Kinesin ATPase: rate-limiting ADP release.

Abstract

The ATPase rate of kinesin isolated from bovine brain by the method of S. A. Kuznetsov and V. I. Gelfand [(1986) Proc. NaWl. Acad. Sci. USA 83,[8530][8531][8532][8533][8534]] is stimulated 1000-fold by interaction with tubulin (turnover rate per 120-kDa peptide increases from "zO.009 sec'-to 9 sec-1).The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that Pi release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (K, < 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound (3, 4). This enzyme has been isolated from a number of sources and contains a large subunit of 110-135 kDa and one or more smaller peptides of kDa. The large subunit contains a nucleotide-binding site, as determined by affinity labeling (5), which may be the active site for ATP hydrolysis. Kuznetsov and Gelfand (6) have described a preparation of this enzyme from bovine brain with a low basal rate of ATP hydrolysis that is greatly stimulated by polymerized tubulin. It is reported here that this preparation hydrolyzes ATP with virtually no extra incorporation of water-derived oxygens over a wide range of tubulin and ATP levels, in striking contrast to myosin. Further analysis indicates that this is not due to a complete lack of rate-limiting product release but rather is due to a marked difference between the Pi and ADP release rates. The release of Pi is rapid and occurs before significant reversal of hydrolysis, whereas the release ofADP is extremely slow and represents the rate-limiting step in the absence of tubulin.EXPERIMENTAL METHODS Bovine brain kinesin was prepared essentially as described by Kuznetsov and Gelfand (6) with batch purification on DEAE-cellulose and phosphocellulose prior to affinity purification by binding to tubulin in the presence of triphosphate in excess of Mg2" and by releasing with MgATP. Modifications included the omission of the second DEAE-cellulose step, the inclusion of bovine serum albumin (0.33 mg/ml) in the ATP release buffer, and the replacement of the final gel-filtration step after ATP-induced release from tubulin with an additional phosphocellulose step that concentrates the enzyme and removes tubulin and excess ATP. In this final step, the supernatant after ATP-induced release from tubulin was loaded on a 1 x 3 cm phosphocellulose column (P-11) equilibrated with buffer A (50 mM imidazole/0.5 mM MgCl2/0.1 mM EDTA/1 mM 2-mercaptoethanol, pH 6.7) of Kuznetsov and Gelfand (6). The column was washed with 5 ml of buffer A and then with 5 ml of buffer A supplemented with 100 mM KCl to remove tubulin, bovine serum albumin, free ATP, and triphosphate, and the kinesin was eluted with buffer A supplemented with 600 mM KCl. The peak fractions were pooled (typically 2-3 ml) and dialyzed ov...

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