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

---

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

Addition of NaCl or KCl in the presence of 50 nM ATP induces a shift in the sedimentation coefficient (apparent S20,w) of kinesin from 9.4 S at low ionic strength to 6.5 S at high ionic strength. The midpoint for the transition occurs at ionic strength values of 0.39, 0.25, and 0.18 for pH values of 6.3, 6.9, and 8.3, respectively. Gel filtration experiments indicate that the transition to the 6.5 S species is accompanied by a decrease in the diffusion coefficient. Under all conditions which were tested, the 64-kDa beta subunits comigrate with the 120-kDa alpha subunits without any evidence for dissociation of the alpha 2 beta 2 complex. These results are consistent with the change in sedimentation coefficient being due to a conformational transition between a folded form at low ionic strength and an extended form at high ionic strength. This conformational transition is not significantly affected by the nature of the nucleotide bound at the active site since similar results are obtained both in the presence of excess EDTA, which removes the bound ADP, and after replacement of the bound ADP with adenosine 5'-(beta,gamma-imino)triphosphate. The alpha 2 form of kinesin, which lacks the beta subunits, undergoes a similar transition between a 6.7 S form at low ionic strength and a 5.1 S form at high ionic strength with a midpoint for the transition at an ionic strength of 0.5 at pH 6.9. Electron microscopic observation also indicates a transition between a folded conformation at low ionic strength and an extended conformation at high ionic strength for both the alpha 2 beta 2 and alpha 2 species.
...
PMID:Kinesin undergoes a 9 S to 6 S conformational transition. 156 10

We have analyzed the effects of various substrates and inhibitors on the rates of microtubule (MT) motility induced by sea urchin egg kinesin using real-time computer analysis and video-enhanced light microscopy. In the presence of magnesium, 10 mM concentrations of all the nucleotides tested supported MT translocation, with velocities in MgATP greater than MgGTP greater than MgTTP approximately equal to MgUTP greater than MgCTP greater than MgITP. The velocity of kinesin-driven MT motility is fairly uniform over approximately 3 pH units, from pH 6 to 9, with almost no motility outside this range. In the presence of ATP, no motility is observed in the absence of divalent cations; addition of Mg2+ but not addition of Ca2+ restores motility. MgATP-dependent MT motility is reversibly inhibited by Mg-free ATP, EDTA, or tripolyphosphate, suggesting that Mg-free ATP is an inactive substrate analogue. MgATP and MgGTP both obey saturable, Michaelis-Menten kinetics, with apparent Km values of approximately 60 microM and 2 mM, and Vmax values of approximately 0.6 and 0.4 microns/s, respectively. MgATP gamma S and MgADP are classic competitive inhibitors of kinesin-driven motility in MgATP, with Ki values of approximately 15 and 150 microM, respectively. Adenosine 5'-(beta, gamma-methylene)-triphosphate and N-ethylmaleimide only inhibit MT motility weakly, while adenyl-5'-yl imidodiphosphate and vanadate strongly inhibit MT motility, but not in a simple competitive manner. Moreover, in contrast to other inhibitors which cause a unimodal decrease in MT mean velocity, vanadate concentrations greater than approximately 10% that of MgATP cause some MTs to become immotile, resulting in a bimodal distribution of MT velocities.
...
PMID:Quantitative analysis of sea urchin egg kinesin-driven microtubule motility. 252 43

Bovine brain kinesin binds ADP tightly and contains a stoichiometric amount of ADP at its active site when isolated in the presence of free Mg2+ (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318). EDTA in excess of Mg2+ weakens ADP binding and nucleotide-free kinesin can be prepared by gel filtration with excess EDTA. On addition of ATP, this nucleotide-free enzyme catalyzes the rapid hydrolysis of a stoichiometric amount of ATP in a burst phase followed by much slower continued ATP hydrolysis limited by the release of ADP from the active site. This burst reaction is evident both by formation of [32P]Pi from [gamma-32P]ATP and by formation of [alpha-32P]ADP from [alpha-32P]ATP. At 1.1 nM kinesin active sites, the observed rate of the burst phase increases linearly with ATP over the 1-20 nM range yielding a bimolecular rate of net ATP binding and hydrolysis of 2.5 microM-1 s-1. The intercept at zero ATP is 0.008 s-1 which equals the ADP release rate at 0.008-0.009 s-1. This predicts a Km for ATP of approximately 3.5 nM and measurements of the dependence on ATP concentration of the steady state rate and amount of bound ADP are consistent with a Km of this magnitude.
...
PMID:Nucleotide-free kinesin hydrolyzes ATP with burst kinetics. 252 42

