Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli
1986; Wiley; Volume: 156; Issue: 2 Linguagem: Inglês
10.1111/j.1432-1033.1986.tb09578.x
ISSN1432-1033
AutoresStuart P. Ballantine, David H. Boxer,
Tópico(s)Polyamine Metabolism and Applications
ResumoEuropean Journal of BiochemistryVolume 156, Issue 2 p. 277-284 Free Access Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli Stuart P. BALLANTINE, Stuart P. BALLANTINE Department of Biochemistry, Medical Sciences Institute, Dundee UniversitySearch for more papers by this authorDavid H. BOXER, Corresponding Author David H. BOXER Department of Biochemistry, Medical Sciences Institute, Dundee UniversityCorrespondence to D. H. Boxer, Department of Biochemistry, Medical Sciences Institute, Dundee University, Dundee DD1 4HN, ScotlandSearch for more papers by this author Stuart P. BALLANTINE, Stuart P. BALLANTINE Department of Biochemistry, Medical Sciences Institute, Dundee UniversitySearch for more papers by this authorDavid H. BOXER, Corresponding Author David H. BOXER Department of Biochemistry, Medical Sciences Institute, Dundee UniversityCorrespondence to D. H. Boxer, Department of Biochemistry, Medical Sciences Institute, Dundee University, Dundee DD1 4HN, ScotlandSearch for more papers by this author First published: April 1986 https://doi.org/10.1111/j.1432-1033.1986.tb09578.xCitations: 114AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract An active tryptic fragment of membrane-bound hydrogenase isoenzyme 2 from anaerobically grown Escherichia coli has been purified. The soluble enzyme derivative was released from the membrane fraction by trypsin cleavage. The purification procedure involved ion-exchange, hydroxyapatite and gel permeation chromatography. The enzyme derivative was purified 100-fold from the membrane fraction and the specific activity of the final preparation was 320 μmol benzyl viologen reduced min−1 mg protein−1 (H2: benzyl viologen oxidoreductase). The native enzyme derivative had an Mr of 180000 and was composed of equimolar amounts of polypeptides of Mr 61000 and 30000. It possessed 12.5 mol Fe, 12.8 mol acid-labile S2− and 3.1 mol Ni/180000 g enzyme. Antibodies were raised to the purified preparation which cross-reacted with hydrogenase isoenzyme 2 but not with isoenzyme 1 in detergent-dispersed preparations. Western immunoblot analysis revealed that isoenzyme 2 which had not been exposed to trypsin contained cross-reacting polypeptides of Mr 61000 and 35000. Trypsin treatment of the membrane-bound enzyme to form the soluble derivative of isoenzyme 2, therefore, cleaves a polypeptide of Mr 35000 to produce the 30000-Mr fragment. Trypsin treatment of the detergent-dispersed isoenzyme 2 produces the same fragmentation of the enzyme. Neither of the subunits of the enzyme revealed any immunological identity with those of hydrogenase isoenzyme 1. Abbreviations SDS sodium dodecyl sulphate HPLC high-pressure liquid chromatography Enzyme Hydrogenase (EC 1.12.–.–) REFERENCES 1 Ballantine, S. P. & Boxer, D. H. (1985) J. Bacteriol. 163, 454– 459. CASPubMedWeb of Science®Google Scholar 2 Sawers, R. G., Ballantine, S. P. & Boxer, D. H. (1985) J. Bacteriol. 164, 1324– 1331. CASPubMedWeb of Science®Google Scholar 3 Sawers, R. G. & Boxer, D. H. (1986) Eur. J. Biochem. 156, 265– 275. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 4 Laemmli, U. K. (1970) Nature (Lond.) 227, 680– 685. 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