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)

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

2,5-Hexanedione (2,5-HD) exposure in rats causes a progressive Sertoli cell injury culminating in testicular atrophy. Morphological injury is preceded by alterations in the assembly characteristics of tubulin isolated from exposed rat testes. This is followed by decreased seminiferous tubule fluid (STF) secretion by Sertoli cells and an increase in the number and size of Sertoli cell vacuoles. The possible involvement of microtubules and microtubule motor-dependent transport processes in STF secretion by Sertoli cells prompted us to examine the immunodistribution of the microtubule motors cytoplasmic dynein and kinesin during and after 2,5-HD exposure in rats. Three weeks following the commencement of exposure (1% 2,5-HD in the drinking water), the intensity of apical Sertoli cell cytoplasmic dynein immunofluorescence declined. This staining deficit became statistically significant by 4 weeks of exposure. Accompanying this change, there was progressive disruption of the immunodistribution of cisternal Golgi elements and associated kinesin immunoreactivity. The decrease in apical Sertoli cell cytoplasmic dynein immunofluorescence and disruption of Golgi and kinesin immunoreactivity suggest that 2,5-HD-induced alterations in Sertoli cell-mediated transport and secretory events could involve deficits in microtubule-dependent motor function.
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PMID:2,5-Hexanedione exposure alters microtubule motor distribution in adult rat testis. 773 29

The effects of selected ligands on the structure of the truncated heavy-chain chemomechanical motor domains of Drosophila ncd and human kinesin were compared using the technique of transient electric birefringence. The 366-amino acid C-terminal motor domain of Drosophila nonclaret disjunctional, ncd(335-700), and the 349-amino acid N-terminal motor domain of human kinesin, kinesin(349), were studied at 4 degrees C in neutral buffers with ionic strength of 100 mM to form complexes with either MgADP or MgADP.Vi. The rotational diffusion time adjusted to 20 degrees C and water, tau 20,W, for ncd(335-700).MgADP is 32.8 ns, and for ncd(335-700).MgADP.Vi is 34.8 ns, suggesting prolate ellipsoids with dimensions 9.40 x 3.77 nm and 9.73 x 3.70 nm, respectively. The specific Kerr constant, Ksp, of ncd is -1.65 x 10(-12) cm2V-2 for the MgADP complex and -1.15 x 10(-12) cm2V-2 for the MgADP.Vi complex. The large negative Ksp for a prolate protein suggests an unusual charge distribution with two long surfaces with opposite charge. The tau 20,W for kinesin(349).MgADP is longer than the corresponding ncd motor and shows a decrease with increased electric field. The kinesin(349).MgADP.Vi complex has a longer tau 20,W. The Ksp for kinesin(349) is 0.36 x 10(-12) cm2V-2 for each complex.
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PMID:Solution structure of two molecular motor domains: nonclaret disjunctional and kinesin. 778 2

Glutaraldehyde-cross-linked microtubules were investigated as substrates for kinesin motility. Microtubules, formed in vitro from chicken brain tubulin, were stabilized with Taxol and chemically fixed with glutaraldehyde. The degree of tubulin monomer cross-linking as a function of time and glutaraldehyde concentration was characterized using polyacrylamide gel electrophoresis. Atomic force microscopy of fixed microtubules indicated that the cross-linking is sufficient to stabilize the gross structure of the microtubules against air drying or a distilled water challenge. Kinesin movement on immobilized, fixed microtubules was determined using a kinesin-coated bead motility assay observed with differential interference contrast microscopy. Within measurement error, kinesin bead movement velocities were independent of the degree of microtubule cross-linking. Binding affinity, however, decreased with increased cross-linking. Although air- and water-challenged microtubules did not support kinesin motility, a dilute suspension of glutaraldehyde-fixed microtubules in buffer supported kinesin motility for at least 2 days without any substantial degradation of activity. Fixed microtubules may be useful for several applications, including affinity purification of microtubule-associated proteins and motility measurements under extreme conditions of temperature and other variables.
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PMID:Kinesin movement on glutaraldehyde-fixed microtubules. 892 59

Biological motors are generally divided into two classes: 1) rotary motors. These include ATP synthase (F0-F1) and the bacterial flagellar motor which are driven by proton and Na+ gradients., 2) linear motors. Myosin, kinesin and dynein are considered to be such motors, F-actin and microtubules serving as passive "tracks". However, data is presented which suggests that the actin filaments rotate in shortening muscle. Microtubules also have been reported to rotate upon interacting with kinesin and dynein. Axial protein rotation thus appears to be a common fundamental characteristic of actin- and of microtubule-based motility systems, in addition to F0-F1 and the bacterial motor. An analysis is carried out of the way ATP hydrolysis and randomly moving protons can induce rotation. It is concluded that all four engines are driven by water jets, thus operating like water turbines.
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PMID:Are rotors at the heart of all biological motors? 961 Mar 53

Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament-binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule-activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild-type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water-mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPASE: The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules.
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PMID:A structural pathway for activation of the kinesin motor ATPase. 1138 96

New crystal structures of the kinesin motors differ from previously described motor-ADP atomic models, showing striking changes both in the switch I region near the nucleotide-binding cleft and in the switch II or 'relay' helix at the filament-binding face of the motor. The switch I region, present as a short helix flanked by two loops in previous motor-ADP structures, rearranges into a pseudo-beta-hairpin or is completely disordered with melted helices to either side of the disordered switch I loop. The relay helix undergoes a rotational movement coupled to a translation that differs from the piston-like movement of the relay helix observed in myosin. The changes observed in the crystal structures are interpreted to represent structural transitions that occur in the kinesin motors during the ATP hydrolysis cycle. The movements of switch I residues disrupt the water-mediated coordination of the bound Mg2+, which could result in loss of Mg2+ and ADP, raising the intriguing possibility that disruption of the switch I region leads to release of nucleotide by the kinesins. None of the new structures is a true motor-ATP state, however, probably because conformational changes at the active site of the kinesins require interactions with microtubules to stabilize the movements.
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PMID:Kinesin: switch I & II and the motor mechanism. 1180 20

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 micro M was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 micro M, and no-effect-concentrations were between 0.001 and 0.005 micro M. Clearly enhanced MN rates were found at 0.1 micro M and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37 degrees C was seen above the no-effect-concentration of 2 mM, with an IC(50) of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 micro M and reaching complete inhibition of motility at 30 micro M, whereas benzonitrile up to 200 micro M did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 micro M. This points to the relevance of interactions with the cellular spindle apparatus.
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PMID:Chromosomal genotoxicity of nitrobenzene and benzonitrile. 1451 6

Although the steps for the forward reaction of ATP hydrolysis by the motor protein kinesin have been studied extensively, the rates for the reverse reactions and thus the energy changes at each step are not as well defined. Oxygen isotopic exchange between water and P(i) was used to evaluate the reverse rates. The fraction of the kinesin x ADP x P(i) complex that reverts to ATP before release of P(i) during net hydrolysis was approximately 0 and approximately 2.6% in the absence and presence of microtubules (MTs), respectively. The rate of synthesis of bound ATP from free P(i) and the MT x kinesin x ADP complex was approximately 1.7 M(-1) x s(-1) (K0.5 ADP = 70 microM) with monomeric kinesin in the absence of net hydrolysis. Synthesis of bound ATP from the ADP of the tethered head of a dimer-MT complex was 20-fold faster than for the monomer-MT complex. This MT-activated ATP synthesis at the tethered head is in marked contrast to the lack of MT stimulation of ADP release from the same site. The more rapid ATP synthesis with dimers suggests that the tethered head binds behind the strongly attached head, because this positions the neck linker of the tethered head toward the plus end of the MT and would thus facilitate its docking on synthesis of ATP. The observed rate of ATP synthesis also puts limits on the overall energetics that suggest that a significant fraction of the free energy of ATP hydrolysis is available to drive the docking of the neck linker on binding of ATP.
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PMID:The tethered motor domain of a kinesin-microtubule complex catalyzes reversible synthesis of bound ATP. 1633 8

In response to volume expansion, red blood cells of the little skate (Raja erinacea) initially swell and then release small organic compounds and osmotically obligated water in what is called a regulatory volume decrease (RVD) to restore cell volume. One of the major intracellular solutes lost during this process is the non-metabolized beta amino acid taurine. This hypoosmotic-induced increase in cell taurine permeability requires the anion exchanger, skAE1. The abundance of this transporter increases on the surface plasma membrane by a process of exocytosis. The second-messenger pathways involved in exocytosis of skAE1 were investigated with the use of inhibitors which affect membrane trafficking. Hypoosmotic-stimulated taurine uptake was significantly decreased by 42% with wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase) inhibitor. Additional evidence for the involvement of PI3K was obtained with a second inhibitor, LY294002, which decreased the hypoosmotic-stimulated taurine uptake by 28%. The state of actin is also involved, as the actin filament depolymerizer latrunculin B decreased hypoosmotic-stimulated taurine uptake by approximately 40%. Although hypoosmotic conditions did not stimulate changes in the distribution of actin between filamentous and globular forms, latrunculin stimulated a decrease in filamentous actin and increase in globular actin in both isoosmotic and hypoosmotic conditions. Disruptors of other potential cytoskeletal factors (myosin, kinesin, dynein, and microtubules) did not affect taurine uptake. The present results suggest that the exocytosis of skAE1 stimulated by hyposmotic-induced cell volume expansion requires activation of PI3 kinase and is regulated by the state of actin filaments.
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PMID:Membrane trafficking factors are involved in the hypotonic activation of the taurine channel in the little skate (Raja erinacea) red blood cell. 1661


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