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)

We have isolated and compared the 116-kilodalton (kDa) kinesin heavy chain from DU 145 human prostatic tumor cells and bovine brain. Comparative sodium dodecyl sulfate - polyacrylamide gel electrophoreses (SDS-PAGE), Western blots, and proteolytic digestion analysis all showed that the 116-kDa polypeptides from both sources were indistinguishable. Polyclonal antibodies raised against sea urchin kinesin cross-reacted with both brain and DU 145 kinesin on Western blots. SDS-PAGE and A-5m chromatographic studies indicated that kinesin forms a quarternary heteropolymer of approximately 400 kDa. DU 145 cells had three proteins of 116, 72, and 64 kDa forming the heteropolymer, in a 2:1:1 ratio, whereas brain cells appeared to have equimolar amounts of the 116-kDa heavy chain and a 64-kDa light chain.
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PMID:Properties of kinesin isolated from human prostatic DU 145 tumor cells and bovine brain. 214 May 13

Microtubule (MT)-binding peptides have been detected in homogenates of bovine brain tissue utilizing a blot overlay assay. Blots were prepared by the electrophoretic transfer to nitrocellulose of proteins separated on polyacrylamide gels. These blots were incubated with taxol stabilized MTs or tubulin, rinsed, and then fixed by air drying. About 17 soluble MT-associated proteins (MAPs) were identified by immunodetection of bound tubulin, including MAP2, kinesin, and tau. The interaction of MTs with these peptides appears to be specific, since MT binding can be displaced by a fluorescent tubulin analog, is competitively inhibited by the addition of exogenous brain MAPs, is decreased by raising the salt concentration, and is diminished by sodium dodecyl sulfate (SDS) denaturation. Only one protein (150 kDa) appears to have an interaction with MTs that is stable in high salt. The specificity of the binding on blots is further illustrated by the interaction of MTs with the MT-binding domains of MAP2 (32-35 kDa fragments) and kinesin (64 kDa fragment). Specific MT-binding peptides or domains can thus be isolated and characterized with this method, which requires little protein and is suitable for use with proteins that are either soluble or insoluble under physiological conditions.
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PMID:Blot overlay identification of microtubule-binding peptides from bovine brain. 238 8

Kinesin was extensively purified from bovine brain cytosol by a microtubule-binding step in the presence of 5'-adenylyl imidodiphosphate (AMP-PNP), followed by gel filtration chromatography and sucrose gradient ultracentrifugation. The products consistently contained 124,000 (124K) and 64,000 (64K) dalton polypeptides. These two polypeptides appear to represent heavy and light chains of kinesin, respectively, because they copurified on sucrose gradients to a constant and equimolar stoichiometry and bound stably to microtubules in the presence of AMP-PNP but not ATP. The mobilities of 124K and 64K in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions were the same as under nonreducing conditions. A diffusion coefficient of (2.24 +/- 0.21) X 10(-7) cm2 s-1 and a sedimentation coefficient of (9.56 +/- 0.34) X 10(-13) s were determined for native kinesin by gel filtration and sucrose gradient ultracentrifugation, respectively. These values were used to calculate a native molecular weight of about 379,000 and suggest that kinesin has an axial ratio of approximately 20. Extensively purified kinesin exhibited microtubule-activated ATPase activity, and only the 124K subunit incorporated ATP in photoaffinity labeling experiments using [32P]ATP. Collectively, these data favor the interpretation that bovine brain kinesin is a highly elongated, microtubule-activated ATPase comprising two subunits each of 124,000 and 64,000 daltons, that the subunits are not linked to one another by disulfide bonds, and that the heavy chains are the ATP-binding subunits.
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PMID:Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide. 313 48

Crystals of the single-headed and double-headed kinesin motor domains of Rattus norvegicus have been grown by vapor diffusion using ammonium sulfate as the precipitant. Both crystal systems belong to the orthorhombic space group P2(1)2(1)2(1). Double-headed kinesin crystallized with unit cell constants of a = 72.2 A, b = 91.9 A, and c = 141.7 A, and so far the best crystals diffracted to a maximum resolution of 2.7 A. Using ammonium sulfate single-headed kinesin crystallized in two different crystal forms with cell constants of a = 73.1 A, b = 73.2 A, c = 84.0 A and a = 73.4 A, b = 74.1 A, c = 74.7 A, respectively. They were found to diffract to 2.1 A resolution. Crystals of monomeric kinesin were also obtained with lithium sulfate as precipitant. They have cell constants of a = 71.6 A, b = 73.7 A, and c = 74.1 A and diffract up to 1.7 A resolution.
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PMID:Crystallization and preliminary X-ray analysis of the single-headed and double-headed motor protein kinesin. 921 86

Many kinds of animal embryos exhibit stereotyped cleavage patterns during early embryogenesis. In the ascidian Halocynthia roretzi, cleavage patterns are invariant but they are complicated by successive unequal cleavages that occur in the posterior region. Here we report the essential roles of a novel structure, called the centrosome-attracting body (CAB), which exists in the posterior pole cortex of cleaving embryos, in generating unequal cleavages. By removing and transplanting posterior egg cytoplasm and by treatment with sodium dodecyl sulfate, we demonstrated that loss of the CAB resulted in abolishment of unequal cleavage, while ectopic formation of the CAB caused ectopic unequal cleavages to occur. Experiments with a microtubule inhibitor demonstrated that the centrosome and nucleus were attracted toward the posterior cortex, where the CAB is located, by shortening of microtubule bundles formed between the centrosome and the CAB. Consequently, the mitotic apparatus was positioned asymmetrically, resulting in unequal cleavage. Immunohistochemistry provided evidence that a microtubule motor protein, a kinesin or kinesin-like molecule, may be associated with the CAB. Formation of the CAB during the early cleavage stage was resistant to treatment with the microtubule inhibitor. In contrast, the integrity of the CAB was lost upon treatment with a microfilament inhibitor. We propose that the CAB plays key roles in the orientation and positioning of cleavage planes during unequal cell division.
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PMID:The centrosome-attracting body, microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo. 1020 44

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

A novel protein with ATPase activity was purified from the cytoplasmic extracts of maize pollen by acetone precipitation, ammonium sulfate fractionation, followed by DEAE-Sephadex A(50) and Mono S ion-exchange chromatography. The molecular weight was about 28 kD as determined by SDS-PAGE and the isoelectric point was pH 8.3 by IEF-PAGE. Western blotting analysis showed the 28 kD protein had no specific immuno-reactions with the anti-kinesin monoclonal or the anti-dynamin polyclonal antibodies. The maximum ultraviolet absorbance was at 278 nm, CD spectrum analysis showed the that 28 kD protein with the feature of a globulin. Pharmacological studies indicated that the enzyme activity was strongly inhibited by Na(3)VO(4) but insensitive to NEM. It was inhibited about 50% by NaF. Oligomycin, KNO(3) and ouabain had no effects on its ATPase activity.
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PMID:Purification of the 28 kD Protein from Maize Pollen and Studies on Its Properties. 1223 28

Crystal structures of the molecular motor kinesin show conformational variability in a structural element called the neck linker. Conformational change in the neck linker, initiated by ATP exchange, is thought to drive the movement of kinesin along the microtubule track. We use site-specific EPR measurements to show that when microtubules are absent, the neck linker exists in equilibrium between two structural states (disordered and 'docked'). The active site nucleotide does not control the position taken by the neck linker. However, we find that sulfate can specifically bind near the nucleotide site and stabilize the docked neck linker conformation, which we confirmed by solving a new crystal structure. Comparing the crystal structures of our construct with the docked or undocked neck linker reveals how microtubule binding may activate the nucleotide-sensing mechanism of kinesin, allowing neck linker transitions to power motility.
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PMID:Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy. 1236 2

The present paper describes two new monoclonal antibodies (MAbs) KN-02 and KN-03 against the heavy chain of conventional kinesin. The kinesin was purified from porcine brain by a combined procedure of ion exchange chromatography, tripolyphosphate-supported microtubule affinity-binding, and gel filtration. Hybridoma cell lines producing antibodies were obtained after immunization of a Balb/c mouse with kinesin and subsequent fusion of the spleen cells with Sp2/0 myeloma cells. The specificity was verified by enzyme-linked immunosorbent assay (ELISA) and further confirmed by immunoblotting and immunoprecipitation analysis. The antibodies recognize different epitopes on the heavy chain of the kinesin molecule as demonstrated by chymotryptic cleavage of kinesin followed by immunoblotting. Differential location of relevant epitopes was also documented by in vitro binding experiments with purified kinesin and taxol-stabilized microtubules. While the KN-03 antibody decorated microtubules, no such staining was observed with KN-02 antibody. The antibodies have a lower affinity to sodium dodecyl sulfate (SDS)-denatured kinesin, but immunofluorescence on fixed cells gave strong dot-like staining characteristic for localization of kinesin on vesicles. The same staining pattern was observed in different cell types. Double-label fluorescence with polyclonal anti-tubulin antibody revealed a co-distribution of stained vesicles with microtubules on the cell periphery. The antibodies KN-02 and KN-03 are therefore valuable tools for localization of kinesins in cells of different tissue origin.
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PMID:Monoclonal antibodies KN-02 and KN-03 against the heavy chain of kinesin. 1257 9

Developing neurons express a motor protein called kinesin-5 (also called kif11 or Eg5) which acts as a 'brake' on the advance of the microtubule array during axonal growth. Pharmacological inhibition of kinesin-5 causes the developing axon to grow at a faster rate, retract less and grow past cues that would otherwise cause it to turn. Here we demonstrate that kinesin-5 is also expressed in adult neurons, albeit at lower levels than during development. We hypothesized that inhibiting kinesin-5 might enable adult axons to regenerate better and to overcome repulsive molecules associated with injury. Using adult mouse dorsal root ganglion neurons, we found that anti-kinesin-5 drugs cause axons to grow faster and to cross with higher frequency onto inhibitory chondroitin sulfate proteoglycans. These effects may be due in part to changes in the efficiency of microtubule transport along the axonal shaft as well as enhanced microtubule entry into the distal tip of the axon. Effects observed with the drugs are further enhanced in some cases when they are used in combination with other treatments known to enhance axonal regeneration. Collectively, these results indicate that anti-kinesin-5 drugs may be a useful addition to the arsenal of tools used to treat nerve injury.
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PMID:Inhibition of Kinesin-5, a microtubule-based motor protein, as a strategy for enhancing regeneration of adult axons. 2116 43


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