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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)

The substrate specificities of dynein, kinesin, and myosin substrate turnover activity and cytoskeletal filament-driven translocation were examined using 15 ATP analogues. The dyneins were more selective in their substrate utilization than bovine brain kinesin or muscle heavy meromyosin, and even different types of dyneins, such as 14S and 22S dynein from Tetrahymena cilia and the beta-heavy chain-containing particle from the outer-arm dynein of sea urchin flagella, could be distinguished by their substrate specificities. Although bovine brain kinesin and muscle heavy meromyosin both exhibited broad substrate specificities, kinesin-induced microtubule translocation varied over a 50-fold range in speed among the various substrates, whereas heavy meromyosin-induced actin translocation varied only by fourfold. With both kinesin and heavy meromyosin, the relative velocities of filament translocation did not correlate well with the relative filament-activated substrate turnover rates. Furthermore, some ATP analogues that did not support the filament translocation exhibited filament-activated substrate turnover rates. Filament-activated substrate turnover and power production, therefore, appear to become uncoupled with certain substrates. In conclusion, the substrate specificities and coupling to motility are distinct for different types of molecular motor proteins. Such nucleotide "fingerprints" of enzymatic activities of motor proteins may prove useful as a tool for identifying what type of motor is involved in powering a motility-related event that can be reconstituted in vitro.
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PMID:Nucleotide specificity of the enzymatic and motile activities of dynein, kinesin, and heavy meromyosin. 182 61

Membrane-bound organelles move bidirectionally along microtubules in the freshwater ameba, Reticulomyxa. We have examined the nucleotide requirements for transport in a lysed cell model and compared them with kinesin and dynein-driven motility in other systems. Both anterograde and retrograde transport in Reticulomyxa show features characteristic of dynein but not of kinesin-powered movements: organelle transport is reactivated only by ATP and no other nucleoside triphosphates; the Km and Vmax of the ATP-driven movements are similar to values obtained for dynein rather than kinesin-driven movement; and of 15 ATP analogues tested for their ability to promote organelle transport, only 4 of them did. This narrow specificity resembles that of dynein-mediated in vitro transport and is dissimilar to the broad specificity of the kinesin motor (Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. Malik, and R. D. Vale. 1991. J. Cell Biol. 112: 1189-1197). Remarkably, anterograde and retrograde organelle transport cannot be distinguished at all with respect to nucleotide specificity, kinetics of movement, and the ability to use the ATP analogues. Since the "kinetic fingerprints" of the motors driving transport in opposite directions are indistinguishable, the same type of motor(s) may be involved in the two directions of movement.
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PMID:Nucleotide specificities of anterograde and retrograde organelle transport in Reticulomyxa are indistinguishable. 182 62

We examined the ability of kinesin to support the movement of adrenal medullary chromaffin granules on microtubules in a defined in vitro system. We found that kinesin and ATP are all that is required to support efficient (33% vesicle motility) and rapid (0.4-0.6 micron/s) translocation of secretory granule membranes on microtubules in the presence of a low-salt motility buffer. Kinesin also induced the formation of microtubule asters in this buffer, with the plus ends of microtubules located at the center of each aster. This observation indicates that kinesin is capable of promoting active sliding between microtubules toward their respective plus ends, a movement analogous to that of anaphase b in the mitotic spindle. The fact that vesicle translocation, microtubule sliding, and microtubule-dependent kinesin ATPase activities are all enhanced in low-salt buffer establishes a functional parallel between this translocator and other motility ATPases, myosin, and dynein.
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PMID:Purified kinesin promotes vesicle motility and induces active sliding between microtubules in vitro. 183 Jun 66

The 'motor' proteins of eukaryotic cells contain specialized domains that hydrolyse ATP to produce force and movement along a cytoskeletal polymer (actin in the case of the myosin family; microtubules in the case of the kinesin family and dyneins). There are motor-protein superfamilies in which each member has a conserved force-generating domain joined to a different 'tail' which conveys specific attachment properties. The minus-end-directed microtubule motors, the dyneins, may also constitute a superfamily of force-generating proteins with distinct attachment domains. Axonemal outer-arm dynein from sea urchin spermatozoa is a multimeric protein consisting of two heavy chains (alpha and beta) with ATPase activity, three intermediate chains and several light chains. Here I report the sequence of cloned complementary DNA encoding the beta heavy chain of a dynein motor molecule. The predicted amino-acid sequence reveals four ATP-binding consensus sequences in the central domain. The dynein beta heavy chain is thought to associate transiently with a microtubule during ATP hydrolysis, but the ATP-dependent microtubule-binding sequence common to the kinesin superfamily is not found in the dynein beta heavy chain. These unique features distinguish the dynein beta heavy chain from other motor protein superfamilies and may be characteristic of the dynein superfamily.
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PMID:Four ATP-binding sites in the midregion of the beta heavy chain of dynein. 183 Sep 24

