REGULATION OF PHOSPHATIDATE PHOSPHATASE-ACTIVITY FROM THE YEAST SACCHAROMYCES-CEREVISIAE BY SPHINGOID BASES

The regulation of Saccharomyces cerevisiae membrane-associated phospha tidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) a ctivity by sphingoid bases was examined using Triton X-100/lipid-mixed micelles. Sphingosine, phytosphingosine, and sphinganine inhibited pu rified preparations of the 104- and 45-kDa forms of phosphatidate phos phatase in a dose-dependent manner. The structural requirements for th e sphingoid base inhibition of phosphatidate phosphatase activity were a free amino group and a long chain hydrocarbon. A detailed kinetic a nalysis was performed to determine the mechanism of phosphatidate phos phatase inhibition by sphingoid bases. The phosphatidate phosphatase d ependence on phosphatidate was cooperative (Hill numbers of approximat ely 2) in the absence and presence of sphingoid bases. Sphingosine, ph ytosphingosine, and sphinganine were parabolic competitive inhibitors of phosphatidate phosphatase activity. This indicated that more than o ne inhibitor molecule contributed to the exclusion of phosphatidate fr om the enzyme. The aK(i) values (inhibitor constants) for sphingosine, phytosphingosine, and sphinganine were 1.5, 0.4, and 0.2 mol %, respe ctively, and the K(m) value for phosphatidate was 2.2 mol %. The cellu lar 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 sphi nganine were in the range of the aK(i) values for these sphingoid base s. These results raised the suggestion that phosphatidate phosphatase activity may be regulated in vivo by sphingoid bases.