Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from bovine adrenocortical mitochondria. I. Physicochemical properties of holo- and apo-NADPH-adrenodoxin reductase and interaction between non-heme iron proteins and the reductase

Abstract

A crystalline NADPH-adrenodoxin reductase was obtained from bovine adrenocortical mitochondria and its properties were investigated. Its molecular weights and isoelectric point were estimated to be 51 000 and 5.4, respectively. Amino acid and sugar contents and the interaction between the apo-reductase and flavin of NADPH-adrenodoxin reductase were investigated. Formation of a complex of bovine NADPH-adrenodoxin reductase with adrenodoxin, its apoadrenodoxin, or other non-heme iron proteins caused quenching of fluorescence of the tryptophanyl residue and bound FAD of the NADPH-adrenodoxin reductase. The results obatined suggest that adrenodoxin and apoadrenodoxin bind functionally to a site close to the tryptophanyl residue and the bound FAD of the reductase. The circular dichroism spectrum of oxidized NADPH-adrenodoxin reductase was measured in the ultraviolet and visible regions. This spectrum showed negative absorption in the visible region and was not appreciably influenced in either the ultraviolet or visible region by formation of a complex with adrenodoxin or apoadrenodoxin.

Protein Phosphorylation and Intermolecular Electron Transfer: A Joint Experimental and Computational Study of a Hormone Biosynthesis Pathway

“…30,31 Protein concentrations were calculated using ε 414 = 9.8 (mM cm) −1 for Adx 32 and ε 450 = 11.3 (mM cm) −1 for AdR. 33 …”

“…Radioactive labeling of Adx using 33 PγATP showed that the phosphate group is stably inserted into the Adx molecule for at least 1 h. 1 Since the relatively fast kinetic reaction takes place within seconds, the phosphate group is not likely to be cleaved off before or during the reaction and hence be the cause of the second phase (also in the seconds regime) that is observed in the kinetic traces for phosphorylated Adx. In addition, the degree of phosphorylation was measured via a luciferase assay prior to each experiment as described in the methods section.…”

Crystallization and properties of reduced nicotinamide adenine dinucleotide phosphate‐adrenodoxin reductase of pig adrenocortical mitochondria

“…Crystalline NADPH-adrenodoxin reductase was purified from pig adrenocortical mitochondria by affinity chromatography using 2'S'-ADP-Sepharose 4B and by a method described previously [2] . Pig adrenodoxin was crystallized by Ichikawa's method 111.…”

“…We have reported previously on crystalline NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria [2,3] . This communication describes the crystallization of pig NADPH-adrenodoxin reductase from pig adrenocortical mitochondria by two methods and the properties of the NADPH-adrenodoxin reductase.…”

Purification and biochemical characterization of hepatic ferredoxin (hepatoredoxin) from bovine liver mitochondria

“…The activity of hepatoredoxin was measured in the presence of NADPH-adrenodoxin reductase and cytochrome c as the activity of NADPHcytochrome-c reductase, by the method of Hiwatashi et al [8].…”