Our detailed measurements of the movements of kinesin- and dynein-coated latex beads have revealed several important features of the motors which underlie basic mechanical aspects of the mechanisms of motor movements. Kinesin-coated beads will move along the paths of individual microtubule protofilaments with high fidelity and will pause at 4 nm intervals along the microtubule axis under low ATP conditions. In contrast, cytoplasmic dynein-coated beads move laterally across many protofilaments as they travel along the microtubule, without any regular pauses, suggesting that the movements of kinesin-coated beads are not an artefact of the method. These kinesin bead movements suggest a model for kinesin movement in which the two heads walk along an individual protofilament in a hand-over-hand fashion. A free head would only be able to bind to the next forward tubulin subunit on the protofilament and its binding would pull off the trailing head to start the cycle again. This model is consistent with the observed cooperativity between the heads and with the movement by single dimeric molecules. Several testable predictions of the model are that kinesin should be able to bind to both alpha and beta tubulin and that the length of the neck region of the molecule should control the off-axis motility. In this article, we describe the technology for measuring nanometer-level movements and the force generated by the kinesin molecule.
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PMID:A model for kinesin movement from nanometer-level movements of kinesin and cytoplasmic dynein and force measurements. 183 66

Kinetochore microtubules result from an interaction between astral microtubules and the kinetochore of the chromosomes after breakdown of the nuclear envelope at the end of prophase. In this process, the end of a microtubule projecting from one of the polar regions contacts the primary constriction of a chromosome. The latter then undergoes rapid poleward movement. Concerning the mechanism of anaphase chromosome movement, the motive force for the chromosome-to-pole movement appears to be generated at the kinetochore or in the region very close to it. It has not been determined whether chromosomes propel themselves along stationary kinetochore microtubules by a motor at the kinetochore, or they are pulled poleward by a traction fiber consisting of kinetochore microtubules and associated motors. As chromosomes move poleward coordinate disassembly of kinetochore microtubules might occur from their kinetochore ends. In diatom and yeast spindles, elongation of the spindle in anaphase (anaphase B) may be explained by microtubule assembly at polar microtubule ends in the spindle mid-zone and sliding of the antiparallel microtubules from the opposite poles. The sliding force appears to be generated through an ATP-dependent microtubule motor. In isolated sea urchin spindles, the microtubule assembly at the equator alone might provide the force for spindle elongation, although, in addition, involvement of microtubule sliding by a GTP-requiring mechanochemical enzyme cannot be excluded. Discussions were made on possible participation in anaphase chromosome movement of such microtubule motors as dynein, kinesin, dynamin and the claret segregation protein.
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PMID:[Cell division and the microtubular cytoskeleton]. 183 52

The protocols described here have proved to be an effective method for preparation of kinesin suitable for biochemical, biophysical, and immunological analyses. Beginning with a 1.2-liter cytosolic extract of bovine brain containing approximately 24 g of protein, 2 mg of approximately 95% pure kinesin can be obtained within 2 days. There are four major enrichment steps, as summarized in Fig. 6 and Table I. Based on quantitative SDS-PAGE, we estimate that these steps result in a purification of more than 300-fold. The ATPase activity in the presence of microtubules is substantial, and the kinetic properties are consistent with cellular levels of ATP (Km approximately 0.2 mM) and microtubules (apparent Km for activation approximately 1.9 microM) in the axon. Minor modifications should allow the procedure to be enlarged or reduced in scale, or adapted to the brains of other vertebrate species. The availability of such procedures will greatly facilitate future studies of the cell and molecular biology of kinesin.
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PMID:Purification of kinesin from bovine brain and assay of microtubule-stimulated ATPase activity. 185 39

An in vitro motility assay has been developed in which single actin filaments move on one or a few heavy meromyosin (HMM) molecules. This movement is slower than when many HMM molecules are involved, in contrast to analogous experiments with microtubules and kinesin. Frequency analysis shows that sliding speeds distribute around integral multiples of a unitary velocity. This discreteness may be due to differences in the numbers of HMM molecules interacting with each actin filament, where the unitary velocity reflects the activity of one HMM molecule. The value of the unitary velocity predicts a step size of 5-20 nm per ATP, which is consistent with the conventional swinging crossbridge model for myosin function.
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PMID:Quantized velocities at low myosin densities in an in vitro motility assay. 185

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.
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PMID:Motor protein independent binding of endocytic carrier vesicles to microtubules in vitro. 191 48

Microtubules are the key elements of the cytoskeleton responsible for cytoplasm organization and intracellular transport. Their functions are realized mainly via microtubule associated proteins (MAP), the minor components bound to the microtubule core. Among MAP there are so-called structural proteins which control tubulin polymerization and provide the "static" interaction of microtubules with other intracellular components and translocator proteins. The latter are capable of moving the material along microtubules; this process is coupled with ATP hydrolysis. The first section summarizes the data on the composition and changes during ontogenesis and functioning, such as influences on tubulin polymerization and promotion of interactions between individual microtubules, between microtubules and microfilaments and neurofilaments as well as between microtubules and membrane organelles (lysosomes, golgi stacks, mitochondria). The second section deals with the description of translocators. The biochemical properties of the following proteins are considered: i) kinesin, the protein translocating particles to the distal end of microtubules and, ii) dynein which promotes translocation in the opposite direction.
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PMID:[Microtubule-associated proteins]. 193 44


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