Purification and characterization of bile salt sulfotransferase from human liver.
Bile salt sulfotransferase, the enzyme responsible for the formation of bile salt sulfate esters, was purified extensively from normal human liver. The purification procedure included DEAE-Sephadex chromatography, taurocholate-agarose affinity chromatography, and preparative isoelectrofocusing. The final preparation had a specific activity of 18 nmol min-1 mg protein-1, representing a 760-fold purification from the cytosol fraction with a overall yield of 15%. The human enzyme has a Mr of 67,000 and a pI of 5.2. DEAE-Sephadex chromatography of the cytosol fraction revealed only a single species of activity. The limiting Km for the sulfuryl donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is 0.7 microM. The limiting Km for the sulfuryl acceptor, glycolithocholate (GLC), is 2 microM. Reciprocal plots were intersecting. Product inhibition studies established that adenosine 3',5'-diphosphate (PAP) was competitive with PAPS (Ki = 0.2 microM) and noncompetitive with respect to GLC. GLC sulfate was competitive with GLC (Ki = 2.2 microM) and noncompetitive with respect to PAPS. Also, 3-ketolithocholate, a dead-end inhibitor, was competitive with GLC (Ki = 0.6 microM) and noncompetitive with respect to PAPS. Iso-PAP (the 2' isomer of PAP) was competitive with PAPS (Ki = 0.3 microM) and noncompetitive with GLC. The cumulative results of the steady-state kinetics experiments point to a random mechanism for the binding of substrates and release of products. The purified enzyme displays no activity toward estrone, testosterone, or phenol. Among the reactive substrates tested, the Vmax/Km values are in the order GLC greater than 3-beta OH-5-cholenic acid greater than glycochenodeoxycholate greater than glycocholate. p-Chloromercuribenzoate inactivated the enzyme. Either PAPS or GLC protected against inactivation, suggesting the presence of a sulfhydryl group at the active site.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008095:Lithocholic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010724:Phosphoadenosine Phosphosulfate / Q000494:pharmacology; D013379:Substrate Specificity; D015238:Sulfotransferases; D013466:Sulfurtransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D010724:Phosphoadenosine Phosphosulfate; D008095:Lithocholic Acid; D000244:Adenosine Diphosphate; D013466:Sulfurtransferases; D015238:Sulfotransferases; C019786:bile-salt sulfotransferase; C027746:glycolithocholic acid