Coupling between ATP hydrolysis and microtubule movement was demonstrated several years ago in flagellar axonemes and subsequent studies suggest that the relevant microtubule motor, dynein, uses ATP to drive microtubule sliding by a cross-bridge mechanism analogous to that of myosin in muscles. Kinesin, a microtubule-based motility protein which may participate in organelle transport and mitosis, binds microtubules in a nucleotide-sensitive manner, and requires hydrolysable nucleotides to translocate microtubules over a glass surface. Recently, neuronal kinesin was shown to possess microtubule-activated ATPase activity although coupling between ATP hydrolysis and motility was not demonstrated. Here we report that sea urchin egg kinesin, prepared either with or without a 5'-adenylyl imidodiphosphate(AMPPNP)-induced microtubule binding step, also possesses significant microtubule-activated ATPase activity when Mg-ATP is used as a substrate. This ATPase activity is inhibited in a dose-dependent manner by addition of Mg-free ATP, by chelation of Mg2+ with EDTA, by addition of Na3VO4, or by addition of AMPPNP with or without Mg2+. Addition of these same reagents also inhibits the microtubule-translocating activities of sea urchin egg kinesin in a dose-dependent manner, supporting the hypothesis that kinesin-driven motility is coupled to the microtubule-activated Mg2+-ATPase activity.
...
PMID:Correlation between the ATPase and microtubule translocating activities of sea urchin egg kinesin. 295 28

The pre-steady-state kinetics of the microtubule-kinesin ATPase were investigated by chemical-quench flow methods using the Drosophila kinesin motor domain (K401) expressed in Escherichia coli [Gilbert, S. P., & Johnson, K. A. (1993) Biochemistry 32, 4677-4684]. The results define a minimal mechanism: M.K + ATP in equilibrium with (M).K.ATP in equilibrium with (M).K.ADP.Pi in equilibrium with M.K.ADP + Pi in equilibrium with M.K + ADP, where M, K, and Pi represent microtubules, kinesin, and inorganic phosphate, respectively, with k+1 = 0.8-3 microM-1 s-1, k-1 = 100-300 s-1, k+2 = 70-120 s-1, k+4 = 10-20 s-1, and k+3 >> k-2 and k+3 >> k+4. Conditions were as follows: 25 degrees C, 20 mM HEPES, pH 7.2 with KOH, 5 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM potassium acetate, 1 mM DTT. The experiments presented do not determine the step in the cycle where kinesin dissociates from the microtubule or the step at which kinesin reassociates with the microtubule; therefore, the steps that may represent kinesin as the free enzyme are indicated by (M). A burst of ADP product formation was observed during the first turnover of the enzyme in the acid-quench experiments that define the ATP hydrolysis transient. The observation of the burst demonstrates that product release is rate limiting even in the presence of saturating microtubule concentrations. The pulse-chase experiments define the time course of ATP binding to the microtubule-K401 complex. At low ATP concentrations, ATP binding limits the rate of the burst. However, at high concentrations of ATP, ATP binding is faster than the rate of ATP hydrolysis with k+2 = 70-120 s-1. The amplitude of the burst of the ATP binding transient reached a maximum of 0.7 per site at saturating concentrations of ATP and microtubules. The amplitude of less than 1 is attributed to the fast k(off) for ATP (k-1 = 100-300 s-1) that leads to a partitioning of the M.K.ATP complex between ATP hydrolysis (k+2) and ATP release (k-1). These results indicate that ATP binds weakly to the M.K complex (Kd,ATP app approximately 100 microM). ADP release (k+4 = 10-20 s-1) is rate limiting during steady-state turnover, indicating that microtubules activate the kinesin ATPase by increasing k(off),ADP from 0.01 s-1 in the absence of microtubules to 10-20 s-1 at saturating microtubule concentrations.
...
PMID:Pre-steady-state kinetics of the microtubule-kinesin ATPase. 811 Aug

