Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

C. elegans contains a microtubule binding protein that resembles both dynein and kinesin. This protein has a MgATPase activity and copurifies on both sucrose gradients and DEAE Sephadex columns with a polypeptide of Mr approximately 400 kd. The ATPase activity is 50% inhibited by 10 microM vanadate, 1 mM N-ethyl maleimide, or 5 mM AMP-PNP; it is enhanced 50% by 0.2% Triton. The 400 kd polypeptide is cleaved at a single site by ultraviolet light in the presence of ATP and vanadate. In these ways, the protein resembles dynein. The protein also promotes ATP-dependent translocation of microtubules or axonemes, "plus" ends trailing. This property is kinesin-like; however, the motility is blocked by 5 microM vanadate, 1 mM N-ethyl maleimide, 0.5 mM ATP-gamma-S, or by ATP-vanadate-UV cleavage of the 400 kd polypeptide, characteristics that differ from kinesin. We propose that this protein is a novel microtubule translocator.
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PMID:Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans. 295 72

Preparations of kinesin, a microtubule-based force-producing protein, have been isolated from Drosophila melanogaster embryos by incubation of microtubules with a nonhydrolyzable ATP analogue and gel filtration of proteins released from the microtubules by ATP. These preparations induced MgATP-dependent microtubule gliding in vitro with a Km for MgATP of 44 microM and a Vmax for gliding of 0.9 micron/sec. Samples of Drosophila proteins that were active in motility assays possessed an average ATPase activity in solution of 17 nmol/min per mg that increased to an average of 106 nmol/min per mg in the presence of microtubules. The major polypeptides that copurified with these activities showed relative molecular masses of 115 kDa and 58 kDa. An antiserum raised against the 115-kDa polypeptide also recognized the 110-kDa component of squid kinesin preparations and the 130-kDa component of sea urchin kinesin preparations.
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PMID:Drosophila kinesin: characterization of microtubule motility and ATPase. 296 38

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.
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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.
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PMID:Kinesin ATPase: rate-limiting ADP release. 297 Jun 38

Several enzyme complexes drive cellular movements by coupling free energy-liberating chemical reactions to the production of mechanical work. A key goal in the study of these systems is to characterize at the molecular level mechanical events associated with individual reaction steps in the catalytic cycles of single enzyme molecules. Ideally, one would like to measure movements driven by single (or a few) enzyme molecules with sufficient temporal resolution and spatial precision that these events can be directly observed. Kinesin, a force-generating ATPase involved in microtubule-based intracellular organelle transport, will drive the unidirectional movement of microscopic plastic beads along microtubules in vitro. Under certain conditions, a few (less than or equal to 10) kinesin molecules may be sufficient to drive either bead movement or organelle transport. Here we describe a method for determining precise positional information from light-microscope images. The method is applied to measure kinesin-driven bead movements in vitro with a precision of 1-2 nm. Our measurements reveal basic mechanical features of kinesin-driven movements along the microtubule lattice, and place significant constraints on possible molecular mechanisms of movement.
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PMID:Tracking kinesin-driven movements with nanometre-scale precision. 312 99

In the present work we have studied the subunit composition of kinesin, the microtubule-activated, mechanochemical ATPase, isolated from bovine brain. Polypeptides with mol. wts of 120 and 62 kd are the major components of the kinesin preparation. These polypeptides could not be separated by electrophoresis under nondenaturing conditions or by FPLC on a MonoQ column, and are therefore assumed to form a tight complex. As shown by immunoblotting with polyclonal and monoclonal antibodies to the 120-kd polypeptide and by one-dimensional peptide mapping, the 62-kd polypeptide does not appear to be a proteolytic product of the 120-kd component. Densitometric scanning of polyacrylamide-SDS gels shows that these polypeptides are present in a complex in a 1:1 molar ratio. The mol. wt of native kinesin was studied by sedimentation equilibrium and was found to be 386 +/- 14 kd. A comparison of the mol. wts of individual polypeptides with the mol. wt of the intact molecule indicates that the native molecule contains two 120-kd subunits and two 62-kd subunits.
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PMID:The quaternary structure of bovine brain kinesin. 313 Feb 48

Kinesin was extensively purified from bovine brain cytosol by a microtubule-binding step in the presence of 5'-adenylyl imidodiphosphate (AMP-PNP), followed by gel filtration chromatography and sucrose gradient ultracentrifugation. The products consistently contained 124,000 (124K) and 64,000 (64K) dalton polypeptides. These two polypeptides appear to represent heavy and light chains of kinesin, respectively, because they copurified on sucrose gradients to a constant and equimolar stoichiometry and bound stably to microtubules in the presence of AMP-PNP but not ATP. The mobilities of 124K and 64K in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions were the same as under nonreducing conditions. A diffusion coefficient of (2.24 +/- 0.21) X 10(-7) cm2 s-1 and a sedimentation coefficient of (9.56 +/- 0.34) X 10(-13) s were determined for native kinesin by gel filtration and sucrose gradient ultracentrifugation, respectively. These values were used to calculate a native molecular weight of about 379,000 and suggest that kinesin has an axial ratio of approximately 20. Extensively purified kinesin exhibited microtubule-activated ATPase activity, and only the 124K subunit incorporated ATP in photoaffinity labeling experiments using [32P]ATP. Collectively, these data favor the interpretation that bovine brain kinesin is a highly elongated, microtubule-activated ATPase comprising two subunits each of 124,000 and 64,000 daltons, that the subunits are not linked to one another by disulfide bonds, and that the heavy chains are the ATP-binding subunits.
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PMID:Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide. 313 48

Kinesin is a force-generating ATPase that drives the sliding movement of microtubules on glass coverslips and the movement of plastic beads along microtubules. Although kinesin is suspected to participate in microtubule-based organelle transport, the exact role it plays in this process is unclear. To address this question, we have developed a quantitative assay that allows us to determine the ability of soluble factors to promote organelle movement. Salt-washed organelles from squid axoplasm exhibited a nearly undetectable level of movement on purified microtubules. Their frequency of movement could be increased greater than 20-fold by the addition of a high speed axoplasmic supernatant. Immunoadsorption of kinesin from this supernatant decreased the frequency of organelle movement by more than 70%; organelle movements in both directions were markedly reduced. Surprisingly, antibody purified kinesin did not promote organelle movement either by itself or when it was added back to the kinesin-depleted supernatant. This result suggested that other soluble factors necessary for organelle movement were removed along with kinesin during immunoadsorption of the supernatant. A high level of organelle motor activity was recovered in a high salt eluate of the immunoadsorbent that contained only little kinesin. On the basis of these results we propose that organelle movement on microtubules involves other soluble axoplasmic factors in addition to kinesin.
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PMID:The role of kinesin and other soluble factors in organelle movement along microtubules. 314 29

Microtubules are involved in several forms of intracellular motility, including mitosis and organelle movement. Fast axonal transport is a highly ordered form of organelle motility that operates in both the anterograde (outwards from the cell body) and retrograde (from the periphery towards the cell body) direction. Similar microtubule-associated movement is observed in non-neuronal cells, and might be involved in secretion, endocytosis and the positioning of organelles within the cell. Kinesin is a mechanochemical protein that produces force along microtubules in an anterograde direction. We recently found that the brain microtubule-associated protein MAP 1C (ref. 7) is a microtubule-activated ATPase and, like kinesin, can translocate microtubules in an in vitro assay for microtubule-associated motility. MAP 1C seemed to be related to the ciliary and flagellar ATPase, dynein, which is thought to produce force in a direction opposite to that observed for kinesin. Here we report that MAP 1C, in fact, acts in a direction opposite to kinesin, and has the properties of a retrograde translocator.
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PMID:Retrograde transport by the microtubule-associated protein MAP 1C. 367 Apr 2

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


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