The microtubule-associated nucleoside diphosphate kinase.
Microtubule protein prepared by cycles of assembly-disassembly contains a nucleoside diphosphate kinase (NDP kinase) activity. We have isolated the NDP kinase responsible for this activity from twice-polymerized bovine brain microtubule protein by a five-step chromatographic procedure. The molecular weight of this enzyme was 103,000 +/- 7,000 daltons as determined by sedimentation equilibrium experiments performed with a Beckman Airfuge. A doublet of subunit bands with molecular masses of about 18,000 daltons was detected by silver staining after gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this preparation. We conclude that the enzyme is a hexamer, although we cannot identify the mix of subunits. We were able to isolate only nanogram quantities of this enzyme, too little for extensive studies, so we isolated the enzyme directly from bovine brain without a preliminary microtubule protein isolation. The whole-brain NDP kinase was isolated by the same chromatographic steps as the enzyme from microtubule protein preparations. Both enzymes had a doublet of subunits at the same molecular weights and both were the same isozyme, chromatofocusing at a pH of 8.0. Both enzymes had similar kinetic properties and similar thermal inactivation profiles. These similar properties of the two enzymes suggest that they are identical. Both subunits of NDP kinase could be reversibly phosphorylated by ATP. Phosphorylation of the native enzyme created multiple, more acidic forms that retained activity. The isolation of this NDP kinase, which can copurify with microtubule protein through cycles of assembly-disassembly, will facilitate future studies on the role of this enzyme in the mechanism and regulation of microtubule assembly.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007525:Isoelectric Focusing; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008869:Microtubule-Associated Proteins; D008970:Molecular Weight; D009701:Nucleoside-Diphosphate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D011506:Proteins / Q000032:analysis*
D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D011506:Proteins; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase