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)

N-Ethylmaleimide, an agent which alkylates free sulfhydryls in proteins, has been used to probe the role of sulfhydryls in kinesin, a motor protein for the movement of membrane-bounded organelles in fast axonal transport. When squid axoplasm is perfused with concentrations of NEM higher than 0.5 mM, organelle movements in both the anterograde and retrograde directions cease, and the vesicles remain attached to microtubules. Incubation of highly purified bovine brain kinesin with similar concentrations of NEM modifies the enzyme's microtubule-stimulated ATPase activity and promotes the binding of kinesin to microtubules in the presence of ATP. These results suggest that alkylation of sulfhydryls on kinesin alters the conformation of the protein in a manner that profoundly affects its interactions with ATP and microtubules. The NEM-sensitive sulfhydryls, therefore, may provide a valuable tool for the dissection of functional domains of the kinesin molecule and for understanding the mechanochemical cycle of this enzyme.
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PMID:Modification of the microtubule-binding and ATPase activities of kinesin by N-ethylmaleimide (NEM) suggests a role for sulfhydryls in fast axonal transport. 248 99

Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane-bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface.
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PMID:Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells. 252 55

Kinesin is a motor protein that uses energy derived from ATP hydrolysis to move organelles along microtubules. Using a new technique for measuring the movement produced in vitro by individual kinesin molecules, it is shown that a single kinesin molecule can move a microtubule for several micrometers. New information about the mechanism of force generation by kinesin is presented.
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PMID:Movement of microtubules by single kinesin molecules. 253 Apr 55

Neurons are highly polarized cells composed of dendrites, cell bodies, and long axons. Because of the lack of protein synthesis machinery in axons, materials required in axons and synapses have to be transported down the axons after synthesis in the cell body. Fast anterograde transport conveys different kinds of membranous organelles such as mitochondria and precursors of synaptic vesicles and axonal membranes, while organelles such as endosomes and autophagic prelysosomal organelles are conveyed retrogradely. Although kinesin and dynein have been identified as good candidates for microtubule-based anterograde and retrograde transporters, respectively, the existence of other motors for performing these complex axonal transports seems quite likely. Here we characterized a new member of the kinesin super-family, KIF3A (50-nm rod with globular head and tail), and found that it is localized in neurons, associated with membrane organelle fractions, and accumulates with anterogradely moving membrane organelles after ligation of peripheral nerves. Furthermore, native KIF3A (a complex of 80/85 KIF3A heavy chain and a 95-kD polypeptide) revealed microtubule gliding activity and baculovirus-expressed KIF3A heavy chain demonstrated microtubule plus end-directed (anterograde) motility in vitro. These findings strongly suggest that KIF3A is a new motor protein for the anterograde fast axonal transport.
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PMID:KIF3A is a new microtubule-based anterograde motor in the nerve axon. 751 68

This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster. Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled-coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.
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PMID:Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport. 752

To further elucidate the mechanism of organelle transport, we cloned a novel member of the mouse kinesin superfamily, KIF1B. This N-terminal-type motor protein is expressed ubiquitously in various kinds of tissues. In situ hybridization revealed that KIF1B is expressed abundantly in differentiated nerve cells. Interestingly, K1F1B works as a monomer, having a microtubule plus end-directed motility. Our rotary shadowing electron microscopy revealed mostly single globular structures. Immunocytochemically, KIF1B was colocalized with mitochondria in vivo. Furthermore, a subcellular fractionation study showed that KIF1B was concentrated in the mitochondrial fraction, and purified K1F1B could transport mitochondria along microtubules in vitro. These data strongly suggested that KIF1B works as a monomeric motor for anterograde transport of mitochondria.
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PMID:KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. 752 8

