Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Kinesin is a mechanochemical ATPase that induces translocation of latex beads along microtubules and microtubule gliding on a glass surface. This protein is thought to be a motor for the movement of membranous organelles in cells. Recently Hollenbeck and Swanson [Hollenbeck, P. J. & Swanson, J. A. (1990) Nature (London) 346, 864-866] showed that kinesin is involved in the positioning of tubular lysosomes in macrophages. However, the role of this protein in the movement of organelles was not yet clear. We used a polyclonal antibody against the kinesin heavy chain that inhibited kinesin-dependent microtubule gliding in vitro to study the role of kinesin in the movement of pigment granules in melanophores of the teleost black tetra (Gymnocorymbus ternetzi). Microinjection of the antibody into cultured melanophores did not produce any specific effect on the aggregation of pigment granules in melanophores, but it did result in a strong dose-dependent inhibition of the dispersion. Immunoblotting of melanophore extracts showed that the kinesin antibody reacted in these cells with a single protein component with a molecular mass of 135 kDa. Thus, kinesin is responsible for the movement of pigment granules from the center to the periphery of the melanophore.
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PMID:Kinesin is responsible for centrifugal movement of pigment granules in melanophores. 182 87

Mutations in the unc-104 gene of the nematode C. elegans result in uncoordinated and slow movement. Transposon insertions in three unc-104 alleles (e2184, rh1016, and rh1017) were used as physical markers to clone the unc-104 gene. DNA sequence analysis of unc-104 cDNAs revealed an open reading frame capable of encoding a 1584 amino acid protein with similarities to kinesin heavy chain. The similarities are greatest in the amino-terminal ATPase and microtubule-binding domains. Although the primary sequence relatedness to kinesin is weak in the remainder of the molecule, the predicted secondary structure and regional isoelectric points are similar to kinesin heavy chain.
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PMID:The C. elegans unc-104 gene encodes a putative kinesin heavy chain-like protein. 184 75

Kinesin is a microtubule-activated ATPase that moves objects toward the plus end of microtubules and makes microtubules glide along a glass surface. Here we investigate a remarkable effect of the nonhydrolyzable analogue of ATP, adenosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppA), on kinesin-driven microtubule gliding. Microtubule gliding that has been blocked by rapid replacement of ATP with p[NH]ppA requires 1-2 min of exposure to ATP before microtubule gliding resumes. This latency is not shortened by prolonged washing of p[NH]ppA-blocked microtubules in nucleotide-free buffer for up to 15 min, suggesting that ATP binding to a second nucleotide binding site on kinesin triggers the release of bound p[NH]ppA. To test this hypothesis, the release of [3H]p[NH]ppA from kinesin-microtubule complexes was followed in parallel biochemical assays. In nucleotide-free buffer, the bound p[NH]ppA was released over several hours from the complexes. However, addition of ATP caused the release of p[NH]ppA from the kinesin-microtubule complexes within 2 min, which was similar to the latent period for start-up of microtubule gliding after p[NH]ppA inhibition. The stoichiometry of p[NH]ppA bound per kinesin heavy chain at saturation was estimated to be approximately 1:2. These results suggest a model in which each molecule of kinesin has at least two nucleotide binding sites that alternately bind nucleotide.
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PMID:Delayed start-up of kinesin-driven microtubule gliding following inhibition by adenosine 5'-[beta,gamma-imido]triphosphate. 214 8

Kinesin is a microtubule-activated, mechanochemical ATPase capable of moving particles along microtubules and making microtubules glide along a solid substrate. In this study we used limited proteolysis to study the structure of bovine brain kinesin, a heterotetramer composed of two heavy (120-kDa) and two light (62-kDa) chains. alpha-chymotrypsin, trypsin, and subtilisin all produced a protease-resistant 45-kDa fragment from the kinesin heavy chain. As isolated by gel-filtration chromatography, this fragment contains both the microtubule-binding site and the ATP catalytic site of the molecule. Proteolytic cleavage stimulated microtubule-dependent Mg2+-ATPase activity 4- to 5-fold up to 75-120 mumol ATP/min/mg. Cleavage also increased the affinity of the fragment for microtubules at least 10-fold. Since the purified fragment does not support the gliding of flagellar axonemes, we propose that cleavage of the heavy chain uncouples ATPase activity from its translocator activity, which may require other parts of the molecule.
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PMID:Isolation of a 45-kDa fragment from the kinesin heavy chain with enhanced ATPase and microtubule-binding activities. 252 Dec 21

The mechanochemical ATPase kinesin is thought to move membrane-bounded organelles along microtubules in fast axonal transport. However, fast transport includes several classes of organelles moving at rates that differ by an order of magnitude. Further, the fact that cytoplasmic forms of kinesin exist suggests that kinesins might move cytoplasmic structures such as the cytoskeleton. To define cellular roles for kinesin, the axonal transport of kinesin was characterized. Retinal proteins were pulse-labeled, and movement of radiolabeled kinesin through optic nerve and tract into the terminals was monitored by immunoprecipitation. Heavy and light chains of kinesin appeared in nerve and tract at times consistent with fast transport. Little or no kinesin moved with slow axonal transport indicating that effectively all axonal kinesin is associated with membranous organelles. Both kinesin heavy chain molecular weight variants of 130,000 and 124,000 M(r) (KHC-A and KHC-B) moved in fast anterograde transport, but KHC-A moved at 5-6 times the rate of KHC-B. KHC-A cotransported with the synaptic vesicle marker synaptophysin, while a portion of KHC-B cotransported with the mitochondrial marker hexokinase. These results suggest that KHC-A is enriched on small tubulovesicular structures like synaptic vesicles and that at least one form of KHC-B is predominantly on mitochondria. Biochemical specialization may target kinesins to appropriate organelles and facilitate differential regulation of transport.
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PMID:Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms. 753 59

