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Simple and fast purification of Escherichia coli adenylate kinase
Author(s) -
Bârzu Octavian,
Michelson Susan
Publication year - 1983
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(83)80624-3
Subject(s) - adenylate kinase , enzyme , sephadex , chemistry , guanidine , escherichia coli , biochemistry , chromatography , mutant , microbiology and biotechnology , biology , gene
Adenylate kinase from E. coli (strains CR341 and CR341 T28, a temperature‐sensitive mutant) was purified by a two‐step chromatographic procedure. The enzyme from crude extracts of both mutant and parent strain was bound to blue—Sepharose at pH 7.5, thereafter specifically eluted with 0.05 mM P 1 , P 5 ‐di(adenosine‐5′)pentaphosphate. A second chromatography on Sephadex G‐100 yielded pure enzyme. E. coli adenylate kinase was strongly inhibited by P 1 , P 5 ‐di(adenosine‐5′)pentaphosphate ( K i 0.6 μM for adenylate kinase of strain CR341 and 2.1 μM in the case of mutant enzyme). After denaturation in 6 M guanidinium hydrochloride both mutant and parent adenylate kinase returned rapidly to the native, active state by dilution of guanidinium hydrochloride.