Dynein-like Mg2+-ATPase in mitotic spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis).

Abstract

Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg 2+-ATPase resembling axonemal dynein, and a Ca t+ -ATPase postulated to be bound in membranes. To examine the nature of the Mgt+ -ATPase, we isolated membranefree mitotic spindles from Strongylocentrotus droebachiensis embryos by rapidly lysing them in a calcium-chelating, low-ionic-strength buffer (5 mM EGTA, 0.5 MM M9Cl 2, 10 mM PIPES, pH 6.8) that contained 1% Nonidet P-40. The fibrous isolated mitotic spindles closely resembled spindles in living cells, both in general morphology and in birefringence. In electron micrographs, the spindles were composed primarily of microtubules, free from membranes and highly extracted of intermicrotubular cytoplasmic ground substance. As analyzed by SDSpolyacrylamide gel electrophoresis (SIDS-PAGE), the pelleted spindles contain 18% tubulin, variable amounts of actin (2-8%), and an unidentified protein of 55 kdaltons in a constant weight ratio to tubulin (1 :2 .5) . The isolated spindles also contained two polypeptides, larger than 300 kdaltons, that comigrated with egg dynein polypeptides, and ATPase activity (0 .02 pmol Pi/mg-min) that closely resembled both flagellar and egg dynein . The spindle Mgt+-ATPase showed a ratio of Cat+ -/Mg 2+ -ATPase = 0.85, had minimal activity in KCI and EDTA, and cleaved GTP at 35% of the rate of ATP. The Mg2+ -ATPase was insensitive to ouabain or oligomycin . The spindle Mg 2+-ATPase was inhibited by sodium vanadate but, like egg dynein, was less sensitive to vanadate than flagellar dynein . The spindle Mg 2+-ATPase does not resemble the mitotic Ca t+-ATPase described by others . We propose that the spindle Mg2 +-ATPase is egg dynein . Bound carbohydrate on the two high-molecular-weight polypeptides of both egg dynein and the spindle enzyme suggest that these proteins may normally associate with membranes in the living cell .

Characterization of a Novel Proteinous Toxin from Sea Anemone Actineria villosa

“…Avt120 cytotoxicity was magnesium and calcium dependent and reduced when these were chelated with EDTA. This effect is similar to that of an ATPase purified from sea urchin embryos [15].…”

A microtubule-activated ATPase from sea urchin eggs, distinct from cytoplasmic dynein and kinesin.

“…This enzyme has received considerable attention because it was found in preparations of isolated mitotic spindles (3) and because some of its pharmacological characteristics were consistent with those reported for reactivated mitotic spindle models (4,5). Cytoplasmic dynein has enzymological characteristics very similar to those of ciliary and flagellar axonemal dynein (6)(7)(8)(9).…”

Isolation of sea urchin egg microtubules with taxol and identification of mitotic spindle microtubule-associated proteins with monoclonal antibodies.

“…Pratt and co-workers have recently described an enzyme present in sea urchin cytosol with enzymatic and physical characteristics similar to axonemal dynein (30,31). Such an enzyme could be involved in the mechanochemistry of the spindle.…”