Characterization of cerebroside sulfotransferase from rat brain.

Cerebroside sulfotransferase, the enzyme which catalyzes the transfer of the sulfate group from d’-phosphoadenosine 5’-phosphosulfate to galactosylceramide to form sulfogalactosylceramide is located primarily in microsomes and possibly in myelin of 17-day-old rat brain. Subfractionation of the microsomes demonstrates the enzyme activity to be present in the lighter, smooth membrane fraction. Virtually all of the cerebroside sulfotransferase activity can be extracted with Triton X-100. The extracted enzyme demonstrates a linear response for 2 hours in the presence of exogenous substrate, galactosylceramide, and has an apparent K, of 8 X lop6 M. Cruder preparations such as homogenate or microsomes without added substrate were linear for only about 15 min. This lack of linearity appears to be related to decreased availability of endogenous substrate. Both cY-hydroxy fatty acid galactosylsphingosine and nonhydroxy fatty acid galactosylsphingosine act as sulfate acceptors. Two further galactosphingolipids, lactosylceramide and galactosylsphingosine, can act as sulfate acceptors in vitro. Small amounts of lactosylceramide have been found in both normal and abnormal brain, whereas naturally occurring galactosylsphingosine has not been described. Glucosylceramide and other lipids examined were unable to act as acceptors for sulfate in this extracted system. [6-3H] Galactosylceramide was utilized as an acceptor for the sulfate group from 3’-phosphoadenosine 5’-phospho[3%]sulfate. The isolated product, sulfogalactosylceramide, contained both the tritium and 35S04 label, indicating that the exogenous galactosylceramide could be utilized as a substrate by cerebroside sulfotransferase of brain.