Human NADH-cytochrome b5 reductases: comparison among those of erythrocyte membrane, erythrocyte cytosol, and liver microsomes.
NADH-cytochrome b5 reductases purified from human red cell membranes and cytosol were compared with those prepared from human liver microsomes. Minimal molecular weights of the membrane and the cytosol enzymes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were 36,000 and 32,000 daltons, respectively, which are comparable to those of the detergent-solubilized reductase (dfp) and the protease-solubilized one (tfp) of liver microsomes, respectively. All the enzymes contained FAD and had essentially the same turnover numbers and apparent Km values for NADH and protease-solubilized cytochrome b5. The membrane enzyme and liver dfp reduced cytochrome c in the presence of detergent-solubilized cytochrome b5 70-80 times faster than in the presence of trypsin-solubilized cytochrome b5, whereas the cytosol enzyme and liver tfp showed essentially the same low activities with both preparations of cytochrome b5. SDS-PAGE mapping of the limited proteolytic products of the reductases obtained by digestion with staphylococcal protease or a-chymotrypsin showed essentially the same patterns of peptides between the red cell membrane enzyme and liver dfp and between the red cell cytosol enzyme and liver tfp. These results suggest that the NADH-cytochrome b5 reductase of human red cell membranes is identical with that of liver microsomes and that the enzyme of red cell cytosol is a proteolytic product of the membrane enzyme.
D003579:Cytochrome Reductases / Q000302:isolation & purification / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D003600:Cytosol / Q000201:enzymology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009928:Organ Specificity
D005182:Flavin-Adenine Dinucleotide; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase