Purification and characterization of bile salt sulfotransferase from human liver

Arch Biochem Biophys. 1985 Sep;241(2):371-9. doi: 10.1016/0003-9861(85)90559-4.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Diphosphate / pharmacology
  • Humans
  • Kinetics
  • Lithocholic Acid / analogs & derivatives
  • Lithocholic Acid / pharmacology
  • Liver / enzymology*
  • Molecular Weight
  • Phosphoadenosine Phosphosulfate / pharmacology
  • Substrate Specificity
  • Sulfotransferases*
  • Sulfurtransferases / antagonists & inhibitors
  • Sulfurtransferases / isolation & purification*


  • Phosphoadenosine Phosphosulfate
  • Lithocholic Acid
  • Adenosine Diphosphate
  • Sulfurtransferases
  • Sulfotransferases
  • bile-salt sulfotransferase
  • glycolithocholic acid