Dynein-like Mg"-ATPase in Mitotic Spindles Isolated from Sea Urchin Embryos (Strongylocentrotus droebachiensis)

Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg2+-ATPase resembling axonemal dynein, and a Cat+ -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 M9Cl2, 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+-/Mg2+-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 Mg2+-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 . Dynein is the mechanochemical cross-bridge protein that reversibly interconnects the outer doublet microtubules ofciliary and flagellar axonemes to produce motility (11, 26, 60) . Recent physiological evidence implicates a dynein-like ATPase as the motive force in chromosome movement (4, 26, 47-49) . Biochemical evidence for ATPase activity within the mitotic spindle has come mainly from analysis ofisolated mitotic apparatus from sea urchin eggs (see references 31, 40, 48 for reviews) . The structure, protein composition, and amount and characteristics of ATPase activity reported for the isolated mitotic apparatus have been as varied as the isolation methods themselves (31, 35, 40, 48) . Two distinctly different ATPases have been reported to be constituents of the mitotic apparatus: a Me'-ATPase, also activated by Ca 2+ (29, 33, 61), and a Ca"-ATPase, not signiftcantly activated by Mg" (30, 37, 39-42) . The Mg2+-ATPase resembled axonemal dynein (61). The mitotic Ca"-ATPase, characterized by Petzelt, Mazia, and co-workers (30, 40), is now postulated to be associated with the smooth endoplasmic reticulum that is integral to the mitotic apparatus, and to be involved in actively sequestering calcium ions (40-42) . Mg2+-ATPase activity was initially identified in the isolated mitotic apparatus by Mazia et al . (29) and Miki (33) in the early 1960s. In 1968, Weisenberg and Taylor (61) purified from isolated mitotic apparatus a soluble Mg2+-ATPase that had a sedimentation coefficient and nucleotide and ionic specificities similarto those ofciliary axonemal dynein. The specific activity of the mitotic apparatus was three times the Mg2+-ATPase specific activity of the whole egg cytoplasm (61 ; see also reference 33) . Weisenberg and Taylor suggested that the cytoTHE JOURNAL OF CELL BIOLOGY " VOLUME 86 SEPTEMBER 1980 738-745 ©The Rockefeller University Press " 0021-9525/80/09/0738/08 $1 .00 on F ebuary 1, 2013 jcb.rress.org D ow nladed fom Published September 1, 1980