Light chains of sea urchin kinesin identified by immunoadsorption.
Previous studies with monoclonal antibodies indicate that sea urchin kinesin contains two heavy chains arranged in parallel such that their N-terminal ends fold into globular mechanochemical heads attached to a thin stalk ending in a bipartite tail [Scholey et al., 1989]. In the present, complementary study, we have used the monoclonal antikinesin, SUK4, to probe the quaternary structure of sea urchin (Strongylocentrotus purpuratus) kinesin. Kinesin prepared from sea urchin cytosol sedimented at 9.6 S on sucrose density gradients and consisted of 130-kd heavy chains plus an 84-kd/78 kd doublet (1 mol heavy chain: 1 mol doublet determined by gel densitometry). Low levels of 110-kd and 90-kd polypeptides were sometimes present as well. The 84-kd/78 kd polypeptides are thought to be light chains because they were precipitated from the kinesin preparation at a stoichiometry of one mol doublet per 1 mol heavy chain using SUK4-Sepharose immunoaffinity resins. The 110-kd and 90-kd peptides, by contrast, were removed using this immunoadsorption method. SUK4-Sepharose immunoaffinity chromatography was also used to purify the 130-kd heavy chain and 84-kd/78-kd doublet (1 mol heavy chain: 1 mol doublet) directly from sea urchin egg cytosolic extracts, and from a MAP (microtubule-associated protein) fraction eluted by ATP from microtubules prepared in the presence of AMPPNP but not from microtubules prepared in ATP. The finding that sea urchin kinesin contains equimolar quantities of heavy and light chains, together with the aforementioned data on kinesin morphology, suggests that native sea urchin kinesin is a tetramer assembled from two light chains and two heavy chains.
Johnson
C S
CS
Department of Zoology, University of California, Davis 95616.
Buster
D
D
Scholey
J M
JM
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D007163:Immunosorbent Techniques; D016547:Kinesins; D046911:Macromolecular Substances; D008868:Microtubule Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009865:Oocytes; D012617:Sea Urchins
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D008868:Microtubule Proteins; D000251:Adenosine Triphosphatases; D016547:Kinesins