Diglyceride kinase activity of microtubules. Characterization and comparison with the protein kinase and ATPase activities associated with vinblastine-isolated tubulin of chick embryonic muscles.
Vinblastine-isolated microtubule protein from chick embryonic muscles has an enzymatic activity which catalyzes the formation of phosphatidic acid from diglycerides and ATP. The pH optimum (6.4), sedimentation on sucrose gradients (Mr = 85 000), and sensitivity to ions of this diglyceride kinase activity are different to those of a similar enzymatic activity present in 150 000 X g supernatants of chick embryonic muscle homogenates, suggesting that it is a different species which is associated specifically with the microtubules. The reaction requires a divalent ion (e.g. 0.4 mM Mg2+ gives half-maximal stimulation), and GTP can replace ATP rather effectively, especially at nucleotide concentrations lower than 50 muM. The sedimentation of the diglyceride kinase on sucrose gradients coincides with that of the microtubules-associated protein kinase (Mr = 75 000); the heat-stability and sensivitity to proteolysis of both activities are also very similar. Stimulation of one reaction by the addition of the corresponding exogenous substrate does not impair the phosphorylation of the other, and no radioactivity is lost from phosphatidic acid or the protein moiety upon incubation of pre-labelled microtubules with a large excess of unlabelled ATP or GTP. In addition to diglyceride and protein kinase activities (0.2 and 0.3 nmol 32P-transferred X min-1 X mg-1 microtubular protein, respectively), microtubules also contain an associated ATPase (2.8 nmol X min-1 X mg-1), which requires either Mg2+ or Ca2+, can hydrolyze GTP quite effectively, and sediments with a molecular weight of 95000. The results obtained are discussed in connection with the possible relationships existing among these enzymatic activities, as well as their probable role in microtubular functions.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent; D002642:Chick Embryo; D004075:Diglycerides; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008870:Microtubules / Q000201:enzymology*; D009132:Muscles / Q000201:enzymology*; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism; D014747:Vinblastine
D002413:Cations, Divalent; D004075:Diglycerides; D014404:Tubulin; D014747:Vinblastine; D010770:Phosphotransferases; D011494:Protein Kinases; D000251:Adenosine Triphosphatases; D008274:Magnesium