Subcellular localization of 3 alpha, 7 alpha-dihydroxy- and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-coenzyme A ligase(s) in rat liver.
Liver peroxisomes from both rat and humans have previously been shown to contain enzymes that catalyze the oxidative cleavage of the C27-steroid side chain in the formation of bile acids. It has not been clear, however, whether the initial step, formation of the CoA-esters of the 5 beta-cholestanoic acids, also occurs in these organelles. In the present work the subcellular localization of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) ligase (THCA-CoA synthetase) and of 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoyl-CoA (DHCA-CoA) ligase in rat liver has been investigated. Main subcellular fractions and peroxisome-rich density gradient fractions from rat liver were incubated with THCA or DHCA, CoA, ATP, and Mg2+. Formation of THCA-CoA and DHCA-CoA was determined after high pressure liquid chromatography of the incubation extracts. The microsomal fraction contained the highest specific (and also relative specific) activity both for the formation of THCA-CoA and DHCA-CoA. The rates of THCA-CoA formation were further increased from 124-159 nmol/mg.hr-1 in crude microsomal fractions to 184-220 nmol/mg.hr-1 when studied in purified rough endoplasmic reticulum fractions. Formation of THCA-CoA in peroxisomal fractions prepared in Nycodenz density gradients could be accounted for by a small contamination (3-7%) by microsomal protein. The distribution of THCA-CoA ligase was different from that of palmitoyl-CoA ligase that was found to be localized also to the peroxisomal fractions.
Prydz
K
K
Institute for Nutrition Research, School of Medicine, University of Oslo, Norway.
Kase
B F
BF
Björkhem
I
I
Pedersen
J I
JI
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003066:Coenzyme A Ligases / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000032:analysis
C057596:3,7,12-trihydroxy-5-cholestanoyl CoA ligase; C057597:3,7-dihydroxy-5-cholestanoyl CoA ligase; D003066:Coenzyme A Ligases