Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by sphingoid bases.
The regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by sphingoid bases was examined using Triton X-100/lipid-mixed micelles. Sphingosine, phytosphingosine, and sphinganine inhibited purified preparations of the 104- and 45-kDa forms of phosphatidate phosphatase in a dose-dependent manner. The structural requirements for the sphingoid base inhibition of phosphatidate phosphatase activity were a free amino group and a long chain hydrocarbon. A detailed kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase inhibition by sphingoid bases. The phosphatidate phosphatase dependence on phosphatidate was cooperative (Hill numbers of approximately 2) in the absence and presence of sphingoid bases. Sphingosine, phytosphingosine, and sphinganine were parabolic competitive inhibitors of phosphatidate phosphatase activity. This indicated that more than one inhibitor molecule contributed to the exclusion of phosphatidate from the enzyme. The aKi values (inhibitor constants) for sphingosine, phytosphingosine, and sphinganine were 1.5, 0.4, and 0.2 mol %, respectively, and the Km value for phosphatidate was 2.2 mol %. The cellular concentrations of free phytosphingosine and sphinganine were 0.16 and 0.53 mol %, respectively, relative to the total phospholipids in S. cerevisiae. The cellular concentrations of phytosphingosine and sphinganine were in the range of the aKi values for these sphingoid bases. These results raised the suggestion that phosphatidate phosphatase activity may be regulated in vivo by sphingoid bases.
Wu
W I
WI
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903.
Lin
Y P
YP
Wang
E
E
Merrill
A H
AH
Carman
G M
GM
D000588:Amines / Q000494:pharmacology; D001667:Binding, Competitive; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D010711:Phosphatidate Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011609:Psychosine / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000588:Amines; D011609:Psychosine; D010711:Phosphatidate Phosphatase; C009317:stearylamine; C012491:phytosphingosine; D013110:Sphingosine; C005682:safingol