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.
...
PMID:Comparison of ncd and kinesin motor domains by circular dichroism spectroscopy. 901 Jul 67

Displacement of the fluorescent substrate analogue methylanthraniloyl ADP (mant-ADP) from kinesin by excess ATP results in a biphasic fluorescent transient. The pH and microtubule dependence of the rates and amplitudes indicates that the two phases are produced by release of bound mant-ADP, with an excess of the 3'-isomer, followed by the subsequent relaxation of the free 2'- and 3'-isomers to their equilibrium distribution. The first phase for release of mant-ADP is accelerated by microtubules and occurs at the same rate as ADP release measured using [32P]ADP. The second phase is subject to base catalysis and occurs at the same rate as the isomerization of isolated 2'- or 3'-mant-ATP over a 100-fold range of rates. The bound mant-ADP isomers undergo isomerization rapidly when bound to kinesin at pH 8.2, whereas mant-ADP isomers interconvert only slowly when bound to myosin. No fluorescence resonance energy transfer occurs between the single tryptophan in the kinesin neck domain and bound mant-ADP, but efficient energy transfer does occur from protein tyrosine groups. The rate of mant-ADP release in the absence of microtubules is minimal (0.005 s-1) at pH 7-8, 2 mM Mg2+, and 25 mM KCl but is accelerated at lower pH (0.04 s-1 at pH 5.5) and either lower or higher [KCl] (0.01 and 0. 06 s-1 at 0 and 800 mM KCl, respectively). The microtubule-stimulated rate of ADP release is accelerated at low pH and inhibited by high concentrations of monovalent salts. Reduction of the free Mg2+ by addition of excess EDTA increases the release of mant-ADP from E.MgADP to 0.03 s-1. This acceleration at low Mg2+ likely represents sequential release of Mg2+ at 0.03 s-1 followed by rapid release of ADP, as the rate of ADP release from Mg-free E.ADP is fast (>0.5 s-1). At high Mg2+, rebinding of Mg2+ to E.ADP forces release of ADP from the E.MgADP complex at 0.005 s-1.
...
PMID:Interaction of mant-adenosine nucleotides and magnesium with kinesin. 954 60

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.
...
PMID:Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport. 1084 36

Puralpha, which is involved in diverse aspects of cellular functions, is strongly expressed in neuronal cytoplasm. Previously, we have reported that this protein controls BC1 RNA expression and its subsequent distribution within dendrites and that Puralpha is associated with polyribosomes. Here, we report that, following treatment with EDTA, Puralpha was released from polyribosomes in mRNA/protein complexes (mRNPs), which also contained mStaufen, Fragile X Mental Retardation Protein (FMRP), myosin Va, and other proteins with unknown functions. As the coimmunoprecipitation of these proteins by an anti-Puralpha antibody was abolished by RNase treatment, Puralpha may assist mRNP assembly in an RNA-dependent manner and be involved in targeting mRNPs to polyribosomes in cooperation with other RNA-binding proteins. The immunoprecipitation of mStaufen- and FMRP-containing mRNPs provided additional evidence that the anti-Puralpha detected structurally or functionally related mRNA subsets, which are distributed in the somatodendritic compartment. Furthermore, mRNPs appear to reside on rough endoplasmic reticulum equipped with a kinesin motor. Based on our present findings, we propose that this rough endoplasmic reticulum structure may form the molecular machinery that mediates and regulates multistep transport of polyribosomes along microtubules and actin filaments, as well as localized translation in the somatodendritic compartment.
...
PMID:Identification of mRNA/protein (mRNP) complexes containing Puralpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor. 1214 88

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(II) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1 microM of complexed Hg(II), and for inhibition of motility it was 0.05 microM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 microM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned.
...
PMID:Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II). 1536 76


1

---