Rat erythrocyte NADH-cytochrome b5 reductase. Quantitation and comparison between the membrane-bound and soluble forms using an antibody against the rat liver enzyme.
The subcellular distribution of rat erythrocyte NADH-cytochrome b5 reductase was determined by radioimmunoassay, using a rabbit antibody against the cathepsin D cleaved water-soluble fragment of rat liver microsomal reductase (I-reductase), which is known to be immunologically similar to the red cell enzyme. Erythrocytes contained approximately 30 ng of reductase/mg of protein, of which 90% were recovered in the hemolysate supernatant and 2.3% in the ghost fraction. After concentration by precipitation with 70% saturated (NH4)2SO4, the NADH-cytochrome c reductase activity of the soluble enzyme could be assayed in the presence of cytochrome b5, and was found to be inhibited by anti 1-reductase antibodies. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobilities of erythrocyte membrane-associated and soluble reductase of the liver microsomal enzyme and its cathepsin D cleaved hydrophilic fragment (I-reductase) were examined in crude fractions by blotting followed by specific and highly sensitive immunostaining. The intact microsomal enzyme and the two erythrocyte reductases all had similar mobilities and migrated behind 1-reductase. However, the ghost-associated reductase, which was not attributable to contaminating leukocyte or reticulocyte membranes, was distinguishable from the soluble form by two criteria: (i) a lower dependence on exogenous cytochrome b5 in the NADH-cytochrome c reductase assay; and (ii) a larger apparent Mr upon gel filtration in the presence of Triton X-100, presumably because of detergent binding. Considering these results, possible biogenetic relations between membrane-bound and soluble erythrocyte reductase are discussed.
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D003579:Cytochrome Reductases / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D042966:Cytochrome-B(5) Reductase; D004912:Erythrocytes / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D000936:Antigen-Antibody Complex; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase