Photoaffinity labeling of the 45-kDa and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae.
The membrane-associated 45- and 55-kDa forms of phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) from Saccharomyces cerevisiae are inhibited by ADP by a competitive mechanism with respect to ATP. We initiated studies toward defining the ATP and ADP sites on the PI 4-kinases using azidonucleotide photoaffinity labeling probes. The photoprobe 8-azido-ATP fulfilled the criteria of a specific photoaffinity label for the 45- and 55-kDa PI 4-kinases. 8-Azido-ATP was a substrate and a competitive inhibitor of the PI 4-kinases with Ki values similar to the Km for ATP. 8-Azido-ATP photoinactivated the enzymes and was photoincorporated into the enzymes in a dose-dependent manner at concentrations similar to the Ki values for the photoprobe. ATP, the true substrate, provided specific protection against photoinactivation and photoincorporation of the PI 4-kinases with 8-azido-ATP, whereas GTP, a nonspecific nucleotide, provided no protection against photoinactivation and photoincorporation. Photoaffinity labeling of the PI 4-kinases with 8-azido-ATP was specifically prevented with ADP. The photoprobe 8-azido-ADP also fulfilled the criteria needed to validate its use as a specific photoprobe for the PI 4-kinases. Photoinactivation of the PI 4-kinases with 8-azido-ADP was prevented specifically with ATP. Taken together, these data supported the conclusion that the ATP and ADP sites on the membrane-associated 45- and 55-kDa PI 4-kinases from S. cerevisiae were the same.
Nickels
J T
JT
Department of Food Science, Cook College, Rutgers University, New Brunswick, New Jersey 08903.
Carman
G M
GM
D019870:1-Phosphatidylinositol 4-Kinase; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels; D001386:Azides / Q000494:pharmacology; D007700:Kinetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010777:Photochemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D000345:Affinity Labels; D001386:Azides; D029701:Saccharomyces cerevisiae Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019870:1-Phosphatidylinositol 4-Kinase; C441760:PIK1 protein, S cerevisiae