Comparative studies on human carboxypeptidases B and N

McKay, Thomas J.; Phelan, Arthur W.; Plummer, Thomas H.

doi:10.1016/0003-9861(79)90271-6

Cited by 58 publications

(19 citation statements)

References 29 publications

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“…(Cbz-Arg); on the whole, the values compare well with those previously reported for mammalian carboxypeptidases; in particular, human carboxypeptidases B and N, and bovine carboxypeptidase B cleaved Bz-Gly-Lys much less effectively than did S. solfataricus carboxypeptidase, but carboxypeptidases B, unlike N, were significantly more effective on BzGly-Arg than the thermophilic enzyme [30]. When using CbzGly-Gly-Phe as a substrate, k J K , determined for the archaebacterial carboxypeptidase was about one order of magnitude lower than that reported for carboxypeptidase A; the difference being almost equally due to the respective k,,,…”

Section: Substrate Specificitysupporting

confidence: 88%

“…Second, it was unique in its broad specificity, in that it could release basic, acidic and aromatic amino acids. In contrast, the mammalian carboxypeptidases cleave either hydrophobic amino acids, as in the case of carboxypeptidase A [44], or basic amino acids, as do carboxypeptidase B, N and enkephalin convertase [30,35,36]. Third, commercially available carboxypeptidases A and B, and thermolysin, did not react with polyclonal antibodies raised against the thermophilic enzyme, as found in immunoblotting experiments (data not shown).…”

Section: Discussionmentioning

confidence: 82%

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Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

Colombo

D’Auria

Fusi

et al. 1992

European Journal of Biochemistry

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A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids. An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus. The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85°C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40°C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B.

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Section: Substrate Specificitysupporting

confidence: 88%

Section: Discussionmentioning

confidence: 82%

Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

Colombo

D’Auria

Fusi

et al. 1992

European Journal of Biochemistry

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“…It is possible that the observed [ 3 H]GEMSA binding in the liver is due to other enzymes, such as carboxypeptidase N (CPN) or carboxypeptidase M. CPN is a serum enzyme which is produced in the liver. Although the reported Ki value for GEMSA is in the micromolar range when CPN is measured at neutral pH (21), this inhibitor is substantially more potent when assayed at pH 6.0 (Fricker, L. D., unpublished observation).…”

Section: Discussionmentioning

confidence: 99%

Isolation and Sequence Analysis of cDNA for Rat Carboxypeptidase E [EC 3.4.17.10], A Neuropeptide Processing Enzyme

Flicker

Adelman

Douglass

et al. 1989

Molecular Endocrinology

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Carboxypeptidase E (CPE) is the carboxypeptidase B-like enzyme associated with the biosynthesis of numerous peptide hormones and neurotransmitters. This enzyme has been previously purified to homogeneity from bovine tissues, and cDNA clones (non-full length) isolated from a bovine pituitary cDNA library. In the present study, cDNA encoding full-length rat CPE has been isolated and sequenced. Both the nucleotide and amino acid sequences of rat CPE show substantial homology with the bovine sequences. The bovine and rat nucleotide sequences are homologous within the entire coding region, as well as within several portions of the 3'-untranslated region. The predicted amino acid sequence of rat CPE is greater than 90% homologous with the bovine enzyme. Northern blot analyses indicate a single species of CPE mRNA approximately 2100 nucleotides in length to be present in many neural and endocrine tissues. High levels of CPE mRNA are present in rat hypothalamus, hippocampus, midbrain, striatum, and cerebral cortex; and moderate levels are present in the brain stem, cerebellum, heart, adrenal, and eye. Low levels are detected in testis and duodenum, but not in liver or thymus. This tissue-specific expression of CPE mRNA is consistent with the proposed role for this enzyme in the production of numerous peptide hormones and neurotransmitters.

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“…Inhibitors of zinc metallopeptidases GPSA, APMSA, BzlSA, MGTA (20)(21)(22), and the carboxypeptidase inhibitor from potatoes (23) (26) had little or no effect on the carboxypeptidase activity. To differentiate the carboxypeptidase in granules from that in lysosomes, the effect of protease inhibitors and divalent cations on lysosomal carboxypeptidase(s) was studied (Table 2).…”

mentioning

confidence: 99%

Carboxypeptidase B-like converting enzyme activity in secretory granules of rat pituitary.

Hook¹,

Loh²

1984

Proc. Natl. Acad. Sci. U.S.A.

126

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Recent amino acid sequence data suggest that trypsin-like and carboxypeptidase B-like activities are required for the processing of pituitary prohormones-e.g., pro-opiocortin (pro-adrenocorticotropin/lipotropin) and provasopressin in secretory granules. In this study the existence of a carboxypeptidase B activity in purified secretory granules from anterior, intermediate, and neural lobes of rat pituitary has been examined. A carboxypeptidase B activity that cleaved the COOH-terminal -Lys-Lys-Arg residues from the adrenocorticotropin fragment ACTH-(1-17) (a potential hormone product liberated from pro-opiocortin by a trypsin-like enzyme) was detected in anterior and intermediate lobe granules. A similar carboxypeptidase B activity was also present in purified secretory granules from rat pituitary neural lobes that The carboxypeptidase B in secretory granules from all three lobes was shown to be active at pH 5.5, but not at pH 7.4. Inhibition by the zinc metallocarboxypeptidase inhibitors guanidinopropylsuccinic acid, aminomercaptosuccinic acid, benzylsuccinic acid, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and the potato carboxypeptidase B inhibitor, and inhibition by the metal chelators EDTA and 1,10-phenanthroline demonstrate metal ion dependence of the pituitary granule carboxypeptidase activities. However, Co2+ stimulated the secretory granule carboxypeptidase B activities. Thiol protease inhibitors such as Cu + and p-chloromercuriphenylsulfonic acid also inhibited the activity. Thus, the secretory granule carboxypeptidase B-like activities in all three lobes of the pituitary appear to be similar thiol-metallopeptidases that differ from other carboxypeptidase activities previously described and may play an exclusive role in hormone biosynthesis in the pituitary.

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Comparative studies on human carboxypeptidases B and N

Cited by 58 publications

References 29 publications

Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

Isolation and Sequence Analysis of cDNA for Rat Carboxypeptidase E [EC 3.4.17.10], A Neuropeptide Processing Enzyme

Carboxypeptidase B-like converting enzyme activity in secretory granules of rat pituitary.

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