Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.4.4 (kinesin)
 5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In murine bone marrow-derived macrophages, lysosomes often form tubulovesicular compartments, whose extended distribution in the cytoplasm depends on the integrity of cytoplasmic microtubules. When macrophages with fluorescently labeled lysosomes were plated onto coverslips opsonized with IgG, they engaged that surface in a phagocytic response (frustrated phagocytosis). The tubular lysosomal compartment of these cells collected in a central, perinuclear region, despite the continued presence of a radiating array of cytoplasmic microtubules. Using methods developed in the study of melanophores, we permeabilized macrophages engaged in frustrated phagocytosis, then re-activated lysosome extension along microtubules. Permeabilization was selective for plasma membranes, in that high molecular weight probes such as trypan blue or IgG could enter cells, while fluorescent probes previously loaded into lysosomes via endocytosis remained contained therein. Addition of 2 mM ATP, GTP or UTP to these permeabilized cell models produced centrifugal extension of tubular lysosomes. Selective depletion of ATP, using Escherichia coli glycerol kinase, inhibited ATP-dependent extension but not that which occurred with GTP or UTP, indicating that the mechanism of radial movement can use any of these three nucleotide triphosphates. Extension was independent of pH between 6.8 and 7.4, and was inhibited by AMP-PNP and by GMP-PNP. Depolymerization of cytoplasmic microtubules with nocodazole prevented subsequent ATP-inducible lysosome extension, whereas preincubation of cells with cytochalasin D did not inhibit the response. These results are consistent with the in vitro mechanochemical properties of kinesin (Cohn et al., 1989), and support earlier evidence, obtained in living cells, that kinesin is the mechanochemical motor of lysosome extension along microtubules in macrophages.
J Cell Sci 1992 Sep
PMID:Radial movement of lysosomes along microtubules in permeabilized macrophages. 142 5

Previous studies have shown that microtubule-based organelle transport requires a membrane receptor but no kinesin-binding membrane proteins have been isolated. Chick embryo brain microsomes have kinesin bound to their surface, and after detergent solubilization, a matrix with an antibody to the kinesin head domain (SUK-4) (Ingold et al., 1988) bound the solubilized kinesin and retained an equal amount of a microsome protein of 160-kD. Similarly, velocity sedimentation of solubilized membranes showed that kinesin and the 160-kD polypeptide cosedimented at 13S. After alkaline treatment to remove kinesin from the microsomes, the same 160-kD polypeptide doublet bound to a kinesin affinity resin and not to other proteins tested. Biochemical characterization localized this protein to the cytoplasmic face of brain microsomes and indicated that it was an integral membrane protein since it was resistant to alkaline washing. mAbs raised to chick 160-kD protein demonstrated that it was absent in the supernatant and concentrated in the dense microsome fraction. The dense microsome fraction also had the greatest amount of microtubule-dependent motility. With immunofluorescence, the antibodies labeled the ER in chick embryo fibroblasts (similar to the pattern of bound kinesin staining in the same cells) (Hollenbeck, P. J. 1989. J. Cell Biol. 108:2335-2342), astroglia, Schwann cells and dorsal root ganglion cells but staining was much less in the Golgi regions of these cells. Because this protein is a major kinesin-binding protein of motile vesicles and would be expected to bind kinesin to the organelle membrane, we have chosen the name, kinectin, for this protein.
J Cell Biol 1992 Sep
PMID:Kinectin, a major kinesin-binding protein on ER. 151 92

