Soluble cytochrome b5 reductase from human erythrocytes

Abstract

I. An enzyme that catalyzes the reduction of erythrocyte cytochrome b 5 has been isolated from the supernatant fraction of erythrocyte hemolysates by chromatography on DEAE-cellulose, Amberlite CG-5 o, and Bio-Gel P-6o. 2. Erythrocyte cytochrome b 5 reductase has been shown to contain FAD. Incubation of the reductase in the absence of EDTA results in both the appearance of flavin fluorescence and the loss of enzymatic activity with time. 3. The reductase catalyzes the reduction of erythrocyte cytochrome b 5 5o times faster with NADH than with NADPH. The apparent Km of NADH was calculated to be 1.6. IO-~ M and the turnover number is 128o moles of cytochrome b 5 per min per mole of flavin. The reduction of electron acceptors proceeded in the following decreasing order of rate: K~Fe(CN)6, 2,6-dichlorophenolindophenol (DCIP), cytochrome b 5, methylene blue, cytochrome c, 03, oxidized glutathione, and methemoglobin. 4-The FAD prosthetic group, the substrate specificity, and the effect of ionic strength, pH, and EDTA on activity suggest that the reductase is the same enzyme as NADH dehydrogenase I, the enzyme lacking in many cases of congenital methemoglobinemia. The properties of the reductase, including its molecular weight, are very similar to those of the cytochrome b 5 reductases solubilized from microsomes and mitochondria of other tissues.

Evidence that two forms of bovine erythrocyte cytochrome b5 are identical to segments of microsomal cytochrome b5.

“…The present and previous data support our hypothesis (13,14) that soluble cytochrome b5 from erythrocytes is derived during erythropoiesis by proteolytic degradation of the membrane-bound cytochrome b5 present in the endoplasmic reticulum of immature erythroid cells. During the early stages of erythroid maturation the endoplasmic reticulum of the proerythroblast disappears and the subsequent cell stages are devoid of this subcellular membrane (26).…”

“…Erythrocyte cytochrome b5 is a hydrophilic protein which participates in the reduction of methemoglobin by transferring electrons from erythrocyte cytochrome b5 reductase to methemoglobin (12,13). Except (14).…”

“…For developmental and biochemical reasons it is of interest to determine the fate of proteins that are associated with the subcellular organelles of immature erythroid cells. We have hypothesized (13) (17). Throughout the procedure the ratio of absorbance at 413 nm to that at 280 nm was used as a measure of cytochrome purity.…”

“…Low-temperature spectra of cytochrome b5 were taken at -196°with a Cary model 13 spectrophotometer adapted for this use. Samples were made 50% in glycerol, reduced with a few crystals of dithionite, and then frozen in liquid nitrogen.…”

“…Cells were immediately sedimented, washed, and lysed as described (11) In a typical purification, hemolysate derived from 3.5 liters of packed erythrocytes was charged onto a 6.5 X 55 cm column of DEAE-cellulose. The column was then washed with 13 liters of 3 mM potassium phosphate buffer, pH 7.2, to remove residual hemoglobin. Several contaminating proteins were then eluted by simultaneously decreasing the pH and increasing the ionic strength, using first 2.5 liters of 10mM KH2PO4/10 mM KCI and then a gradient prepared with 4 liters of 10 mM KH2PO4/10 mM KCI in the mixer and 4 liters of 50 mM KH2PO4 in the reservoir.…”