Method of preparing an enzyme participating in C-terminal amidat

Details Method of preparing an enzyme participating in C-terminal amidat Method of preparing an enzyme participating in C-terminal amidat

1998-10-14

2000-12-05

Naff, David M.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

435 18, 435 24, 435 691, 435195, 435227, 435228, 435471, 435814, 435815, C12N 948, C12N 914, C12N 978, C12N 980, C12N 1500

Patent

active

061565555

ABSTRACT:
A purified enzyme-I is obtained that participates in C-terminal amidation by acting on a peptide C-terminal glycine adduct to form a peptide C-terminal .alpha.-hydroxyglycine adduct. The enzyme has an optimum pH of about 5 to 7, an optimum temperature of 25 to 40.degree. C. and a molecular weight of about 25 kDa or about 36 kDa, and metal ions and ascorbic acid act as a cofactor. A purified enzyme-II is obtained that participates in C-terminal amidation by acting on the peptide C-terminal .alpha.-hydroxyglycine adduct to produce a C-terminal amidated compound. The enzyme has an optimum pH of about 5 to 6, an optimum temperature of 15 to 35.degree. C. and a molecular weight of about 40 kDa or about 43 kDa. Enzyme-I does not act on the peptide C-terminal .alpha.-hydroxyglycine adduct and enzyme-II does not act on the peptide C-terminal glycine adduct. The enzymes may be purified from a biological material such as horse serum by affinity chromatography using a peptide C-terminal glycine adduct as a ligand. The enzymes may also be obtained from host cells transformed with a plasmid containing a cDNA coding for the enzymes. Assay of activity of the enzymes is carried out by measuring the C-terminal .alpha.-hydroxyglycine adduct or the C-terminal amidated compound that has been isolated such as by high performance liquid chromatography with the use of an acetonitrile-containing buffer.

REFERENCES:
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