Activation of rat liver cytosolic phosphatidic acid phosphatase by nucleoside diphosphates.
Phosphatidic acid phosphatase (EC 3.1.3.4) was purified 30-fold by ammonium sulfate fractionation and hydroxyapatite chromatography from the soluble fraction of rat liver. ADP was found to stimulate the enzyme activity with half-maximal stimulation at 0.2 mM. Similar effects were seen when ADP was replaced by GDP or CDP. In contrast, ATP inhibited the enzyme; half-maximal inhibition observed at 0.2 mM. Again, the degree of inhibition did not differ when GTP or CTP replaced ATP. Thus, the structure of the base part of the nucleotide was not critical for mediating these effects. The positions of the phosphate groups in the nucleotide structure were however found to be of importance for the enzyme activity. Variations in the structure of the phosphate ester bound at the 5'-position had a pronounced effect on phosphatidic acid phosphatase activity. The effect of nucleotides depended on pH, and the inhibition by ATP was more pronounced at pH levels lower than 7.0, whereas the stimulatory effect of ADP was virtually the same from pH 6.0 to pH 8.0. The enzyme showed substrate saturation kinetics with respect to phosphatidic acid, with an apparent Km of 0.7 mM. Km increased in the presence of ATP, whereas both apparent Vmax and Km increased in the presence of ADP, suggesting different mechanisms for the action of the two types of nucleotides. The results indicated that physiological levels of nucleotides with a diphosphate or a triphosphate ester bound at the 5'-position of the ribose moiety influenced the activity of phosphatidic acid phosphatase. The possibility is discussed that these effects might be of importance for the regulation of triacylglycerol biosynthesis.
Berglund
L
L
Department of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, Sweden.
Björkhem
I
I
Angelin
B
B
Einarsson
K
K
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D003565:Cytidine Diphosphate / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D009711:Nucleotides / Q000494:pharmacology*; D010711:Phosphatidate Phosphatase / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D009711:Nucleotides; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D003565:Cytidine Diphosphate; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D010711:Phosphatidate Phosphatase; D008274:Magnesium