Abstract
Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by nucleotides was examined using pure enzyme and Triton X-100/phosphatidate-mixed micelles. Adenosine, guanosine, cytidine, and uridine nucleotides inhibited phosphatidate phosphatase activity in a dose-dependent manner. ATP and CTP were the most potent inhibitors of the enzyme. A kinetic analysis was performed to determine the mechanism of enzyme inhibition by nucleotides. The mechanism of inhibition by ATP and CTP with respect to phosphatidate (the substrate) was complex. The dependence of phosphatidate phosphatase activity on phosphatidate was cooperative, and nucleotides affected both Vmax and Km. ATP did not inhibit phosphatidate phosphatase activity by binding to the enzyme or to phosphatidate. Phosphatidate phosphatase dependence on Mg2+ ions (the cofactor) followed saturation kinetics, and the mechanism of nucleotide inhibition with respect to Mg2+ ions was competitive. Thus, the mechanism of enzyme inhibition by nucleotides was the chelation of Mg2+ ions. The inhibitor constant for ATP was lower than its cellular concentration in glucose-grown cells. However, the inhibitor constant for ATP was higher than its cellular concentration in glucose-starved cells. Changes in the cellular concentration of ATP affected the proportional synthesis of triacylglycerols and phospholipids. These results were consistent with the regulation of phosphatidate phosphatase activity by ATP through a Mg2+ ion chelation mechanism.
Highlights
Enzyme.A kinetic analysis wapserformed to determine The substrate andproduct of the PA phosphatase reaction are the mechanism of enzyminehibitionby nucleotides.The mechanism ofinhibitionby ATPand CTP with respect phosphatidate(thesubstrate) was compleTxh. e dependence of phosphatidatephosphataseactivityonphosphatidatewascooperative,andnucleotidesaffected both V, and K
On Mg2‘ ions followed saturation kinetics, The nucleotides ATP and CTP are substrates in the pathways andthemechanism of nucleotide inhibition with re- leading to the synthesisof the energy-rich intermediates CDP
The mechaniestmhanolamine and CDP-choline [2] (Fig. 1).These intermediof enzymeinhibitionby nucleotides was the chelatiofn ates combine with theproduct of the PA phosphatase reaction, Mg2‘ ions
Summary
Enzyme Assays and Protein Determination-PA phosphatase activ- were maintained on 1% yeast extract, 2% peptone, 2%glucose medium ity was measuredfor 20 min by following the release of water-soluble plates containing 2% Bacto-agar. To label lipids,cells were grown in the m~ 2-mercaptoethanol, 2 mM MgCl,, 1 m~ Triton X-100, 0.1 mM PA, presence of [2-14C]acetate(0.5mCi/ml). Half of the culture was washed and anappropriate dilutionof PA phosphatase ina total volume of 0.1 twice with growth medium containing glucose, and half of the culture ml. Preparation of Diton X-IOOIPA-mixed Micelles-PA in chloroform ids on the chromatograms were determinedby fluorography and comwas transferred tao test tube, and solvent waresmoved in uacuo for 40 pared with standard lipids after exposure to iodvinaepor. Sephacryl S-200 Chromatography-A Sephacryl S-200 column (1X 11cm) was equilibrated with 50 m~ Tris-maleate buffer (pH7.0) containing 10 m~ 2-mercaptoethanol, 2 mM MgCl,, and 0.35 mM Triton X-100 at room temperature.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
