Subcellular Distribution of Bile Acid Conjugating Enzymes in Rat Liver

Abstract

The subcellular distribution of bile acid conjugating enzymes in rat liver was studied. Liver fractions were prepared by differential and zonal centrifugation. Golgi, plasma membranes and rough and smooth microsomes were prepared by sucrose gradient centrifugation. Fractions purity was assessed by the distribution of marker enzymes and by electron microscopy. Bile acid conjugating enzymes as determined by the synthesis of bile acid conjugates from [l4C]-cholic acid were recovered mainly in the differential mitochondrial fractions but were in microsomal-soluble fractions obtained by zonal centrifugation. The activating enzyme choloyl-S-CoA ligase determined by the formation of choloylhydroxamate showed a distribution consistent with a microsomal localization in zonal fractions. Bile acyl-S-CoA was chemically synthesized and an assay for bile acyl-S-CoA transferase activities in soluble and membrane fractions was developed. The bile acyl-S-CoA transferase had a dual mitochondrial-lysosomal localization but was mainly recovered in zonal microsomal-soluble fractions. Rough microsomes with approximately 3.5 times more RNA, than smooth microsomes contained choloyl-S-CoA transferase comparable to the soluble fraction and approximately 4 times more than smooth microsomes. It is thought likely that the preferential adsorption of the bile acyl-S-CoA transferase to rough microsomes coupled with the artefactual sedimentation of rough microsomes with large granules gave rise to the anomalous transferase distribution during differential pelleting. High concentrations of KCl (1.0 M) completely solubilized the choloyl-S-CoA transferase and cations were also effective solubilizers suggesting enzyme adsorption by coulombic attraction. Coulombic attraction alone however cannot account for sedimentable choloyl-S-CoA transferase activity since the more basic glutathione transferase demonstrated less adsorption (less than 10% of total liver activity). Sedimentable glutathione transferase may arise from membrane adsorbed activities and from a particulate component insoluble in basic solutions containing 1.0 m KCl. Choloyl-S-CoA transferase specific activities in plasma membranes purified 5-18 times were 10-52% of initial homogenates. Golgi membranes purified 120 times contained choloyl-S-CoA transferase specific activities lO-20% of that of the initial homogenates. Choloyl-S-CoA ligase did not co-purify with plasma membranes and could not be detected in golgi membranes. Taurine and glycine dependent choloyl-S-CoA transferase co-elute during ion-exchange chromatography and isoelectric focusing suggesting their common identity. The choloyl-S-CoA thiolase activity observed in the soluble fraction may be an artefact of the assay method since the thiolase and transferase co-elute during Sephadex, Agarose and ion-exchange chromatography and isoelectric focusing. Evidence presented suggests that non-specific bile acyl-S-CoA thiolase activity may be restricted to the lumen of subcellular organelles.