Both the outer mitochondrial membrane and the microsomal forms of cytochrome b5 reductase contain covalently bound myristic acid. Quantitative analysis on the polyvinylidene difluoride-immobilized proteins.
NADH-cytochrome b5 reductase is known to be located on two distinct membranes, i.e. endoplasmic reticulum and outer mitochondrial membranes. The endoplasmic-reticulum-associated form of the enzyme contains myristic acid in an amide linkage to its N-terminal glycine [Ozols, Carr & Strittmatter (1984) J. Biol. Chem. 259, 13349-13354]. To investigate whether the dual subcellular localization of the reductase corresponds to a difference in fatty acylation, the enzyme was purified from well-characterized rat liver microsomal and mitochondrial fractions and analysed by a new quantitative analytical procedure. The purified reductases were run on SDS/polyacrylamide gels and blotted on to polyvinylidene difluoride membranes. The reductase-containing bands were treated with hydroxylamine, and amide-linked fatty acids were then detached by acid hydrolysis. The detached fatty acids were extracted, derivatized and analysed as phenylacyl esters by reverse-phase h.p.l.c., and the protein content of the samples was determined by amino acid analysis of the acid hydrolysates. Myristic acid was found in both the microsomal and mitochondrial reductases in a molar ratio of 1:1 with protein. These results demonstrate for the first time the presence of a myristylated protein on outer mitochondrial membranes, and show that the microsomal and mitochondrial reductases are also identical in their fatty acylation.
Borgese
N
N
C.N.R. Center of Cytopharmacology, Faculty of Pharmacy, University of Milan, Italy.
Longhi
R
R
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003579:Cytochrome Reductases / Q000032:analysis / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D011145:Polyvinyls; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D019814:Myristic Acid; D009227:Myristic Acids; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers