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 processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results show that the release of two molecules of ADP from dimeric kinesin (K401) after the binding of kinesin ADP to the microtubule is a sequential process leading to biphasic kinetics. The maximum rate of release of mantADP from the first motor domain of K401 or monomeric K341 is fast (300 s-1) and independent of added nucleotide. The rate of mantADP release from the second motor domain of K401 is slow in the absence of added nucleotide (0.4 s-1) and reaches a maximum rate of 300 s-1 at saturating concentrations of ATP. High concentrations of ADP stimulate mantADP release from the second head to a maximum rate of 3.8 s-1. The nonhydrolyzable analog AMP-PNP and ATP-gamma S also stimulate ADP release from the second head (maximum rate of 30 s-1), suggesting that ATP hydrolysis is not necessary to stimulate the ADP release. These experiments establish an alternating site mechanism for dimeric kinesin whereby ATP binding to one kinesin active site stimulates the release of ADP from the second site such that the reactions occurring at the active sites of the two monomer units are kept out of phase from each other by interactions between the heads. These results define the steps of the ATPase pathway that lead to the efficient coupling of ATP hydrolysis to force production in a processive reaction whereby force production in forming a tight microtubule complex by one head is coupled to the rate-limiting release of the other head from the microtubule.
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PMID:Alternating site mechanism of the kinesin ATPase. 945 68

In Drosophila melanogaster the staufen gene encodes an RNA-binding protein that is essential for the correct localization of certain nurse cell-derived transcripts in oocytes. Although the mechanism underlying mRNA localization is unknown, mRNA-staufen complexes have been shown to move in a microtubule-dependent manner, and it has been suggested that staufen associates with a motor protein which generates the movement. We have investigated this possibility using Notonecta glauca in which nurse cells also supply the oocytes with mRNA, but via greatly extended nutritive tubes comprised of large aggregates of parallel microtubules. Using a staufen peptide antibody and RNA probes we have identified a staufen-like protein, which specifically binds double-stranded RNA, in the nutritive tubes of Notonecta. We show that while the staufen-like protein does not co-purify with microtubules from ovaries using standard procedures it does so under conditions of motor-entrapment, specifically in the presence of AMP-PNP. We also show that the staufen-like protein is subsequently removed by ATP and GTP, but not ADP. Nucleotide-dependent binding to microtubules is typical of a motor-mediated interaction and the pattern of attachment and detachment of the staufen-like protein correlates with that of a kinesin protein within the ovaries. Our findings indicate that the staufen-like RNA-binding protein attaches to, and is transported along, Notonecta ovarian microtubules by a kinesin motor.
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PMID:A staufen-like RNA-binding protein in translocation channels linking nurse cells to oocytes in Notonecta shows nucleotide-dependent attachment to microtubules. 1044 89

Kinesin is a microtubule-based motor protein responsible for anterograde transport of vesicles and organelles in nerve axons and other cell types. The energy necessary for this transport is derived from the hydrolysis of ATP which is thought to induce conformational changes in the protein. We have solved the X-ray crystal structures of rat brain kinesin in three conditions intended to mimic different nucleotide states: (1) with ADP bound to the nucleotide-binding site, (2) with bound ADP in the presence of AIF(-)4, and (3) with ADP hydrolyzed to AMP by apyrase. In contrast to analogous cases observed in GTP-binding proteins or the muscle motor myosin, the structure of kinesin remained nearly unchanged. This highlights the stability of kinesin's ADP state in the absence of microtubules. Surprisingly, even after hydrolysis of ADP to AMP by apyrase a strong density peak remains at the position of the beta-phosphate which is compatible either with a phosphate or a sulfate from the solvent and appears to stabilize the nucleotide-binding pocket through several hydrogen bonds.
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PMID:The structure of the nucleotide-binding site of kinesin. 1049 51

Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes.
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PMID:Identification and characterization of a novel microtubule-based motor associated with membranous organelles in tobacco pollen tubes. 1100 43

Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor-ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by approximately 300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end-directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 microM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized.
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PMID:Microtubule and motor-dependent endocytic vesicle sorting in vitro. 1101 63

