Correlation between the ATPase and microtubule translocating activities of sea urchin egg kinesin

Abstract

Coupling between ATP hydrolysis and microtubule movement was demonstrated several years ago in flagellar axonemes and subsequent studies suggest that the relevant microtubule motor, dynein, uses ATP to drive microtubule sliding by a cross-bridge mechanism analogous to that of myosin in muscles. Kinesin, a microtubule-based motility protein which may participate in organelle transport and mitosis, binds microtubules in a nucleotide-sensitive manner, and requires hydrolysable nucleotides to translocate microtubules over a glass surface. Recently, neuronal kinesin was shown to possess microtubule-activated ATPase activity although coupling between ATP hydrolysis and motility was not demonstrated. Here we report that sea urchin egg kinesin, prepared either with or without a 5'-adenylyl imidodiphosphate(AMPPNP)-induced microtubule binding step, also possesses significant microtubule-activated ATPase activity when Mg-ATP is used as a substrate. This ATPase activity is inhibited in a dose-dependent manner by addition of Mg-free ATP, by chelation of Mg2+ with EDTA, by addition of Na3VO4, or by addition of AMPPNP with or without Mg2+. Addition of these same reagents also inhibits the microtubule-translocating activities of sea urchin egg kinesin in a dose-dependent manner, supporting the hypothesis that kinesin-driven motility is coupled to the microtubule-activated Mg2+-ATPase activity.

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains.

“…General biochemical procedures were carried out as described previously (Scholey et al, 1984(Scholey et al, , 1985Porter et al, 1987Porter et al, , 1988Cohn et al, 1987). Immunological procedures were carried out following the advice of colleagues, in particular Drs.…”

“…The ability of test antibodies to inhibit MT motility was assayed using videoenhanced microscopy and computer-assisted velocity analysis of kinesin/MT samples as described previously (Cohn et al, 1987). 14 I.tl of 100-300 t.tg/ml kinesin solutions were adsorbed onto coverslips for 10-20 min at room temperature in a humidified chamber before addition of the test antibodies.…”

“…Purification of kinesin was carried out essentially as described previously (Scholey et al, 1985;Cohn et al, 1987). Sea urchin eggs were homogenized in PMEG extraction buffer (0.9 M glycerol, 0.I M Pipes, pH 6.9, 5 mM EGTA, 2.5 mM MgSO4, 0.5 mM EDTA, 1 mM DTT, 1 mg/ml p-tosyl-L-arginine methyl ester hydrochloride, 100 gg/ml soybean trypsin inhibitor, 10 gg/ml leupeptin, pepstatin, and aprotinin), and centrifuged to yield the "high speed extract supernatant" (HSS), which was treated with hexokinase, glucose, taxol/GTP, plus AMPPNP and the resulting microtubules were pelleted.…”

“…One such enzyme, kinesin (Vale et al, 1985a), binds microtubules in a nucleotide sensitive fashion, and displays a microtubule-activated ATPase activity that drives movement of kinesin-coated objects towards the plus end of microtubules (MTs) ~ in vitro (Vale et al, 1985a, b;Scholey et al, 1985;Brady, 1985; Kuznetsoy and Gelfand, 1986;Porter et al, 1987;Cohn et al, 1987;Saxton et al, 1988;Gelles et al, 1988). Kinesin from a variety of animals contains a major polypeptide with an apparent molecular mass of 110-140 kD, encoded by a single gene in Drosophila (Yang et al, 1988), plus copurifying light chains of Mr = 40-80 kD (Vale et al, 1985a,b;Amos, 1987;Kuznetsov and Gelfand, 1986;Saxton et al, 1988).…”

“…Chromatography resins and antibody conjugates were usually from Bio-Rad Laboratories, Richmond, CA. Cellulose phosphate (Pll) and DEAE cellulose (DE 52) resins were from Whatman Inc., Hillsboro, OR.General biochemical procedures were carried out as described previously (Scholey et al, 1984(Scholey et al, , 1985Porter et al, 1987Porter et al, , 1988Cohn et al, 1987). Immunological procedures were carried out following the advice of colleagues, in particular Drs.…”