Purification and characterization of the enzymes of bile acid conjugation from fish liver.

1. The two steps in bile acid conjugation have been studied in subcellular fractions of liver from three species of fish; vermillion rockfish, canary rockfish and ling codfish. 2. The bile acid: coenzyme A (CoA) ligase activity in homogenates and isolated microsomes is undetectable due to indeterminate factors. 3. A purification scheme is presented which eliminates the interfering factors. The purified ligase was found to have a lower affinity for bile acids as compared to the mammalian form and to be present in much lower titer. 4. Since it appears to be the rate controlling enzyme in all species, it is expected that the rate of bile acid conjugation is much slower in non-mammalian liver as compared to mammalian liver. 5. The bile acid-CoA:taurine N-acyltransferase was found to exist as a dimer of molecular weight 100,000, in contrast to the monomeric mammalian forms. 6. The only major kinetic difference is that the fish liver forms have rates of glycine conjugation which are only 1-2% of the rate with taurine, in part due to a very high Km for glycine.

Purification and properties of squirrel monkey (Saimiri sciureus) corticosteroid binding globulin.

Corticosteroid binding globulin (CBG), a serum glycoprotein which binds glucocorticoids and progestins with high affinity, is widely distributed throughout the animal world. Although its charge and size characteristics have largely been conserved across species, we found the behavior of CBG in squirrel monkey (Saimiri sciureus) serum during fractionation by polyacrylamide gel electrophoresis or Sephadex chromatography was consistent with a molecule about twice the size of that found in most species. To more fully understand the basis for this difference, we purified the protein by sequential affinity and DEAE-Sepharose chromatographies. The final product was obtained in greater than 60% yield and was found to migrate as a single homogeneous band when examined by electrophoresis at pH 8.3 in polyacrylamide gels varying total acrylamide concentration or under conditions of severe protein overload. The steroid binding specificity of the purified protein was identical with that of the protein in the starting serum. The ultraviolet absorption spectrum of the isolated CBG-steroid complexes revealed that the protein had no pyridine nucleotide cofactor or nucleic acid. Amino acid analyses showed that the composition of the squirrel monkey protein is quite similar to that of CBG molecules from other species but distinct from albumins, hemoglobin, or rabbit progesterone receptor. In contrast to the single protein band observed following electrophoresis under normal conditions, separations in the presence of sodium dodecyl sulfate (SDS) resolved the pure protein into two bands: one at 54,000 daltons and one at 57,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)