Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae.
Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity was associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.
Lin
Y P
YP
Department of Food Science, Rutgers University, New Brunswick, New Jersey 08903.
Carman
G M
GM
D019826:Cholic Acid; D002793:Cholic Acids; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D005989:Glycerides / Q000494:pharmacology; D006882:Hydroxyapatites; D007700:Kinetics; D008566:Membranes / Q000201:enzymology; D010711:Phosphatidate Phosphatase / Q000302:isolation & purification* / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D002793:Cholic Acids; D005989:Glycerides; D006882:Hydroxyapatites; D010743:Phospholipids; D017886:Durapatite; D010744:Phosphoric Monoester Hydrolases; D010711:Phosphatidate Phosphatase; D019826:Cholic Acid