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Dynamin’s basal (unassembled) GTPase activity.
On several occasions we have measured WT dynamin’s GTPase activity in the absence of lipid nanotubes (whilst otherwise maintaining our normal assaying conditions). We have found the activity to range between 1/200 and 1/1000 of that observed when lipid nanotubes are included. (This variability is probably in part due to the low signal that we are measuring and consequent high relative background and error values in addition to variations in dynamin preparations and degree of self assembly.) This corresponds to Kcats in the approximate range of 0.002-0.01s-1. This is lower but roughly comparable to the figure of 0.04s-1 quoted by Sever et. al. 1, although their value was determined under lower ionic strength conditions (50mM as compared to 150mM). It is however similar to values reported by Binns et. al. 7 (0.0046 to 0.0061s-1) under high salt assay conditions. When we assay dynamin’s GTPase activity under low ionic strength conditions (50mM) we have obtained Kcat values of up to 0.4s-1. We believe that the increase in activity at low ionic strength is due to spontaneous dynamin oligomerisation as has been previously reported6 8 9. Variability in this basal activity, most likely represents variable amounts of spontaneous oligomerisation in different dynamin preparations. |
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References
1. Sever, S., Muhlberg, A. B. & Schmid, S. L. Impairment of dynamin's GAP domain stimulates receptor-mediated endocytosis. Nature 398, 481-486 (1999). 2. Sever, S., Damke, H. & Schmid, S. L. Dynamin:GTP controls the formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis. J Cell Biol 150, 1137-1148 (2000). 3. Muhlberg, A. B., Warnock, D. E. & Schmid, S. L. Domain structure and intramolecular regulation of dynamin GTPase. EMBO J. 16, 6676-6683 (1998). 4. Okamoto, P. M., Tripet, B., Litowski, J., Hodges, R. S. & Vallee, R. B. Multiple distinct coiled-coils are involved in dynamin self-assembly. J Biol Chem 274, 10277-10286 (1999). 5. Herskovits, J. S., Burgess, C. C., Obar, R. A. & Vallee, R. B. Effects of mutant rat dynamin on endocytosis. J. Cell Biol. 122, 565-578 (1993). 6. Owen, D. J. et al. Crystal structure of the Amphiphysin-2 SH3 domain and its role in prevention of dynamin ring formation. EMBO J. 17, 5273-5285 (1998). 7. Binns, D. D. et al. The mechanism of GTP hydrolysis by dynamin II: a transient kinetic study. Biochemistry 39, 7188-7196 (2000). 8. Hinshaw, J. E. & Schmid, S. L. Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature 374, 190-192 (1995). 9. Warnock, D. E., HInshaw, J. E. & Schmid, S. L. Dynamin self-assembly stimulates its GTPase activity. J. Biol. Chem. 271, 22310-22314 (1996). |
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