Abstract
The oxidation of the side chain of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) into chenodeoxycholic acid has been studied in subcellular fractions of rat liver. The product was separated from the substrate by high pressure liquid chromatography and identified by gas-liquid chromatography-mass spectrometry. The highest specific rate of conversion was found in the heavy (M) and the light (L) mitochondrial fractions with the highest enrichment in the L fraction. Washing the M fraction reduced the side chain cleavage activity by 90%. The peroxisomal marker enzyme urate oxidase was reduced to the same extent. The activity found in the M fraction may thus be due to peroxisomal contamination. After centrifugation of the L fraction on a Nycodenz density gradient, the highest specific activity for side chain cleavage of DHCA (31 nmol X mg-1 X h-1) was found in the fraction with the highest peroxisomal marker enzyme activity. This fraction also catalyzed conversion of 3 alpha,7 alpha,12 alpha-5 beta-cholestanoic acid (THCA) into cholic acid at the highest rate (32 nmol X mg-1 X h-1). The peroxisomal oxidation of DHCA into chenodeoxycholic acid required the presence of ATP, CoA, Mg2+, and NAD in the incubation medium. The reaction was not inhibited by KCN. It is concluded that rat liver peroxisomes contain enzymes able to catalyze the cleavage of the side chain of both DHCA and THCA. The enzymes involved are similar to, but not necessarily identical to, those involved in the peroxisomal beta-oxidation of fatty acids.
Highlights
5/3-cholestanoic acid (DHCA) into chenodeoxycholic acid has been studied in subcellular fractions of rat liver
The reaction was not inhibited by KCNlIt is concluded that rat liver peroxisomes contain enzymes able to catalyze the cleavage of the side chain of both dihydroxy-5/3-cholestanoic acid (DHCA) and THCA
The R- and the s- isomers of THCA and DHCA were not separated since it was found that both isomers were well converted into the corresponding primary bile acids. ["C]Cholic acid and ['*C]chenodeoxycho1ic acid used as standards were from the Radiochemical Centre, Amersham, England
Summary
5/3-cholestanoic acid (DHCA) into chenodeoxycholic acid has been studied in subcellular fractions of rat liver. After centrifugation of the L fracdeoxycholic acid in the bile fistula rat [7].H e concluded that the mechanism for conversion of DHCA to chenodeoxycholic acid appeared to be the same as that of THCA to cholic acid [7].In vivo studies by Cass, Williams, and tion on a Nycodenz density gradient, the highest specific activity Hanson [8] showed that T H C A had the ability to comfor side chain cleavage of DHCA (31 nmol x mg-' x h-I) was found in the fraction with the highest peroxisomal marker enzyme activity. For the Supplementary key words bile acids cholic acid 3a,7a,12a-trihydroxy-5&cholestanoic acid HPLC isolation of peroxisomes in Nycodenz gradient
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