ISSN:
0886-1544
Keywords:
kinesin
;
molecular structure
;
immunoaffinity purification
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
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 bipartitetail [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 cyto-sol 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 densitomctry). 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 equi-molar quantities of heavy und 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.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/cm.970160307
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