Roy G. Burns

Multiple phosphorylation sites of microtubule-associated protein (MAP2) observed at high ATP concentrations

Microtubule protein,purified by cycles of assembly and disassembly, contains associated non-tubulin proteins, which include two high-molecular weight proteins termed MAP, and MAP* [l-4]. MAP, is of particular interest, as it is the preferred substrate for phosphorylation by an endogenous CAMP-dependent protein kinase [S-9]. In [3-51 the MAP, protein was reported to be phosphorylated to the extent of 3 mol/mol protein in the presence of CAMP. In [7, 1 OJ 1 ] a phosphoprotein phosphatase has been reported and the protein kinase/phosphoprotein phosphatase suggested to determine the final extent of phosphorylation [7]. The existence of such an equilibrium hinders any calculation of the maximum number of phosphorylation sites. Here, we examine the pattern and stoichiometry of phosphorylation of microtubule protein as a function of ATP concentration, and observe that the stoichiometry of MAP, phosphorylation is significantly higher than in earlier reports.

Direct incorporation of microtubule oligomers at high GTP concentrations

Chick brain microtubule protein consists primarily of a mixture of MAP2 : tubulin oligomers and dimeric tubulin. The assembly of this protein is described by a single pseudofirst‐order reaction at 20 μM GTP, but by the summation of two pseudofirst‐order reactions at 1 mM GTP. The protein contains two GTP‐binding species, corresponding to the tubulin dimers and the oligomers, and conditions which alter the dimer : oligomer equilibrium, affect the kinetics of microtubule assembly. The results indicate that the oligomers are only direct assembly intermediates at high GTP concentrations.

Modulation of the Kinetic Parameters of Microtubule Assembly by MAP‐2 Phosphorylation, the GTP/GDP Occupancy of Oligomers, and the Tubulin Tyrosylation Status

in which [S] is the concentration of free “subunit,” [MI is the number concentration of microtubule ends, and k,, and k-, are the respective association and dissociation rate constants. One clear consequence of this analysis, and also of the subsequent consideration of the assembly and disassembly at the two polymer ends,’v4 is that the cell could regulate the rate of assembly by controlling either the effective subunit concentration of k+,, but could only regulate the rate of disassembly by controlling

Assembly of microtubules with ATP: evidence that only a fraction of the protein is assembly-competent

Chick brain microtubule protein can be assembled in vitro with ATP, although the extent of assembly is less than that with GTP. The ATP‐induced assembly is not the result of generation of GTP by the co‐purifying nucleoside diphosphate kinase. Neither an observed increase in the critical concentration nor the phosphorylation of MAP2 can account for the decreased extent of assembly. However, whereas microtubules are formed with both ATP and GTP, incubation with ATP yields additional filaments and polymorphic aggregates. The results demonstrate that of the total protein which can be assembled into microtubules by GTP, about 25–35% is assembled into other structural forms in the presence of ATP.

The GTP Concentration Modulates the Association Rate Constant for Microtubule Assembly

Guanosine triphosphate (GTP) binds to a readily exchangeable E-site on the tubulin (Tu) dimer. Microtubule (MT) assembly affects the site and renders it relatively nonexchangeable, and also promotes the hydrolysis of bound GTP to guanosine diphosphate (GDP). Unfractionated cold-dissociated MT protein is, though, a mixture of Tu dimers, free MAP-2, and MAP-2:Tu oligomers, and both Tu dimers and the

Purification of a soluble monoisozyme of nucleoside diphosphate kinase from chick brain: exploitation of ionic characteristics

A procedure for the rapid purification of nucleoside diphosphate kinase, 24 h with a single operator, from the chick brain soluble fraction is described. The influence of the ionic conditions on the association-disassociation properties of the enzyme are exploited to obtain yields of 30% from the crude homogenate. The enzyme has been purified 500-fold with a maximal specific activity of 1500 mumol/min/mg at 25 degrees C (using thymidine diphosphate as the phosphate acceptor and ATP as the donor) and is demonstrated to be monoisozymic.

Specificity and biological significance of microtubule-associated protein-DNA interactions in chick

The interactions between chick brain microtubule associated proteins (MAPs) and chick DNA have been examined using DNA-cellulose chromatography, cross-blotting, and nitrocellulose filter-binding. Comparison of nitrocellulose filter-binding and cross-blotting results show that while MAPs and a minor, Mr 48,000, protein show significant binding at 50 mM NaCl, only the latter continues to bind a significant amount of DNA at 150 mM NaCl, suggesting an ionic basis for the MAP-DNA interactions. MAP-DNA interactions also show weak preference for AT-rich fractions, and are sensitive to S1 nuclease digestion. We suggest that the MAPs bind preferentially to single-stranded DNA. The binding may involve an interaction between the DNA phosphates and the highly cationic tubulin-binding domain of the MAPs. Repetitive fractions of the chick genome prepared both by hydroxyapatite chromatography and by S1 nuclease digestion show binding to a number of minor proteins present in preparations of microtubule proteins, as well as to the MAPs. We conclude that the MAPs probably do not bind specifically to repetitive DNA, in contrast to earlier reports using mouse DNA. MAP-DNA interactions are therefore unlikely to be involved in the attachment of microtubules to mitotic chromosomes.