Fatty acid hydroxylation in rat kidney cortex microsomes.
Rat kidney microsomes have been found to catalyze the hydroxylation of medium-chained fatty acids to the omega- and (omego-1)-hydroxy derivatives. This reaction, which requires NADPH and molecular oxygen, is a function of monooxygenase system present in the kidney microsomes, containing NADPH-cytochrome c reductase and cytochrome P-450K. NADH is about half as effective as an electron donor as NADPH and there is an additive effect in the presence of both nucleotides. Cytochrome P-450K absorbs light maximally at 452-3 nm, when it is reduced and bound to carbon monoxide. The extinction coefficient of this complex is 91 mM(-1) cm(-1). Electrons from NADPH are transferred to cytochrome P-450K via the NADPH-cytochrome c reductase. The reduction rate of cytochrome P-450K is stimulated by added fatty acids and the reduction kinetics reveal the presence of endogenous substrates bound to cytochrome P-450K. Both cytochrome P-450K concentration and fatty acid hydroxylation activity in kidney microsomes are increased by starvation. On the other hand, phenobarbital treatment of the rats has no effect on either the hemoprotein or the overall hydroxylation reaction and 3,4-benzpyrene administration induces a new species of cytochrome P-450K not involved in fatty acid hydroxylation. Cytochrome P-450K shows, in contrast to liver P-450, high substrate specificity. The only substances forming enzyme-substrate complexes with cytochrome P-450K are the medium-chained fatty acids and certain derivatives of these acids. The chemical requirements for substrate binding include a carbon chain of medium length and at the end of the chain a carbonyl group and a free electron pair on a neighbouring atom. The distance between the binding site for the carbonyl group and the active oxygen is suggested to be in the order of 16 A. This distance fixes the ratio of omega- and (omega-1)-hydroxylated products formed from a certain fatty acid by the single species of cytochrome P-450K involved. The membrane microenvironment seems also to be of importance for the substrate specificity of cytochrome P-450K, since removal of the cytochrome from the membrane lowers its binding specificity to some extent. A comparison between the liver and kidney cytochrome P-450 systems suggests that the kidney cytochrome P-450K system is specialized for fatty acid hydroxylation.
D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide; D002374:Catalase / Q000378:metabolism; D002849:Chromatography, Gas; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005227:Fatty Acids; D006454:Hemoglobins / Q000378:metabolism; D007672:Kidney Cortex / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D007850:Lauric Acids; D008297:Male; D008861:Microsomes / Q000201:enzymology* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009243:NAD; D009249:NADP; D011485:Protein Binding; D051381:Rats; D013053:Spectrophotometry; D013217:Starvation; D013329:Structure-Activity Relationship
D005227:Fatty Acids; D006454:Hemoglobins; D007850:Lauric Acids; D009243:NAD; D009249:NADP; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D002374:Catalase