Journal article
Photoaffinity labeling of the 45-kDa and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae
The Journal of biological chemistry, v 268(32), pp 24083-24088
15 Nov 1993
PMID: 8226954
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
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.
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Details
- Title
- Photoaffinity labeling of the 45-kDa and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae
- Creators
- G M Carman - Rutgers, The State University of New JerseyJoseph T Nickels - Biochemistry and Molecular Biology
- Publication Details
- The Journal of biological chemistry, v 268(32), pp 24083-24088
- Publisher
- Elsevier
- Grant note
- GM-35655 / NIGMS NIH HHS
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology
- Web of Science ID
- WOS:A1993MF29400058
- Scopus ID
- 2-s2.0-0027428748
- Other Identifier
- 991021229993504721
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- Web of Science research areas
- Biochemistry & Molecular Biology