Identity and origin of the ATPase activity associated with neuronal microtubules. II. Identification of a 50,000-dalton polypeptide with ATPase activity similar to F-1 ATPase from mitochondria.
We determined that the ATPase activity contained in preparations of neuronal microtubules is associated with a 50,000-dalton polypeptide by four different methods: (a) photoaffinity labeling of the pelletable ATPase fraction with [gamma-32P]-8-azido-ATP; (b) analysis of two-dimensional gels (native gel X SDS slab gel) of an ATPase fraction solubilized by treatment with dichloromethane; (c) ATPase purification by glycerol gradient sedimentation and gel filtration chromatography of a solvent-released ATPase fraction, (d) demonstration of the binding of affinity-purified antibody to the 50-kdalton polypeptide to ATPase activity in vitro. Beginning with preparations of microtubules we have purified the ATPase activity greater than 700-fold and estimate that the purified enzyme has a specific activity of 20 mumol Pi x mg-1 x min-1 and comprises 80-90% of the total ATPase activity associated with neuronal microtubules. With affinity-purified antibody we also demonstrate cross-reactivity to the 50-kdalton subunits of mitochondrial F-1 ATPase and show that the antibody specifically labels mitochondria in PtK-2 cells. Biochemical comparisons of the enzymes reveal similar but not identical subunit composition and sensitivity to mitochondrial ATPase inhibitors. These studies indicate that the principal ATPase activity associated with microtubules is not contained in high molecular weight proteins such as dynein or MAPs and support the hypothesis that the 50-kdalton ATPase is a membrane protein and may be derived from mitochondria or membrane vesicles with F-1-like ATPase activity.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D008297:Male; D008752:Methylene Chloride / Q000494:pharmacology; D008854:Microscopy, Electron; D008870:Microtubules / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009474:Neurons / Q000201:enzymology* / Q000648:ultrastructure; D010455:Peptides / Q000302:isolation & purification*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D013552:Swine
D010455:Peptides; D008752:Methylene Chloride; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases