Localization and biosynthesis of NADH-cytochrome b5 reductase, an iontegral membrane protein, in rat liver cells. III. Evidence for the independent insertion and turnover the enzyme in various subcellular compartments.
The biosynthesis and turnover of rat liver NADH-cytochrome b(5) reductase was studied in in vivo pulse-labeling and long-term, double-labeling experiments. Rats under thiopental anesthesia were injected into the portal vein with [(3)H]L-leucine and sacrificed at various times after the injection. NADH-cytochrome b(5) reductase was extracted from liver cell fractions by cathepsin D-catalyzed cleavage and was then immunoadsorbed onto antireductase-bearing affinity columns in the presence of excess unlabeled rat serum. After elution of the enzyme from the columns with a pH-2.2 buffer, the amount of the reductase protein in the samples was determined by radioimmunoassay, and the radioactivity in reductase was determined on SDS polyacrylamide gel reductase bands. The specific radioactivity of the reductase extracted from the homogenate as well as from rough and smooth microsomal, mitochondrial, and Golgi fractions, estimated at the end of the pulse (10 min after the injection) and at various time points thereafter, remained approximately constant over a 6-h period. These data suggest tha tth eenzyme is independently inserted into the various membranes where it is located. Moreover, the specific radioactivity of the mitochondrial reductase was lower than that of the other fractions, suggesting that it turns over at a slower rate. The lower turnover rate of the mitochondrial enzyme was confirmed by long-term, double-labeling experiments carried out according to the technique of Arias et al. (J. Biol. Chem. 244: 3303-3315.). The relevance of these findings in relation to the understanding of membrane biogenesis and turnover is discussed.
D000818:Animals; D003579:Cytochrome Reductases / Q000096:biosynthesis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D051381:Rats
D008565:Membrane Proteins; D003579:Cytochrome Reductases