PHOSPHATIDATE PHOSPHATASE - A KEY ENZYME IN GLYCEROLIPID BIOSYNTHESIS, STUDIES ON THE YEAST ENZYME

Phosphatidate phosphatase is an important enzyme in glycerolipid biosy nthesis, but difficult to purify. A purified preparation of N-ethylmal eimide-sensitive phosphatidate phosphatase from the yeast Saccharomyce s cerevisiae was obtained by a five-step protocol, using chromatograph y on DE-53/DEAE FF, Affi-Gel Blue, hydroxyapatite, Mono-Q, and Superde x 200. A protease-deficient yeast strain gave preparations similar to those of the wild-type strain. In exclusion chromatography, the enzyme activity of all preparations eluted at approximately the same positio n as albumin. However, the behavior on SDS/PAGE differed considerably among preparations, suggesting a multimeric subunit structure or degra dation during purification. A 35-kDa and a 40-kDa protein band which c oincided with activity were found in all preparations. Glycerol in the buffers could be excluded without rapid loss of enzyme activity, and Tris could be substituted for ammonium bicarbonate, while at least 0.6 % sodium cholate in the buffers was essential.