Purification and activation of brain sulfotransferase.
Galactosylceramide sulfotransferase (EC 2.8.2.11) catalyzes the biosynthesis of sulfatide from galactocerebroside and adenosine 3'-phosphate 5'-phosphosulfate (PAPS). This enzyme is developmentally controlled, reaching a maximum activity in the brains of mice corresponding to that of maximum myelination. The product, sulfatide, is an important component of myelin. This transferase from mouse brain has been purified 2600-fold using a combination of pyridoxal 5'-phosphate- and ATP-ligated columns. The purified enzyme yielded a single band following SDS-polyacrylamide gel electrophoresis with an apparent M(r) of 31,000. The entire purification procedure can be completed in 1 day. The pH optimum for the enzyme is 7.0. The Km for PAPS is 1.2 x 10(-6) M, and the Km for cerebroside is 2.6 x 10(-5) M. Cerebroside concentrations > 80 pmol/ml are inhibitory. Enzyme preparations were associated with several lipids. Vitamin K+P(i) activated purified preparations of the sulfotransferase and maintained enzyme activity during storage at -80 degrees C.
Sundaram
K S
KS
Department of Microbiology, City University of New York Medical School/Sophie Davis School of Biomedical Education, New York 10031.
Lev
M
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008861:Microsomes / Q000201:enzymology*; D015238:Sulfotransferases; D013466:Sulfurtransferases / Q000302:isolation & purification* / Q000378:metabolism*
D000255:Adenosine Triphosphate; D013466:Sulfurtransferases; D015238:Sulfotransferases; C020556:galactosylceramide sulfotransferase