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:3.6.4.4 (kinesin)
5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin-ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin.
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PMID:Pathway of processive ATP hydrolysis by kinesin. 785 36

In this study we report that phospholipid vesicles activate ATP hydrolysis by cytoplasmic dynein but not kinesin, consistent with reported differences in the organelle/vesicle binding of these motor proteins. Dynein activation by phospholipids was comparable with that seen in the presence of microtubules but was not sensitive to moderate salt concentrations and was independent of the net charge of the phospholipid, suggesting that the means of interaction between dynein and the lipid vesicle was not strictly ionic in nature. Based on this result, previous data that show that the interaction between dynein and vesicles is not ATP sensitive, and the concentration dependence observed for lipid activation of cytoplasmic dynein, it is likely that the binding interaction between dynein and liposomes is a stable one. In contrast to a previous report, microtubules increased the hydrolysis rate of all naturally occurring nucleotides tested, whereas only ATPase activity was stimulated by phospholipids. As ATP is the physiologically relevant substrate and is the only nucleotide to promote motility, the activation of only the ATPase by phospholipids may represent a means of discriminating between coupled and uncoupled nucleotide hydrolysis in vitro.
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PMID:Microtubule-independent phospholipid stimulation of cytoplasmic dynein ATPase activity. 787 16

In previous studies (Bulinski and Borisy (1979). Proc. Nat. Acad. Sci. 76, 293-297; Weatherbee et al. (1980). Biochemistry 19, 4116-4123) a microtubule-associated protein (MAP) of M(r) approximately 125,000 was identified as a prominent MAP in HeLa cells. We set out to perform a biochemical characterization of this protein, and to determine its in vitro functions and in vivo distribution. We determined that, like the assembly-promoting MAPs, tau, MAP2 and MAP4, the 125 kDa MAP was both proteolytically sensitive and thermostable. An additional property of this MAP; namely, its unusually tight association with a calcium-insensitive population of MTs in the presence of taxol, was exploited in devising an efficient purification strategy. Because of the MAP's tenacious association with a stable population of MTs, and because it appeared to contribute to the stability of this population of MTs in vitro, we have named this protein ensconsin. We examined the binding of purified ensconsin to MTs; ensconsin exhibited binding that saturated its MT binding sites at an approximate molar ratio of 1:6 (ensconsin:tubulin). Unlike other MAPs characterized to date, ensconsin's binding to MTs was insensitive to moderate salt concentrations (< or = 0.6 M). We further characterized ensconsin in immunoblotting experiments using mouse polyclonal anti-ensconsin antibodies and antibodies reactive with previously described MAPs, such as high molecular mass tau isoforms, dynamin, STOP, CLIP-170 and kinesin. These experiments demonstrated that ensconsin is distinct from other proteins of similar M(r) that may be present in association with MTs. Immunofluorescence with anti-ensconsin antibodies demonstrated that ensconsin was detectable in association with most or all of the MTs of several lines of human epithelial, fibroblastic and muscle cells; its in vivo properties and distribution, especially in response to drug or other treatments of cells, were found to be different from those of MAP4, the predominant MAP found in these cell types. We conclude that ensconsin, a MAP found in a variety of human cells, is biochemically - and perhaps functionally - distinct from other MAPs present in non-neuronal cells.
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PMID:Purification and characterization of ensconsin, a novel microtubule stabilizing protein. 787 51

