Abstract
A procedure for the purification of the enzyme bile acid:CoA ligase from guinea pig liver microsomes was developed. Activity toward chenodeoxycholate, cholate, deoxycholate, and lithocholate co-purified suggesting that a single enzyme form catalyzes the activation of all four bile acids. Activity toward lithocholate could not be accurately assayed during the earlier stages of purification due to a protein which interfered with the assay. The purified ligase had a specific activity that was 333-fold enriched relative to the microsomal cell fraction. The purification procedure successfully removed several enzymes that could potentially interfere with assay procedures for ligase activity, i.e. ATPase, AMPase, inorganic pyrophosphatase, and bile acid-CoA thiolase. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified ligase gave a single band of approximately 63,000 Mr. A molecular size of 116,000 +/- 4,000 daltons was obtained by radiation inactivation analysis of the ligase in its native microsomal environment, suggesting that the functional unit of the ligase is a dimer. The purified enzyme was extensively delipidated by adsorption to alumina. The delipidated enzyme was extremely unstable but could be partially stabilized by the addition of phospholipid vesicles or detergent. However, such additions did not enhance enzymatic activity. Kinetic analysis revealed that chenodeoxycholate, cholate, deoxycholate, and lithocholate were all relatively good substrates for the purified enzyme. The trihydroxy bile acid cholate was the least efficient substrate due to its relatively low affinity for the enzyme. Bile acid:CoA ligase could also be solubilized from porcine liver microsomes and purified 180-fold by a modification of the above procedure. The final preparation contains three polypeptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three peptides range in size from 50,000 to 59,000, somewhat smaller than the guinea pig enzyme. The functional size of the porcine enzyme in its native microsomal environment was determined by the technique of radiation inactivation analysis to be 108,000 +/- 5,000 daltons. Thus, the functional form of the porcine enzyme also appears to be a dimer.
Highlights
IntroductionOn sodium dodecylsulfate- the bile acid-CoAreacts with either glycine or taurine toform polyacrylamide gel electrophoresis the purified ligase the respective conjugated bile acid
From the Liver Study Unit, Veterans Administration Medical Center, San Francisco, California94121, the Departments of Medicine and Phmmacology, University ofCalifornia,San Francisco, California94143, and the Laboratory of Physical Biology, National Institute of Arthritis and Musculoskeletal andSkin Diseases, National Institutes of Health, Bethesda, Maryland 20892
A procedure for the purification of the enzyme bile jugated bile acids returned to the liver via the enterohepatic acid:CoA ligase from guinea pig liver microsomes was circulation are conjugated prior to resecretion
Summary
On sodium dodecylsulfate- the bile acid-CoAreacts with either glycine or taurine toform polyacrylamide gel electrophoresis the purified ligase the respective conjugated bile acid This reaction is catalyzed gave a single band of approximately 63,000 Mr A by the soluble fraction enzyme bile acid-CoA:glycine/taurine molecular size of 116,000 2 4,000 daltons was ob- N-acyltransferase. Bile acid:CoA ligase could be solubilized from porcine liver microsomes and purified 180-fold by a conjugation [14], and itsactivity appears to be controlled by the size of the recirculating pool of bile acid [11] Further study of this enzyme has been limited by the lack of a suitable scheme for the extraction of the enzyme in stable form and its subsequent purification. The final prepa- rification of the ligase from porcine liver and its structural ration contains three polypeptides as judged by sodium characterization is reported
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