PURIFICATION AND CHARACTERIZATION OF AN NADH-HEXACYANOFERRATE(III) REDUCTASE FROM SPINACH LEAF PLASMA-MEMBRANE

Plasma membranes were purified from spinach (Spinacea oleracea L.) lea ves by aqueous two-phase partitioning. The NADH-hexacyanoferrate(III) reductase was released from the membrane by Chaps solubilization and p urified 360-fold by ion-exchange chromatography followed by affinity c hromatography and size-exclusion chromatography on FPLC. A major band of 45 kDa and a minor contaminant of 66 kDa were detected by sodium do decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The band at 45 kDa cross-reacted with antibodies raised against an NADH-hexacya noferrate(III) reductase from potato tuber microsomes. The native size of the enzyme was 160 kDa as determined by size-exclusion chromatogra phy indicating that it is a tetramer. Two-dimensional gel electrophore sis, isoelectric focusing, followed by SDS-PAGE revealed three main ba nds of identical molecular weight with pi of 5.3-5.6. The enzyme conta ined about one flavin adenine dinucleotide (FAD) per 45-kDa subunit as determined by fluorescence spectroscopy, was specific for the beta-hy drogen of NADH, preferred NADH over NADPH as electron donor, and prefe rred hexacyanoferrate(III) as electron acceptor, e.g., it reduced Fe3-EDTA, cytochrome c, oxygen, and duroquinone at <10% of the rate with hexacyanoferrate(III). p-Chloromercurobenzoate, mersalyl, and dicumaro l inhibited the activity by >70%, whereas FAD, flavin mononucleotide, duroquinone, and ubiquinone(0) did not affect the activity. (C) 1995 A cademic Press, Inc.