The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes.
An NADPH-specific disulfide reductase that is active with bis-gamma-glutamylcystine has been purified 1,900-fold from Halobacterium halobium to yield a homogeneous preparation of the enzyme. Purification of this novel reductase, designated bis-gamma-glutamylcystine reductase (GCR), and purification of halobacterial dihydrolipoamide dehydrogenase (DLD) were accomplished with the aid of immobilized-metal-ion affinity chromatography in high-salt buffers. Chromatography of GCR on immobilized Cu2+ resin in buffer containing 1.23 M (NH4)2SO4 and on immobilized Ni2+ resin in buffer containing 4.0 M NaCl together effected a 120-fold increase in purity. Native GCR was found to be a dimeric flavoprotein of Mr 122,000 and to be more stable to heat when in buffer of very high ionic strength. DLD was chromatographed on columns of immobilized Cu2+ resin in buffer containing NaCl and in buffer containing (NH4)2SO4, the elution of DLD differing markedly in the two buffers. Purified DLD was found to be a heat-stable, dimeric flavoprotein of Mr 120,000 and to be very specific for NAD. The utility of immobilized-metal-ion affinity chromatography for the purification of halobacterial enzymes and the likely cellular function of GCR are discussed.
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis / Q000302:isolation & purification*; D004151:Dipeptides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006217:Halobacterium / Q000201:enzymology*; D006358:Hot Temperature; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis / Q000302:isolation & purification*; D013379:Substrate Specificity; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D004151:Dipeptides; C056782:bis-gamma-glutamylcystine; C007409:dihydrolipoamide; D008063:Thioctic Acid; D009247:NADH, NADPH Oxidoreductases; C056781:bis-gamma-glutamylcystine reductase; D008058:Dihydrolipoamide Dehydrogenase