of acyl-enzyme of carboxypeptidase A].

Semi-empirical calculation of conformational properties of covalent complexes of carboxypeptidase A with ester and peptide substrates are presented. There is a favourable form of transient tetrahedral and covalent acyl-enzyme intermediates in the active site of carboxypeptidase A. The ester and peptide substrates bind in the active site of carbopeptidase A in the same mode. The transition from one stage of the reaction to the other is followed by a small conformational change. The atom coordinates of the substrate component of these complexes are presented.

Binding Sites↗
PubMed1995

Methotrexate-alpha-phenylalanine: optimization of methotrexate prodrug for activation by carboxypeptidase A-monoclonal antibody conjugate.

Methotrexate-alpha-phenylalanine (MTX-Phe), a second-generation prodrug in the MTX alpha-peptide series designed for activation to MTX by carboxypeptidase-mAb conjugates, was synthesized by reaction of the p-nitrophenyl ester of 4-amino-4-deoxy-10-methylpteroic acid with L-glutamyl-alpha-L-phenylalanine. Production of MTX from MTX-Phe, catalyzed by bovine pancreas carboxypeptidase A (CPA), was 250-fold faster than the corresponding reaction involving methotrexate-alpha-alanine, previously the best MTX peptide substrate for the enzyme. The amount of CPA required to make MTX-Phe equitoxic with MTX, when tested against UCLA-P3 human lung adenocarcinoma cells in vitro, was more than 10-fold lower than that required to achieve the same result with MTX-alpha-alanine. When the lung tumor cells were treated with CPA conjugated to KS1/4 (a mAb targeted to these cells) and excess conjugate removed by extensive washing, the ID50 for MTX-Phe improved from 2.2 x 10(-6) M (no enzyme present) to 6.3 x 10(-8) M; the latter value was comparable to that of the parent drug MTX (4.5 x 10(-8) M). [3H]MTX-Phe was synthesized and used to investigate the mechanism by which the prodrug exerts its cytotoxic effect in the presence and absence of CPA. The present results demonstrate that, for use in conjunction with CPA-mAb conjugates, the alpha-phenylalanine derivative is the optimal prodrug form of MTX (and probably other antifols that contain the glutamate moiety).

Antibodies, Monoclonal↗
PubMed1994

C-tail valine is a key residue for stabilization of complex between potato inhibitor and carboxypeptidase A.

Although the carboxypeptidase A-potato carboxypeptidase inhibitor (CPA-PCI) complex is a well known example of protein-protein interaction, little was known about the basis of its thermodynamic stability. Site-directed mutagenesis has been used to identify key residues in the PCI tail and estimate the contribution of their chemical groups to the binding to CPA. Two deletion mutants were created, one lacking the C-terminal residue of the tail (Gly39) and another one lacking the two C-terminal residues (Val38, Gly39). The last mutant had an inhibition constant for CPA 10(4)-fold higher than that of wild-type PCI, indicating that Val38 is a key residue. The interactions of Val38 with CPA residues contribute 5.4-5.7 kcal mol-1 to the overall stability of the CPA-PCI complex (11.9-12.1 kcal mol-1). A series of PCI point mutants at valine 38 were created, and their inhibition constant for CPA was measured. Two of these mutants with smaller side chains, V38G and V38A, allowed us to estimate that the contribution of the three side chain aliphatic groups of valine 38 to the overall stability of the complex is 3.4-4 kcal mol-1. Another two mutants with larger side chains, V38L and V38I, were constructed, the first being a significantly worse inhibitor than the wild type. These results suggest that only aliphatic groups in positions beta and gamma of residue 38 in PCI (but not those in delta) can establish van der Waals interactions with atoms of the active center of CPA and participate in binding. The energetic contribution of each methyl/methylene group in those positions can be estimated as 1-1.5 kcal mol-1. Our hypothesis is supported by computer simulation analysis.

Carboxypeptidases↗
PubMed1994

Regulation of carboxypeptidase E. Effect of Ca2+ on enzyme activity and stability.

Carboxypeptidase E (CPE), an enzyme that functions in the post-translational processing of bioactive peptides, is a member of the metallocarboxypeptidase gene family. A 12-residue region of CPE has 70% amino acid identity with the bacterial enzyme carboxypeptidase T (CPT); in CPT, this region has been identified previously as the Ca(2+)-binding region (Teplyakov, A., Polyakov, K., Obmolova, G., Strokopytov, B., Kuranova, I., Osterman, A., Grishin, N., Smulevitch, S., Zagnitko, O., Galperina, O., Matz, M., and Stepanov, V. (1992) Eur. J. Biochem. 208, 281-288). Using 45Ca2+ binding, we determined that CPE binds Ca2+. To investigate the potential function for the interaction of CPE with Ca2+, we investigated the effect of Ca2+ on aggregation, thermostability, and enzyme activity of CPE. CPE does not aggregate under a variety of Ca2+ concentrations at either pH 5.5 or 7.5, and with protein concentrations ranging from 10 to 100 micrograms/ml. Whereas Ca2+ generally stabilizes proteins to thermal denaturation, CPE was destabilized by Ca2+ and stabilized by low concentrations of EGTA. The Ca(2+)-induced destabilization of CPE was more pronounced at pH 8 than at lower pH values. At pH 8, CPE was unstable even at 37 degrees C, with approximately 40% loss of activity upon incubation for 30 min in the absence of added Ca2+ and 70% loss of activity upon incubation in the presence of 10 mM CaCl2. Enzyme activity was not influenced by added Ca2+, but was stimulated by micromolar concentrations of EGTA; kinetic analysis showed this stimulation to be due to a change in Vmax, and not Km. Taken together, these data suggest that Ca2+ plays a role in the regulation of CPE activity.

Amino Acid Sequence↗
PubMedDate not supplied

[The effect of emotional stress on carboxypeptidase H activity in sections of the rat brain with varying sensitivity to it].

The activity of carboxypeptidase H involved in metabolism of neuropeptides was studied in rats resistant and predisposed to emotional stress under normal and stress conditions. The enzymatic activity was higher in the striatum and hypothalamus of the rats predisposed to stress than that of stress-resistant animals. Emotional stress resulted in activation of the enzyme in these both groups of animals, while the dynamics of the enzyme activity depended on the resistance of animals to stress and the poststress interval. There was a correlation between the activity of carboxypeptidase H and the content of neuropeptides in animals with varying stress-resistance.

Amino Acid Sequence↗
PubMed1995

[Mechanisms of regulation and the biological role of carboxypeptidase H--a neuropeptide-processing enzyme].

The review summarizes contemporary views on the biological role, levels and mechanisms of regulation of carboxypeptidase H--the exopeptidase of secretory vesicles removing arginine and lysine residues from C-termini of peptides. It is concluded that although carboxypeptidase participates at the terminal stages of biological active peptide generation, it does not determine the levels of active neuropeptides in the organism.

Allosteric Regulation↗