Nucleotide interconversions in microtubule protein preparations, a significant complication for accurate measurement of GTP hydrolysis in the presence of adenosine 5'-(beta, gamma-imidotriphosphate).
Pursuing the observation of Carlier and Pantaloni [Carlier, M.-F., & Pantaloni, D. (1982) Biochemistry 21, 1215-1224] that adenosine 5'-(beta, gamma-imidotriphosphate) (pNHppA) strongly inhibited tubulin-independent phosphatases in microtubule protein preparations, we observed with a number of commercial preparations of pNHppA that a major proportion of the terminal phosphate of [gamma-32P]GTP added to microtubule protein preparations was rapidly converted into ATP. Initially postulating degradation of pNHppA to AMP followed by stepwise conversion of AMP to ATP, we isolated two nucleoside monophosphate kinase activities from microtubule protein capable of generating ATP from AMP + GTP. The amounts of these enzymes in microtubule protein preparations, however, are probably too low to account for rapid ATP formation. Instead, ATP formation most likely is caused by nucleoside diphosphate kinase acting on ADP contaminating commercial pNHppA preparations. Such ADP contamination was demonstrated by high-performance liquid chromatography, with the amount of ATP formed with different pNHppA preparations proportional to the amount of ADP contamination. Repurification of commercial pNHppA until it was free of contaminating ADP also resulted in the elimination of ATP formation. The repurified pNHppA potently inhibited GTP hydrolysis in microtubule protein preparations. In addition, especially when supplemented with equimolar Mg2+, the repurified pNHppA strongly inhibited GTP hydrolysis and microtubule assembly in reaction mixtures containing purified tubulin and heat-treated microtubule-associated proteins (which contain negligible amounts of tubulin-independent phosphatase activity). We conclude that studies of microtubule-dependent GTP hydrolysis which make use of pNHppA must be interpreted with extreme caution.
Batra
J K
JK
Laboratory of Pharmacology and Experimental Therapeutics, National Cancer Institute, Bethesda, Maryland 20892.
Lin
C M
CM
Hamel
E
E
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000263:Adenylate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000302:isolation & purification / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D051528:Guanylate Kinases; D006868:Hydrolysis; D007700:Kinetics; D008868:Microtubule Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D009703:Nucleoside-Phosphate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*
D008868:Microtubule Proteins; D000266:Adenylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000263:Adenylate Kinase; D009703:Nucleoside-Phosphate Kinase; D009701:Nucleoside-Diphosphate Kinase; D051528:Guanylate Kinases