Kinesin, a mechanochemical enzyme that translocates membranous organelles, was initially identified and purified from soluble extracts from vertebrate brains. However, immunocytochemical and morphological approaches have demonstrated that kinesin could be associated to intracellular membranous organelles. We used an antibody raised against the head portion of the Drosophila kinesin heavy chain to reveal the presence of this protein in membranous organelles from rat brain. By using differential centrifugation and immunoblotting we observed a 116 kDa protein that crossreacts with this antibody in microsomes, synaptic vesicles, and mitochondria. This protein could be extracted from mitochondria with low salt concentrations or ATP. The 116 kDa solubilized protein has been identified as conventional kinesin based on limited sequence analysis. We also show that a polyclonal antibody raised against mitochondria-associated kinesin recognizes soluble bovine brain kinesin. The soluble and mitochondrial membrane-associated kinesins show a different isoform pattern. These results are consistent with the idea that kinesin exists as multiple isoforms that might be differentially distributed within the cell. In addition digitonin fractionation of mitochondria combined with KI extraction revealed that kinesin is a peripheral protein, preferentially located in a cholesterol-free outer membrane domain; this domain has the features of contact points between the mitochondrial outer and inner membranes. The significance of these observations on the functional regulation of the mitochondria-associated kinesin is discussed.
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PMID:Structural and biochemical properties of kinesin heavy chain associated with rat brain mitochondria. 804 52

The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules.
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PMID:The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study. 853 27

Kinesin is a two-headed motor protein that powers organelle transport along microtubules. Many ATP molecules are hydrolysed by kinesin for each diffusional encounter with the microtubule. Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy in the absence of attachment of the motor to a cargo (for example, an organelle or bead). The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a approximately 1% probability of detachment per mechanical cycle. Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl. Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin's two force-generating heads operate by a hand-over-hand mechanism.
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PMID:Direct observation of single kinesin molecules moving along microtubules. 860 45

ncd is a microtubule motor protein from Drosophila, having a 40 kDa domain homologous to the kinesin motor domain. In the present study, we investigated the circular dichroism (CD) spectra of the ncd motor domain in comparison with those of the kinesin motor domain. Although the two are about 40% identical in amino acid sequence, and recent X-ray crystallographic studies [Sablin, Kull, Cooke, Vale, and Fletterick (1996) Nature 380, 555-559; Kull, Sablin, Lau, Fletterick, and Vale (1996) Nature 380, 550-555] indicate that their core structures are nearly identical, the far UV CD spectra of ncd and kinesin motor domains, both being monomeric, were considerably different from each other, suggesting a significant difference in the secondary, especially loop structures. The motor domain of ncd, like that of kinesin, contains tightly associating ADP even after purification. We removed ADP from the ncd motor domain by gel filtration in the presence of EDTA and high salt. The resultant protein, however, was likely to be in an inactive state, since it bound ATP slowly. The far UV CD spectrum of the ncd motor domain devoid of ADP was nearly identical to that of the ncd motor domain with bound ADP. This indicated that the removal of ADP did not affect the backbone structure in the presence of high salt. On the other hand, the near UV CD spectrum of the ADP-free ncd motor domain differed from that of the ncd motor domain. ADP complex, one possibility being that the local conformation was changed upon removal of bound ADP. The near UV CD spectra of kinesin motor domain also showed a difference between the ADP-bound form and the nucleotide-free form, although the difference was much smaller.
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PMID:Comparison of ncd and kinesin motor domains by circular dichroism spectroscopy. 901 Jul 67

We studied the effect of alkaline-earth metal ions on the kinesin-driven gliding of microtubules, using a narrow glass chamber enabling the exchange of buffer components without interrupting microscopic observation. Under standard conditions (0.5 mM Mg2+), microtubules were found to glide at a mean velocity of about 0.6 micron/s. Motility was widely ceased after removing Mg2+. Subsequent addition of Ca2+ restored motility (maximal mean gliding velocity measured: 0.26 micron/s at 2.5 mM Ca2+). Also in the presence of Sr2+ or Ba2+ a slow gliding could be observed (0.025 micron/s and 0.014 micron/s, respectively, at 0.5 mM). After removal of Ca2+, Sr2+, or Ba2+ and re-addition of Mg2+, the gliding velocities reached approximately the values determined under standard conditions. Motility was not changed when 0.5 mM Ca2+, Sr2+, or Ba2+ were applied together with Mg2+. Microtubule gliding stopped after substitution of 0.5 mM BeCl2 for Mg2+. When both BeCl2 and Mg2+ were present, the mean gliding velocity was reduced to 0.29 micron/s. In addition, many microtubules were released from the kinesin coated glass surface, indicating that the beryllium salt disorders the binding between kinesin and microtubules. Our results confirm that Mg2+ is the most suitable cofactor for kinesin driven microtubule motility. However, they also demonstrate that brain kinesin can generate motility when Ca2+ was substituted for Mg2+.
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PMID:Kinesin-driven microtubule motility in the presence of alkaline-earth metal ions: indication for a calcium ion-dependent motility. 922 52

