Purification and characterization of an NADH-hexacyanoferrate(III) reductase from spinach leaf plasma membrane.
Plasma membranes were purified from spinach (Spinacea oleracea L.) leaves by aqueous two-phase partitioning. The NADH-hexacyanoferrate(III) reductase was released from the membrane by Chaps solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size-exclusion chromatography on FPLC. A major band of 45 kDa and a minor contaminant of 66 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The band at 45 kDa cross-reacted with antibodies raised against an NADH-hexacyanoferrate(III) reductase from potato tuber microsomes. The native size of the enzyme was 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Two-dimensional gel electrophoresis, isoelectric focusing, followed by SDS-PAGE revealed three main bands of identical molecular weight with pI of 5.3-5.6. The enzyme contained about one flavin adenine dinucleotide (FAD) per 45-kDa subunit as determined by fluorescence spectroscopy, was specific for the beta-hydrogen of NADH, preferred NADH over NADPH as electron donor, and preferred 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 dicumarol inhibited the activity by > 70% whereas FAD, flavin mononucleotide, duroquinone, and ubiquinone0 did not affect the activity.
Bérczi
A
A
Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged.
Fredlund
K M
KM
Møller
I M
IM
D002462:Cell Membrane / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional; D007120:Immunochemistry; D007526:Isoelectric Point; D008970:Molecular Weight; D009243:NAD / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D018724:Spinacia oleracea / Q000201:enzymology; D013379:Substrate Specificity
D009243:NAD; D009247:NADH, NADPH Oxidoreductases; C014227:ferricyanide reductase