Regulation of the microtubule steady state in vitro by ATP.
ATP increases microtubule steady state assembly and disassembly rates in vitro in a concentration-dependent manner. Bovine brain microtubules, composed of 75% tubulin and 25% high molecular weight microtubule-associated proteins (MAPs), were purified by three cycles of assembly and disassembly in the absence of ATP. When assembled to steady state, these microtubules add dimers at one end and lose them at the other in a unidirectional assembly-disassembly process. In the presence of 1.0 mM ATP the unidirectional flow of tubulin from one end of the microtubules to the other increases as much as 20 fold, as revealed by loss of 3H-GTP from uniformly labeled microtubules under GTP chase conditions and by the rate of disassembly following addition of 50 microM podophyllotoxin. UTP, CTP and 5' adenylylimidodiphosphate (AMP-PNP) cannot substitute for ATP in producing this effect. Furthermore, the increase in steady state flow rate persists afer ATP is removed. Thus microtubules assembled in ATP and centrifuged through sucrose cushions to separate them from nucleotides continue to exhibit increased rates in the next assembly cycle in the absence of ATP. It is possible that an ATP-dependent microtubule protein kinase is responsible for the observed increase in tubulin flow rate. A kinase activity associated with brain MAPs has been reported to be cAMP-dependent (Sloboda et al., 1975). We have found an adenylate cyclase activity associated with these microtubules. Whether the adenylate cyclase is a contaminant or due to a specific microtubules-associated protein, and whether its activity is functionally linked to the increased rate of assembly and disassembly in the presence of ATP, remain to be determined.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D014404:Tubulin; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D000262:Adenylyl Cyclases