Many cell types contain a subset of long-lived, 'stable' microtubules that differ from dynamic microtubules in that they are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). Elevated Glu tubulin does not stabilize the microtubules and the mechanism for the stability of Glu microtubules is not known. We used detergent-extracted cell models to investigate the nature of Glu microtubule stability. In these cell models, Glu microtubules did not incorporate exogenously added tubulin subunits on their distal ends, while >70% of the bulk microtubules did. Ca(2+)-generated fragments of Glu microtubules incorporated tubulin, showing that Glu microtubule ends are capped. Consistent with this, Glu microtubules in cell models were resistant to dilution-induced breakdown. Known microtubule end-associated proteins (EB1, APC, p150(Glued) and vinculin focal adhesions) were not localized on Glu microtubule ends. ATP, but not nonhydrolyzable analogues, induced depolymerization of Glu microtubules in cell models. Timelapse and photobleaching studies showed that ATP triggered subunit loss from the plus end. ATP breakdown of Glu microtubules was inhibited by AMP-PNP and vanadate, but not by kinase or other inhibitors. Additional experiments showed that conventional kinesin or kif3 were not involved in Glu microtubule capping. We conclude that Glu microtubules are stabilized by a plus-end cap that includes an ATPase with properties similar to kinesins.
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PMID:Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap. 1105 78

The motility of kinesin motors is explained by a "hand-over-hand" model in which two heads of kinesin alternately repeat single-headed and double-headed binding with a microtubule. To investigate the binding mode of kinesin at the key nucleotide states during adenosine 5'-triphosphate (ATP) hydrolysis, we measured the mechanical properties of a single kinesin-microtubule complex by applying an external load with optical tweezers. Both the unbinding force and the elastic modulus in solutions containing AMP-PNP (an ATP analog) were twice the value of those in nucleotide-free solution or in the presence of both AMP-PNP and adenosine 5'-diphosphate. Thus, kinesin binds through two heads in the former and one head in the latter two states, which supports a major prediction of the hand-over-hand model.
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PMID:Nucleotide-dependent single- to double-headed binding of kinesin. 1115 81

Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.
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PMID:Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules. 1160 96

The unicellular green alga Micrasterias denticulata performs a two-directional postmitotic nuclear migration during development, a passive migration into the growing semicell, and a microtubule mediated backward migration towards the cell centre. The present study provides first evidence for force generation by motor proteins of the kinesin family in this process. The new kinesin specific inhibitor adociasulfate-2 causes abnormal nuclear displacement at 18 microM. AMP-PNP, a non hydrolyseable ATP analogue or the general ATPase inhibitors calyculin A and sodium orthovanadate also disturb nuclear migration. In addition kinesin-like proteins are detected by means of immunoblotting using antibodies against brain kinesin, plant derived antibodies to kinesin-like proteins and a calmodulin binding kinesin-like protein. Immunoelectron microscopy suggests a correlation of conventional kinesin-like proteins, but not of the calmodulin binding kinesin-like protein to the microtubule apparatus associated with the migrating nucleus.
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PMID:Kinesin-like proteins are involved in postmitotic nuclear migration of the unicellular green alga Micrasterias denticulata. 1217 72

Kinesin is a processive motor protein that "walks" on a microtubule toward its plus end. We reported previously that the distribution of unbinding force and elastic modulus for a single kinesin-microtubule complex was either unimodal or bimodal depending on the nucleotide states of the kinesin heads, hence showing that the kinesin may bind the microtubule either with one head or with both heads at once. Here, we found that the shape of the unbinding-force distribution depends both on the loading rate and on the manner of loading not only in the presence of AMP-PNP but also in the absence of nucleotides. Irrespective of the nucleotide state and the loading conditions examined here, the unbinding force obtained by loading directed toward the minus end of microtubule was 45% greater than that for plus end-directed loading. These results could be explained by a model in which equilibrium exists between single- and double-headed binding and the load (F) dependence of lifetime, tau(F), of each binding is expressed by tau(F) = tau(0)exp(-Fd/k(B)T), where tau(0) is the lifetime without external load and d a characteristic distance, both of which depend on single- or double-headed binding, k(B), the Boltzmann constant and T, the absolute temperature. The model analysis showed that the forward and backward rates of transition from single- to double-headed binding are 2 and 0.2/s for the AMP-PNP state, and 70 and 7/s for the nucleotide-free state. Moreover, in the presence of AMP-PNP, we detected the moment of transition from single- to double-headed binding through an abrupt increase in the elastic modulus and estimated the transition rate to be approximately 1/s, which is consistent with the model analysis.
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PMID:Equilibrium and transition between single- and double-headed binding of kinesin as revealed by single-molecule mechanics. 1254 91


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