Does carboxypeptidase Y have intrinsic endopeptidase activity?

Lee, How-Ming; Riordan, James

doi:10.1016/0006-291x(78)90660-5

Cited by 36 publications

(9 citation statements)

References 6 publications

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“…The concentration of the enzyme was determined spectrophotometrically using i% E280nm = 14.8. This enzyme preparation was free of Protease A activity as checked by the assay of LEE and RIORDAN (14).…”

Section: Methodsmentioning

confidence: 99%

Influence of the substrate structure on carboxypeptidase Y catalyzed peptide bond formation

Breddam

Widmer

Johansen

1980

Carlsberg Res. Commun.

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It is shown that for carboxypeptidase Y catalyzed peptide synthesis the coupling yields using peptide esters as substrates and amino acids as nucleophiles are strongly dependent on the C-terminal amino acid residue. A dependence on the length of the acyl donating peptide chain is also observed, indicating the influence of residues other than the one directly involved in acylation of the enzyme. Using amino acid amides as nucleophiles, the yields are generally high (80 %-100 96). No peptide ester substrate with C-terminal D-amino acid residue can be used in this method of peptide synthesis since they are not substrates of the enzyme. However, the presence of Damino acids in the penultimate position does not prevent peptide synthesis. No dependence on the size of the leaving group of ester-and depsipeptide substrates could be observed.

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Section: Methodsmentioning

confidence: 99%

Influence of the substrate structure on carboxypeptidase Y catalyzed peptide bond formation

Breddam

Widmer

Johansen

1980

Carlsberg Res. Commun.

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“…The pellet was resuspended in 1 ml of 10 mM sodium acetate, pH 5. Pepstatin A (1 /zl of 2 mM in 20% dimethylsulfoxide) was added and then incubated at 22~ for 2 rain (Lee & Riordan, 1978). Carboxypeptidase Y (10 tzl of 500/xg/ml) and norleucine (40/xl of 50/zM) were added to the suspension.…”

Section: Reconstituted Proteoliposomes Containing Purified Lactose Camentioning

confidence: 99%

Transport by reconstituted lactose carrier from parental and mutant strains ofEscherichia coli

Seto-Young

Bédu

1984

J. Membrain Biol.

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The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lower Km but similar Vmax compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.

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“…CPD-Y was isolated as previously described (7). It was checked that the preparation was free of Protease A by the assay of LEE and RIORDAN (9). Bz-Lys-Ala-OH was prepared by enzymatic synthesis (5).…”

Section: Methodsmentioning

confidence: 99%

Semisynthesis of human insulin utilizing chemically modified carboxypeptidase Y

Breddam

Johansen

1984

Carlsberg Res. Commun.

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It has previously been demonstrated that the C-terminal alanyl residue in the B-chain of porcine insulin can be exchanged with a threonyl residue in a carboxypeptidase Y catalyzed transpeptidation reaction using threonine amide as nucleophile. However, with this procedure the transpeptidation product, human insulin amide, was obtained in relatively low yields. In the present paper it is demonstrated that mercury halide derivatives of CPD-Y catalyze the transpeptidation reaction with porcine insulin effectively and human insulin amide could be isolated in high yield. It is also demonstrated that deamidation of human insulin amide to yield essentially homogeneous human insulin can be achieved using CPD-Y modified with methyl mercuric iodide (Me-Hg-CPD-Y). Springer-Verlag

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Does carboxypeptidase Y have intrinsic endopeptidase activity?

Cited by 36 publications

References 6 publications

Influence of the substrate structure on carboxypeptidase Y catalyzed peptide bond formation

Influence of the substrate structure on carboxypeptidase Y catalyzed peptide bond formation

Transport by reconstituted lactose carrier from parental and mutant strains ofEscherichia coli

Semisynthesis of human insulin utilizing chemically modified carboxypeptidase Y

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