The identification, purification, and characterization of a pancreatic beta-cell form of the microtubule adenosine triphosphatase kinesin.
Microtubules have been implicated as being necessary for the secretion of insulin from beta-cells, although the mechanism by which cytoplasmic microtubules contribute to the release of insulin is unknown. Kinesin is a microtubule-dependent adenosine triphosphatase (ATPase) that is thought to be responsible for the intracellular transport of vesicles and organelles. In this manuscript, the purification and preliminary characterization of a beta-cell form of kinesin is described. A 120-kilodalton antikinesin-reactive polypeptide was identified on blots when cultured insulinoma tumor cell lines were subjected to immunoblot analysis using monoclonal antibodies specific for the heavy chain of mammalian kinesin. The beta-cell form of kinesin was isolated from solid rat insulinoma tumors by cosedimentation of the kinesin with microtubules from tissue homogenates in the presence of adenylyl-imidodiphosphate. The beta-cell kinesin was further purified by gel filtration chromatography, and then the pure enzyme was characterized using in vitro assays. Although beta-cell kinesin showed little ATPase activity alone, the enzyme exhibited considerable ATP hydrolysis activity in the presence of taxol-stabilized microtubules. Moreover, in motility assays beta-cell kinesin was able to translocate microtubules across microscope coverslips in the presence of Mg(2+)-ATP. In summary, we report the identity of a novel islet beta-cell form of the microtubule-dependent ATPase kinesin and suggest a possible contribution of the microtubule cytoskeleton in insulin secretion.
Balczon
R
R
Department of Structural and Cellular Biology, University of South Alabama, Mobile 36688.
Overstreet
K A
KA
Zinkowski
R P
RP
Haynes
A
A
Appel
M
M
D000818:Animals; D001921:Brain / Q000201:enzymology; D015151:Immunoblotting; D007340:Insulinoma / Q000201:enzymology; D007515:Islets of Langerhans / Q000201:enzymology*; D016547:Kinesins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D010190:Pancreatic Neoplasms / Q000201:enzymology; D051381:Rats; D014407:Tumor Cells, Cultured
D016547:Kinesins