The ATPase mechanism for a monomeric Drosophila kinesin construct, K341, was determined by pre-steady-state kinetic methods and compared to dimeric kinesin, K401. We directly measured the kinetics of binding mantATP (a fluorescent ATP analog) to the microtubule K341 complex, the dissociation of K341 from the microtubule, and release of phosphate and ADP from K341. Measurements of phosphate release kinetics at low salt concentration show that K341 hydrolyzes 18 molecules of ATP per kinesin monomer prior to release from the microtubule. At a higher salt concentration the amplitude of the pre-steady-state burst of phosphate release was reduced to 8 molecules per kinesin monomer. The maximum rate of dissociation of K341 from the microtubule following the addition of ATP was 22 s-1. The rate of mantADP release from the M.K341.mantADP complex increased as a function of tubulin concentration with a second-order rate constant of 11 microM-1 s-1 for K341 binding to the microtubule and reached a maximum rate of mantADP release of 303 s-1. ADP release kinetics were also determined by monitoring the binding of mantATP to K341.ADP and K401.ADP after mixing with microtubules. We show that monomeric kinesin remains associated with the microtubule through multiple rounds of ATP hydrolysis. This apparent processivity implies that one of the functions of the cooperative interaction between the two kinesin heads in dimeric kinesin is for the reactions occurring on one kinesin head to facilitate the release of the adjacent head from the microtubule.
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PMID:Pathway of ATP hydrolysis by monomeric and dimeric kinesin. 945 69

The motor protein kinesin is a heterotetramer composed of two heavy chains of approximately 120 kDa and two light chains of approximately 65 kDa protein. Kinesin motor activity is dependent on the presence of ATP and microtubules. The kinesin light chain-binding site in human kinesin heavy chain was determined by reconstituting in vitro a complex of recombinant heavy and light chains. The proteins expressed in bacteria included oligohistidine-tagged fragments of human ubiquitous kinesin heavy chain, spanning most of the stalk and all of the tail domain (amino acids 555-963); and untagged, essentially full-length human kinesin light chain (4-569) along with N-terminal (4-363) and C-terminal (364-569) light chain fragments. Heavy chain fragments were attached to Ni2+-charged beads and incubated with untagged light chain fragments. Analysis of eluted complexes by SDS-PAGE and immunoblotting mapped the light chain-binding site in heavy chain to amino acids 771-813, a region close to the C-terminal end of the heavy chain stalk domain. In addition, only the full-length and N-terminal kinesin light chain fragments bound to this heavy chain region. Within this heavy chain region are four highly conserved contiguous heptad repeats (775-802) which are predicted to form a tight alpha-helical coiled-coil interaction with the heptad repeat-containing N-terminus of the light chain, in particular region 106-152 of human light chain. This predicted hydrophobic, alpha-helical coiled-coil interaction is supported by both circular dichroism spectroscopy of the recombinant kinesin heavy chain fragment 771-963, which displays an alpha-helical content of 70%, and the resistance of the heavy/light chain interaction to high salt (0.5 M).
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PMID:The C-terminal region of the stalk domain of ubiquitous human kinesin heavy chain contains the binding site for kinesin light chain. 984 34


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