Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli.
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 mumol benzyl viologen reduced min-1 mg protein-1 (H2:benzyl viologen oxidoreductase). The native enzyme derivative had an Mr of 180,000 and was composed of equimolar amounts of polypeptides of Mr 61,000 and 30,000. It possessed 12.5 mol Fe, 12.8 mol acid-labile S2- and 3.1 mol Ni/180,000 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 61,000 and 35,000. Trypsin treatment of the membrane-bound enzyme to form the soluble derivative of isoenzyme 2, therefore, cleaves a polypeptide of Mr 35,000 to produce the 30,000-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.
D000693:Anaerobiosis; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003067:Coenzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006864:Hydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D006868:Hydrolysis; D007120:Immunochemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification*; D012995:Solubility; D014357:Trypsin
D003067:Coenzymes; D007527:Isoenzymes; D010446:Peptide Fragments; D006864:Hydrogenase; D014357:Trypsin