Characterization of Liver Cholic Acid Coenzyme A Ligase Activity

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.


Conjugated
bile acids, which aid in the digestion of dietary lipids (l), are synthesized in the liver. Their formation is thought to be the principal pathway of cholesterol catabolism (2). The major bile acids of the rat are cholic acid and deoxycholic acid, which are conjugated to taurine and glycine to form bile salts. The de nova synthesis of bile salts from cholesterol involves enzymatic processes which occur in the mitochondrial and cytoplasmic compartments of the liver cell ( When a standard assay mixture without microsomes (see "Experimental Procedures") containing [3H]cholic acid was acidified with perchloric acid, over 99% of the ["Hlcholic acid was removed from the aqueous phase by four 4-ml extractions with diethyl ether. When chemically synthesized ["Hlcholoyl-CoA, purified by paper chromatography, was subjected to the same procedure over 99% of the "H label remained in the aqueous phase. Thus, cholic acid CoA ligase activity could be assayed by monitoring the conversion of ["Hlcholic acid to a non-ether-extractable product. When the non-ether-extractable labeled material from a reaction mixture containing 100 pg of rat liver microsomal protein was chromatographed on Whatman No. 1 paper in propanol-l/H,0 (60/40, v/v), over 91% of the label migrated similar to chemically synthesized ["Hlcholoyl-CoA.

Subcellular
Fractionation -The subcellular distribution of the cholic acid CoA ligase was determined in rat liver which had been fractionated as follows. The livers were minced and homogenized in 5 volumes of Medium I (10 to 12 strokes).
A 375 x g for 10 min pellet (Pl) was resuspended in 3 volumes of Medium I and collected again by centrifugation.
The resulting pellet was suspended again in 3 volumes of Medium I and collected. This pellet was designated Pl. The combined supernatants were centrifuged at 33,000 x g for 10 min and the pellet was suspended in 3 volumes of Medium I and centrifuged at 33,000 x g for 10 min. The pellet was again collected, suspended in an additional 3 volumes of Medium I, and centrifuged for 10 min at 33,000 x g. This pellet was designated P2. The combined supernatants were centrifuged at 200,000 x g for 60 min to separate the microsomal pellet from the soluble material. Cholic acid CoA ligase was predominately (70%) associated with the microsomal fraction.
The recovery of activity from the homogenate was 85%. These data on the subcellular distribution of the cholic acid CoA ligase agreed well with more extensive studies done by Killenberg.' The distribution of a microsomal marker activity, NADPH cytochrome c reductase, assayed spectrophotometrically (181, was essentially identical with the cholic acid CoA ligase. The long chain fatty acid CoA ligase activity of rat liver was predominately localized in the microsomal fraction in agreement with previous data (19), with most of the remaining activity associated with the P2 fraction.
The amount of [3H1choloyl-CoA produced in 10 min at 23" was proportional to the amount of microsomal protein added up to 100 pg ( Fig. 1). When '75 pg of microsomal protein was employed, the amount of ["Hlcholoyl-CoA produced was proportional with time for 20 min (Fig. 1). The dependencies of the microsomal cholic acid CoA ligase activity measured under conditions where the quantity of ["Hlcholoyl-CoA produced was proportional to the time and amount of protein employed, are presented in Table I. The reaction was completely dependent upon addition of microsomal protein, ATP, CoA, and Mg'+. The addition of 1 mg/ml of Triton W-1339 stimulated the activity about 3-fold and thus was included routinely in the* assay. The addition of up to 0.5 M sodium chloride to the assay mixture had little effect on the activity. Potassium bromide at 0.5 M inhibited about 60%. The addition of ethanol and acetone at 2.5% (v/v) increased activity 116 and 160%, respectively. Higher concentrations, 15% (v/v), of either ethanol or acetone led to nearly a complete loss of activity. The assay was linear with protein up to 3 yg and with time up to 12 min when 2 Fg of liver microsomal protein was added.
In the following sections, the highly sensitive assay, validated above, has been employed to characterize the cholic acid CoA ligase activity. A side by side comparison of cholic acid CoA ligase activity and of fatty acid CoA ligase activity has been performed to evaluate whether these activities are catalyzed by a single microsomal enzyme.
Assay for Cholic Acid CoA Ligase -Cholic acid CoA ligase activity was assayed in a manner similar to the fatty acid CoA ligase as outlined above, except that 350 mM Tris, pH 7.8, was employed, and 60 ELM ["Hlcholic acid (10 ~Ci/~mol) replaced the labeled palmitic acid (see Results" for validation of the assay).

Substrate
Dependencies of Liver Microsomal Fatty Acid and Cholic Acid CoA Ligase Activities-The substrate dependencies of the liver microsomal fatty acid and cholic acid CoA ligase activities were investigated on the same day with the same microsomal preparation. The apparent K,,, values for ATP derived from the double reciprocal plots shown in Fig. 2, were similar for the fatty acid and cholic acid CoA ligase activities. However, cholic acid CoA ligase activity was the same when assayed using 10 mM ATP or dATP while fatty acid CoA ligase activity was 60% lower with the deoxynucleotide. The cholic acid CoA ligase activity had an apparent K,,, for dATP only 50% higher than for ATP while the apparent K,,, for dATP increased 389% relative to ATP for the fatty acid CoA ligase activity.  pH Dependence -An investigation of the pH dependencies of the fatty acid CoA ligase and cholic acid CoA ligase activities (Fig. 3)  The addition of up to 60 PM unlabeled palmitic acid to a tigated. When up to 100 PM unlabeled cholic acid was added to standard assay for the cholic acid CoA ligase activity resulted a standard assay for the fatty acid CoA ligase activity, no in less than 10% inhibition (Fig. 4A) The remaining data on the characterization of the cholic acid CoA ligase activity will be discussed in relation to the hypothesis that the cholic acid and fatty acid CoA ligase activities are dual activities of a single microsomal enzyme. The subcellular distribution and the apparent K,,, values for ATP, CoA,* and choli? (palmitic) acid for the liver microsomal fatty acid and cholic acid CoA ligase activities were similar. These observations and the approximately lo-fold lower specific activity for the cholic acid CoA ligase than the fatty acid CoA ligase were consistent with either a single enzyme with two activities or two independent microsomal enzymes.
Several lines of evidence, however, suggest that the fatty acid CoA ligase and the cholic acid CoA ligase activities are separate microsomal enzymes. Cholic acid was not an inhibitor of the fatty acid CoA ligase activity and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities were different in their pH dependencies, their ability to use dATP as a substrate, their thermolability at 41", and their sensitivity to phospholipase C. Moreover, the tissue survey demonstrated that cholic acid CoA ligase activity was present only in liver while the fatty acid CoA ligase activity was present in every tissue examined. The best interpretation of the available data is that separate cholic acid and fatty acid CoA ligases exist in liver microsomal preparations. The absence of cholic acid CoA ligase from all other organs examined appears reasonable physiologically since the uptake and activation of bile acids in e Recently a preliminary communication on the characterization of cholic acid CoA ligase in guinea pig liver microsomes appeared (10). The specific activity and apparent K,,, values for CoA and cholic acid reported agree closely with the data presented.
non-hepatic tissues could lead to accumulation of bile acids which could have deleterious effects on cellular processes.