Glucosyceramide synthase of mouse kidney: further characterization with an improved assay method

The synthesis of glucosylceramide from ceramide and UDP-[3H]glucose by mouse kidney homogenates is very sensitive to the concentration of tissue. This was shown to be due to the presence of a UDP-glc pyrophosphatase, which could be blocked by adding NAD to the medium. A new solvent partitioning system is described, containing t-butyl methyl ether, isopropyl alcohol, and aqueous sodium sulfate, which separates the original substrate (UDP-[3H]glc) from the enzyme product, [3H]cerebroside. A particular advantage of the solvent system is that only a single partitioning step is needed, without backwashes, and the enzyme product appears in the upper phase, making transfer to a counting vial more reliable. A novel incubation device, a thermostatically controlled ultrasonic bath, is used to produce highly uniform enzyme reaction rates. Ca2+, as well as Mg2+ and Mn2+, was found to be a good stimulator of the glucosyltransferase. The enzyme activity in kidney of 22-day old mice, approximately 240 pmol/h/mg tissue, is significantly greater than previously demonstrated. The enzyme was stable in intact kidneys stored at -70 degrees C but unstable at 4 degrees C. The enzyme, when acting on endogenous ceramides, showed no demonstrable glucosylation of the C24 family of ceramides although this family is the predominant one in kidney.