During the first cell cycle, the vegetal cortex of the fertilized frog egg is translocated over the cytoplasm. This process of cortical rotation creates regional cytoplasmic differences important in later development, and appears to involve an array of aligned microtubules that forms transiently beneath the vegetal cortex. We have investigated how these microtubules might be involved in generating movement by analyzing isolated cortices and sections of Xenopus laevis and Rana pipiens eggs. First, the polarity of the cortical microtubules was determined using the "hook" assay. Almost all microtubules had their plus ends pointing in the direction of cortical rotation. Secondly, the association of microtubules with other cytoplasmic elements was examined. Immunofluorescence revealed that cytokeratin filaments coalign with the microtubules. The timing of their appearance and their position on the cytoplasmic side of the microtubules suggested that they are not involved directly in generating movement. ER was visualized with the dye DiIC16(3) and by immunofluorescence with anti-BiP (Bole, D. G., L. M. Hendershot, and J. F. Kearney, 1986. J. Cell Biol. 102:1558-1566). One layer of ER was found closely underlying the plasma membrane at all times. An additional, deeper layer formed in association with the microtubules of the array. Antibodies to sea urchin kinesin (Ingold, A. L., S. A. Cohn, and J. M. Scholey. 1988. J. Cell Biol. 107:2657-2667) detected antigens associated with both the ER and microtubules. On immunoblots they recognized microtubule associated polypeptide(s) of approximately 115 kD from Xenopus eggs. These observations are consistent with a role for kinesin in creating movement between the microtubules and ER, which leads in turn to the cortical rotation.
J Cell Biol 1991 Sep
PMID:Evidence for the involvement of microtubules, ER, and kinesin in the cortical rotation of fertilized frog eggs. 171 12

Growth cones are intimately involved in determining the direction and extent of neurite elongation during development. They are able to monitor their environment and respond to it by undergoing directed motility. We have isolated a fraction enriched in growth cone particles from embryonic chick brain. Assayed by immunoblots, this fraction is enriched in GAP-43, and contains the cytoskeletal proteins actin, myosin II, neurofilament protein, tubulin, kinesin, and dynamin. All of the major components of focal adhesions are also present: alpha-actinin, vinculin, talin, and integrin. In addition to integrin, we also identify the cell adhesion molecules A-CAM, L1, fibronectin, and laminin in these particles. This preparation of isolated growth cone particles may be a useful model system for studying growth cone adhesion and motility.
J Neurosci Res 1991 Sep
PMID:Identification of cytoskeletal, focal adhesion, and cell adhesion proteins in growth cone particles isolated from developing chick brain. 183 41

We have established an in vitro assay to characterize the binding of endocytic carrier vesicles to microtubules. Magnetic beads coated with microtubules were used as an affinity matrix. A fraction from nocodazole-treated cells enriched in endocytic carrier vesicles, labeled with internalized horseradish peroxidase, was used in the binding experiments. Binding of the endocytic carrier vesicles to microtubules in vitro was cytosol-dependent. This activity of cytosolic factors was saturable, heat-sensitive, and insensitive to N-ethyl-maleimide. Binding was sensitive to GTP and ATP. Addition of neuronal microtubule-associated proteins completely abolished binding of the endocytic organelles to microtubules. This binding was independent of the cytosolic microtubule-based motor proteins kinesin and cytoplasmic dynein, since cytosol depleted of these proteins remained fully active. Microtubule-binding proteins from HeLa cells, however, stimulated the interaction of endocytic carrier vesicles with microtubules. Trypsinized vesicles could no longer bind to microtubules in the presence of cytosol. These results suggest that cytosolic microtubule-binding proteins, other than the known microtubule-based motor proteins, as well as membrane proteins are involved in the nucleotide-dependent interaction of endocytic carrier vesicles with microtubules.
J Biol Chem 1991 Sep 25
PMID:Motor protein independent binding of endocytic carrier vesicles to microtubules in vitro. 191 48

The nod gene is required for the distributive segregation of nonexchange chromosomes during meiosis in D. melanogaster. Loss-of-function nod mutations cause nondisjunction and loss of nonrecombinant chromosomes both at meiosis I and during subsequent mitotic divisions. We have cloned the nod locus, examined its expression patterns, and determined its coding sequence. In adults the nod transcript is only present in females, consistent with the observation that males do not use the distributive segregation system. However, the nod locus is also transcribed in the embryonic, larval, and pupal stages of development, and possibly in all dividing cells. Finally, the N-terminal domain of the predicted nod protein has amino acid similarity to the mechanochemical domain of kinesin heavy chain; however, the C-terminal domain is unlike that of kinesin heavy chain or of any previously reported protein. Thus, the nod protein is a member of the kinesin superfamily and may be a microtubule motor.
Cell 1990 Sep 21
PMID:A kinesin-like protein required for distributive chromosome segregation in Drosophila. 214 92

