Affinity purification and subcellular localization of kinesin in human neutrophils.
Studies of granule-microtubule interactions in human neutrophils have suggested that mechanochemical ATPases such as kinesin or dynein may play a role in granule mobilization during neutrophil activation by inflammatory signals. In this study we show that proteins extracted from the surface of neutrophil granules, found previously to contain microtubule-dependent ATPase activity, caused microtubules polymerized from phosphocellulose-purified rat brain tubulin to move across glass slides. Antibodies were generated against peptides based on two regions of the amino acid sequence of Drosophila kinesin: the ATPase active site (amino acids 86-99) in the head of the kinesin heavy chain and the tail of the heavy chain (residues 913-933). These antibodies were found to recognize kinesin in rat brain extracts as well as kinesin-like polypeptides in extracts of human neutrophils. Furthermore, when used in immunoaffinity chromatography, these antibodies permitted the isolation of a protein from neutrophil granule extracts that was recognized by Drosophila kinesin antibodies. Subcellular localization by immunofluorescence microscopy showed this protein to be associated principally with the cytoplasmic granules of neutrophils.
Rothwell
S W
SW
Department of Hematology, Walter Reed Army Institute of Research, Washington, DC 20307-5100.
Deal
C C
CC
Pinto
J
J
Wright
D G
DG
D000818:Animals; D000906:Antibodies; D001921:Brain / Q000502:physiology; D002417:Cattle; D002645:Chickens; D002846:Chromatography, Affinity; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000502:physiology; D005455:Fluorescent Antibody Technique; D006801:Humans; D016547:Kinesins / Q000032:analysis / Q000097:blood* / Q000302:isolation & purification; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D008970:Molecular Weight; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000737:chemistry / Q000648:ultrastructure
D000906:Antibodies; D016547:Kinesins