Possible treadmilling in zinc(II)-induced sheet polymers of bovine brain microtubule protein.
We have examined the ability of zinc(II)-induced sheet polymers, formed from thrice-cycled bovine brain microtubule protein prepared in the absence of glycerol, to exchange with tubulin subunits at steady state. By a rapid filtration assay in which labeled GTP was used as a marker for tubulin addition and loss, we found that steady-state sheet polymers, formed in 0.5 mM-ZnCl2, 1 mM-dithiothreitol, and 100 mM-2-(N-morpholino)ethanesulfonic acid (pH 6.75) in the presence of a GTP-regenerating system at 37 degrees C, incorporated the label in a time-dependent manner to a maximum level. The steady-state uptake of label was inhibited by colchicine, podophyllotoxin and vinblastine. In pulse-chase experiments, we observed that label added onto sheet polymers in a short pulse was retained for a period equal to that required by the polymers to become fully labeled in a continuous pulse; thereafter, the label was lost gradually to a baseline level. An average of 82% of the label was retained in the sheet polymers after a "cold" chase of equal duration to the time of the pulse. Sheet polymers assembled from microtubule protein prepared in the presence of glycerol gave similar results. Using a double-labeling procedure to analyze tubulin addition and loss simultaneously, we found that the rates of steady-state addition and loss were similar. Sheet polymers retained their structural integrity throughout these experiments, as determined by electron microscopy. We believe that the data are consistent with a "treadmilling" mechanism of polymerization and depolymerization, analogous to that documented to occur in steady-state microtubules in vitro. Such a mechanism is discussed in the context of recent findings from structural studies, and a model consistent with established structural data is offered.
D000818:Animals; D001704:Biopolymers; D001923:Brain Chemistry; D002417:Cattle; D005990:Glycerol; D006160:Guanosine Triphosphate; D007700:Kinetics; D008854:Microscopy, Electron; D014404:Tubulin / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology*
D001704:Biopolymers; D014404:Tubulin; D006160:Guanosine Triphosphate; D015032:Zinc; D005990:Glycerol