A 100-kDa polypeptide with microtubule-interacting properties was identified in a Golgi vesicle-enriched fraction from Corylus avellana pollen. The k71s23 antibody (directed to the kinesin heavy chain from bovine brain) [Tiezzi et al., 1992: Cell Motil. Cytoskeleton 21:132-137] localized the polypeptide on the external surface of membrane-bounded organelles. Some 100-kDa-containing vesicles copelleted with microtubules (polymerized from purified bovine brain tubulin) either in presence or absence of 5 mM AMPPNP, but they could be released by 10 mM ATP or 0.5 M KCl. The pollen microtubule-interacting protein, salt-extracted from membranes and partially purified by gel filtration, exhibited an ATPase activity (16.2 nmolPi/mg/min) which could be stimulated about 2-fold (32.5 nmolPi/mg/min) by addition of bovine brain microtubules. We suppose that the 100-kDa polypeptide is part of a molecular complex showing properties of the kinesin class.
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PMID:Kinesin-related polypeptide is associated with vesicles from Corylus avellana pollen. 782 Aug 65

The N-terminal residues of the two heavy chains of the motor enzyme kinesin form two globular "heads"; the heads are attached to a "rod" domain which is a two-stranded alpha-helical coiled-coil. Interaction between the heads is thought to be important to kinesin function. The rod may not be necessary for head-head interactions because a heavy chain N-terminal fragment containing only residues from the head and adjacent region forms dimers (Huang, T.-G., Suhan, J., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16502-16507). However, the nature and stability of the subunit-subunit interactions in such derivatives are unclear. To examine the physical properties of heavy chain interaction in and near the head domains, we characterized the self-association behavior of two dimeric kinesin derivatives predicted (Lupas, A., van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164) to lack the rod. Derivative K448-BIO contains the 448 N-terminal residues of Drosophila kinesin heavy chain fused at the C terminus to a 2-residue linker and a C-terminal fragment from Escherichia coli biotin carboxyl carrier protein; derivative K448-L is the same except that it lacks the biotin carboxyl carrier protein fragment. Both derivatives expressed in insect cells display microtubule-stimulated ATPase activity; K448-BIO also displays microtubule motility. Equilibrium sedimentation and gel filtration indicate that purified K448-BIO and K448-L at 0.02-0.4 mg/ml form homogeneous solutions of homodimers with no detectable formation of monomers or higher order oligomers. Derivative self-association is non-covalent but extremely stable with an association constant > or = 2 x 10(8) M-1. Stable subunit-subunit association induced by structures in and near the kinesin heads may be necessary for full mechanochemical function.
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PMID:Subunit interactions in dimeric kinesin heavy chain derivatives that lack the kinesin rod. 787 39

Complementary DNAs of two kinesin-related genes, katB and katC, were isolated from Arabidopsis thaliana and sequenced. The carboxyl-terminal regions of the polypeptides encoded by these genes, especially the presumptive ATP-binding and microtubule-binding domains, share significant sequence homology with the mechanochemical motor domain of the kinesin heavy chain. The predicted secondary structures of KatB and KatC proteins include a large globular domain in the carboxyl-terminal region and a small globular domain in the amino-terminal region that are separated by a long alpha-helical coiled-coil with heptad repeats. A truncated KatC polypeptide (KatC(207-754)), which includes the carboxyl-terminal region of KatC, was expressed in Escherichia coli and was shown to possess microtubule-stimulated ATPase activity and to bind to microtubules in an ATP-sensitive manner, both of which are characteristics of kinesin and kinesin-like proteins.
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PMID:Sequencing and characterization of the kinesin-related genes katB and katC of Arabidopsis thaliana. 807 2

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.
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PMID:Affinity purification and subcellular localization of kinesin in human neutrophils. 848 17

The Drosophila kinesin heavy-chain gene was truncated to obtain the N-terminal 401 amino acid motor domain (designated K401) containing both the microtubule and ATP binding sites. The plasmid construct with the truncated kinesin gene was used to transform Escherichia coli. After induction, K401 was expressed as soluble kinesin protein at high levels and purified to homogeneity in milligram quantities. The purified protein was active and behaved as native kinesin with respect to its steady-state kinetic properties: K401 demonstrated a very low ATPase activity (kcat = 0.01 s-1) which was stimulated approximately 1000-fold by the addition of microtubules (kcat = 10 s-1; K0.5,MT = 0.9 microM tubulin; Km,ATP = 31 microM). Like native kinesin, K401 when purified contained ADP tightly bound at its active site, and the release of ADP from the active site occurred at a rate equal to the steady-state ATPase kcat. Active-site measurements using [alpha-32P]ATP demonstrated a stoichiometry of one ATPase site per K401 molecule. Like native kinesin, K401 can also hydrolyze MgGTP, and in the presence of microtubules, the rate of hydrolysis was increased dramatically from 0.03 to 16 s-1 (K0.5,MT = 2 microM tubulin; Km,GTP = 3.5 mM). These results establish that an active kinesin motor domain can be bacterially expressed and that this domain, the N-terminal 401 amino acids of the Drosophila kinesin heavy chain without light chains or additional eukaryotic factors, has full catalytic activity with microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression, purification, and characterization of the Drosophila kinesin motor domain produced in Escherichia coli. 848 45


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