Biosynthesis of microsomal nicotinamide-adenine dinucleotide phosphate-cytochrome c reductase by membrane-bound and free polysomes from rat liver.
1. NADPH-ferricytochrome c oxidoreductase (EC 1.6.2.3) was purified from the endoplasmic reticulum of rat liver cells. The methods, which involved digestion of membrane with Steapsin, a crude pancreatic extract containing diastase and trypsin, gel filtration and preparative electrophoresis on polyacrylamide, provided an enzyme with a high specific activity in good yield. 2. The incorporation of (14)C-labelled amino acids into the purified reductase by the incubation of various subcellular fractions was studied. The microsome fraction, bound polysomes, free polysomes and detergent-treated polysomes effected the synthesis of the enzyme. 3. The reductase that had been synthesized by the polysomes was tightly bound to preparations of smooth-surfaced endoplasmic reticulum that were added to the incubation medium. 4. Reductase activity could be detected on both free and detergent-treated polysomes. Evidence is presented to show that this activity was due, at least in part, to the presence on the ribosomes of nascent enzyme. The association of enzyme with detergent-treated polysomes did not appear to be due to contamination of the ribosomes with either membrane or cell sap but it is possible for such ribosomes to adsorb some enzyme. 5. The amount of reductase activity associated with the detergent-treated polysomes was increased when the rats from which the polysomes were derived had been previously injected with phenobarbitone. 6. The results are discussed with respect to their relevance for the question of the existence of two functionally different groups of polysomes in the liver and for current ideas on the biogenesis of membranes.
D000596:Amino Acids / Q000378:metabolism; D000681:Amylases; D000818:Animals; D002247:Carbon Isotopes; D002499:Centrifugation, Density Gradient; D003580:Cytochromes; D003864:Depression, Chemical; D003902:Detergents; D004721:Endoplasmic Reticulum; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D008566:Membranes / Q000201:enzymology*; D008722:Methods; D008861:Microsomes / Q000378:metabolism; D009249:NADP; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000302:isolation & purification; D010634:Phenobarbital / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D011691:Puromycin / Q000494:pharmacology; D051381:Rats; D012270:Ribosomes / Q000201:enzymology*; D013268:Stimulation, Chemical; D014357:Trypsin
D000596:Amino Acids; D002247:Carbon Isotopes; D003580:Cytochromes; D003902:Detergents; D011506:Proteins; D011691:Puromycin; D009249:NADP; D010088:Oxidoreductases; D000681:Amylases; D014357:Trypsin; D010634:Phenobarbital