Nucleotides and microtubule-associated proteins are important in the regulation of microtubule-mediated functions in cells. Continued effort will be made to clarify the role of nucleotides in brain microtubule assembly by turbidimetric measurements, electron microscopy and 31P nuclear magnetic resonance studies using purified tubulin, microtubule-associated proteins and phosphorothioate analogues of guanosine nucleotides. Two enzymes, nucleoside diphosphate kinase (NDPK) and tubulin-stimulated ATPase, which are found in brain microtubule protein fractions prepared by several cycles of assembly-disassembly will be studied in detail. These enzymes will be purified and used to answer the following questions: 1) What are the mechanisms by which NDPK and ATPase effect in vitro microtubule polymerization? 2) Does NDPK phosphorylate GDP bound to the exchangeable site of tubulin? 3) Does NDPK or ATPase interact with tubulin, microtubules and/or microtubule-associated proteins? 4) Do thiophosphate analogues of GTP, GDP, ATP and/or ADP interact with microtubule-associated NDPK or ATPase? 5) Does NDPK or ATPase have an effect on a proposed ATP binding site of tubulin? In addition, monoclonal and/or polyclonal antibodies raised to microtubule-associated NDPK and ATPase will be used for immunocytochemical localization in different cell types and to determine tissue specificity. These studies should help to elucidate the role of nucleotides and microtubule-associated enzymes on the cytoskeleton and increase our understanding of microtubule functions. They should also contribute to our understanding of age-dependent alterations of microtubule-associated enzyme activities in brain.