Bovine brain kinesin binds ADP tightly and contains a stoichiometric amount of ADP at its active site when isolated in the presence of free Mg2+ (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318). EDTA in excess of Mg2+ weakens ADP binding and nucleotide-free kinesin can be prepared by gel filtration with excess EDTA. On addition of ATP, this nucleotide-free enzyme catalyzes the rapid hydrolysis of a stoichiometric amount of ATP in a burst phase followed by much slower continued ATP hydrolysis limited by the release of ADP from the active site. This burst reaction is evident both by formation of [32P]Pi from [gamma-32P]ATP and by formation of [alpha-32P]ADP from [alpha-32P]ATP. At 1.1 nM kinesin active sites, the observed rate of the burst phase increases linearly with ATP over the 1-20 nM range yielding a bimolecular rate of net ATP binding and hydrolysis of 2.5 microM-1 s-1. The intercept at zero ATP is 0.008 s-1 which equals the ADP release rate at 0.008-0.009 s-1. This predicts a Km for ATP of approximately 3.5 nM and measurements of the dependence on ATP concentration of the steady state rate and amount of bound ADP are consistent with a Km of this magnitude.
J Biol Chem 1989 Sep 25
PMID:Nucleotide-free kinesin hydrolyzes ATP with burst kinetics. 252 42

Kinesin was prepared from bovine brain as described previously for studies of translocation. A major component of kinesin, (116 kDa) was shown to undergo specific photocrosslinking with [alpha-32P]ATP, indicating it was an ATP-binding polypeptide. A low ATPase activity associated with kinesin was stimulated up to 5-fold by microtubules to a specific activity of 14 nmol . min-1 . mg-1. N-Ethylmaleimide inhibited both [alpha-32P]ATP binding to the 116 kDa polypeptide and microtubule-stimulated ATPase activity, suggesting that the 116 kDa polypeptide was the catalytic subunit of kinesin. Though the ATPase activity associated with kinesin is low, it may be sufficient to support motility assuming it is coupled to the velocity of translocation.
FEBS Lett 1987 Sep 28
PMID:Evidence that the 116 kDa component of kinesin binds and hydrolyzes ATP. 295 62

Kinesin was purified from bovine adrenal medulla. The sedimentation coefficient was 8.8 S. Sedimentation equilibrium ultracentrifugation studies showed the molecular weight of kinesin to be 300,000. The calculated axial ratio was 1:16. The Stokes radius was estimated to be 8.9 nm by gel filtration. Circular dichroism showed the alpha-helix content to be about 50%. Purified kinesin preparation contained a major polypeptide with a molecular weight of 120,000 and minor ones with molecular weights of 71,000, 68,000, and 65,000. Bovine adrenal kinesin had an ATPase activity which was stimulated severalfold by microtubules to a specific activity of about 0.1 mumol/min.mg. Kinesin molecules adsorbed to a glass slide promoted the movement of microtubules on the glass surface at a rate of about 0.5 micron/s. Immunostaining of EBTr (bovine embryonic trachea fibroblast) cells and bovine adrenal chromaffin cells in interphase with an affinity-purified antibody against the major polypeptide of kinesin showed that some kinesin was located on microtubules and the rest distributed throughout the cytoplasm in a diffuse manner. EBTr cells in mitotic phase gave a staining pattern showing that kinesin was present throughout the cytoplasm with higher concentration in the region of mitotic apparatus.
J Biol Chem 1988 Sep 05
PMID:Purification and characterization of kinesin from bovine adrenal medulla. 297 Apr 64

The ATPase rate of kinesin isolated from bovine brain by the method of S.A. Kuznetsov and V.I. Gelfand [(1986) Proc. Natl. Acad. Sci. USA 83, 8530-8534)] is stimulated 1000-fold by interaction with tubulin (turnover rate per 120-kDa peptide increases from approximately equal to 0.009 sec-1 to 9 sec-1). The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that Pi release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (Ki less than 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by [14C]ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.
Proc Natl Acad Sci U S A 1988 Sep
PMID:Kinesin ATPase: rate-limiting ADP release. 297 Jun 38


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