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. 1988 Aug;170(8):3459-67.
doi: 10.1128/jb.170.8.3459-3467.1988.

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

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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

A R Sundquist et al. J Bacteriol. .
Free PMC article

Abstract

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.

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