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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 from porcine brain was prepared by a procedure based on the strong binding of the protein to microtubules in the presence of sodium fluoride and ATP. The protocol reduces the requirement for taxol and AMP-PNP. The kinesin is active in terms of its ability to move microtubules on glass slides and its ATPase. The ATPase of this kinesin is about 8 nmol/min/mg; it is activated to 19 nmol/min/mg in the presence of microtubules. The relationship between gliding velocity and ATP concentration follows Michaelis-Menten kinetics. Using the motility assay, the maximal velocity is 0.78 micron/sec, and the Km value is 150 microM for ATP. For GTP the corresponding values are 0.38 micron/sec and 1.7 mM. ADP is a competitive inhibitor (Ki = 0.29 mM). Crude preparations of kinesin do not support motility on glass slides, whereas gel-filtered kinesin does. A search for potential inhibitory factors showed that one of them is MAP2; however, its inhibitory effect becomes visible only in certain conditions. MAP2 bound to microtubules does not inhibit kinesin-induced motility. However, when MAP2 and kinesin are preadsorbed to the glass surface independently of microtubules, MAP2 prevents the interaction of kinesin with microtubules, as if it formed a "lawn" that acted as a spacer and thus repelled the MAP-free microtubules or crosslinked the MAP-containing ones. The repelling effect of MAP2 domains (projection or assembly fragments obtained by chymotryptic cleavage) added separately is less pronounced and can be overcome by kinesin. These results reinforce the view of MAP2 as a spacer molecule.
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PMID:Interaction between kinesin, microtubules, and microtubule-associated protein 2. 253 84

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

We have demonstrated the presence of kinesin in the secretory pancreatic tissue using SDS-PAGE, immunoblot and immunoelectron microscopy techniques. Polyclonal antibodies were raised against the rat brain kinesin heavy chain and affinity-purified. Immunoblot studies showed that these antibodies were bound to a 116 kDa protein found in rat pancreas crude extracts and in partially purified kinesin fractions. Kinesin identification was also performed by a cosedimentation procedure based on its strong binding to microtubules in the presence of sodium fluoride. The microtubule-kinesin complex was observed by immunoelectron microscopy gold staining. The reversible association of kinesin with microtubules was generated by MgATP.
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PMID:Rat pancreas kinesin: identification and potential binding to microtubules. 833 81

To investigate the possibility that kinesin transports vesicles bearing proteins essential for ion channel activity, the effects of kinesin (Khc) and ion channel mutations were compared in Drosophila using established tests. Our results show that Khc mutations produce defects and genetic interactions characteristic of paralytic (para) and maleless (mle) mutations that cause reduced expression or function of the alpha-subunit of voltage-gated sodium channels. Like para and mle mutations, Khc mutations cause temperature-sensitive (TS) paralysis. When combined with para or mle mutations, Khe mutations cause synthetic lethality and a synergistic enhancement of TS-paralysis. Furthermore, Khc: mutations suppress Shaker and ether-a-go-go mutations that disrupt potassium channel activity. In light of previous physiological tests that show that Khc mutations inhibit compound action potential propagation in segmental nerves, these data indicate that kinesin activity is required for normal inward sodium currents during neuronal action potentials. Tests for phenotypic similarities and genetic interactions between kinesin and sodium/potassium ATPse mutations suggest that impaired kinesin function does not affect the driving force on sodium ions. We hypothesize that a loss of kinesin function inhibits the anterograde axonal transport of vesicles bearing sodium channels.
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PMID:Mutation of the axonal transport motor kinesin enhances paralytic and suppresses Shaker in Drosophila. 877 May 97

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

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

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

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


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