Kinesin is known as a representative cytoskeletal motor protein that is engaged in cell division and axonal transport. In addition to the mutant assay, recent advances using the PCR cloning technique have elucidated the existence of many kinds of kinesin-related proteins in yeast, Drosophila, and mice. We previously cloned five different members of kinesin superfamily proteins (KIFs) in mouse brain (Aizawa, H., Y. Sekine, R. Takemura, Z. Zhang, M. Nangaku, and N. Hirokawa. 1992. J. Cell Biol. 119:1287-1296) and demonstrated that one of them, KIF3A, is an anterograde motor (Kondo, S., R. Sato-Yashitake, Y. Noda, H. Aizawa, T. Nakata, Y. Matsuura, and N. Hirokawa. J. Cell Biol. 1994. 125:1095-1107). We have now characterized another axonal transport motor, KIF2. Different from other KIFs, KIF2 is a central type motor, since its motor domain is located in the center of the molecule. Recombinant KIF2 exists as a dimer with a bigger head and plus-end directionally moves microtubules at a velocity of 0.47 +/- 0.11 microns/s, which is two thirds that of kinesin's. Immunocytological examination showed that native KIF2 is abundant in developing axons and that it accumulates in the proximal region of the ligated nerves after a 20-h ligation. Soluble KIF2 exists without a light chain, and KIF2's associated-vesicles, immunoprecipitated by anti-KIF2 antibody, are different from those carried by existing motors such as kinesin and KIF3A. They are also distinct from synaptic vesicles, although KIF2 is accumulated in so-called synaptic vesicle fractions and embryonal growth cone particles. Our results strongly suggest that KIF2 functions as a new anterograde motor, being specialized for a particular group of membranous organelles involved in fast axonal transport.
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PMID:KIF2 is a new microtubule-based anterograde motor that transports membranous organelles distinct from those carried by kinesin heavy chain or KIF3A/B. 753 3

We have previously demonstrated that the in vivo vitreal injection of an antisense oligonucleotide directed to the kinesin heavy chain inhibits retinal kinesin synthesis by 82% and concomitantly inhibits rapid transport of total protein into the optic nerve by 70%. These results establish a major role for kinesin in rapid axonal transport in vivo. Recently, the cloning of a family of kinesin-like molecules from the mammalian brain has been reported, and some of these proteins are also expressed in neurons. To assign to specific function to the kinesin heavy chain we inhibited the kinesin synthesis with an antisense kinesin oligonucleotide and assessed the axonal transport into the optic nerve of representative proteins from each of three vesicle classes that contain rapidly transported proteins. Marker proteins used were substance P for peptide-containing synaptic vesicles, the amyloid protein for plasma membrane precursor vesicles, and several integral synaptic vesicle proteins. Our results indicate that the major anterograde motor protein for all three vesicle classes utilizes kinesin heavy chain, although we discuss alternative explanations.
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PMID:Inhibition of kinesin synthesis in vivo inhibits the rapid transport of representative proteins for three transport vesicle classes into the axon. 753 13

We have used monoclonal antibodies to perform confocal light microscopic immunolocalization of KRP(85/95), a heterotrimeric plus-end-directed microtubule motor protein, in dividing cells of sea urchin embryos. Embryos were stained during the first division cycle, and dissociated blastomeres were stained at the 32- to 64-cell stages. Double labeling of the dividing cells with anti-tubulin and anti-KRP(85/95) showed a clear concentration of the motor protein in the mitotic apparatus; KRP(85/95) appeared to associate with pericentriolar regions during prophase, with kinetochore-to-pole microtubules during metaphase, and, in a striking fashion, with the spindle interzone during anaphase. KRP(85/95) began to accumulate in the interzone immediately following chromosome separation and the area of concentration expanded with the lengthening of the interzonal region during anaphase. During telophase KRP(85/95) appeared to disperse with the establishment of the cleavage furrow and did not concentrate in the midbody. KRP(85/95) staining in the mitotic apparatus was punctate and detergent-sensitive, suggesting an association with membranous vesicles, but unlike kinesin, KRP(85/95) did not appear to codistribute with calsequestrin-containing endoplasmic reticulum. Finally, KRP(85/95) appears to be present in dividing blastomeres up to at least the blastula stage, but, unlike kinesin, it is not expressed in terminally differentiated, nonmitotic coelomocytes of the adult animal. These results suggest that the expression and targeting of KRP(85/95) and kinesin differ and that KRP(85/95) may play a role in vesicle transport during embryonic cell division.
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PMID:Immunolocalization of the heterotrimeric kinesin-related protein KRP(85/95) in the mitotic apparatus of sea urchin embryos. 755 95

We cloned a new member of the murine brain kinesin superfamily, KIF3B, and found that its amino acid sequence is highly homologous but not identical to KIF3A, which we previously cloned and named KIF3 (47% identical). KIF3B is localized in various organ tissues and developing neurons of mice and accumulates with anterogradely moving membranous organelles after ligation of nerve axons. Immunoprecipitation assay of the brain revealed that KIF3B forms a complex with KIF3A and three other high molecular weight (approximately 100 kD)-associated polypeptides, called the kinesin superfamily-associated protein 3 (KAP3). In vitro reconstruction using baculovirus expression systems showed that KIF3A and KIF3B directly bind with each other in the absence of KAP3. The recombinant KIF3A/B complex (approximately 50-nm rod with two globular heads and a single globular tail) demonstrated plus end-directed microtubule sliding activity in vitro. In addition, we showed that KIF3B itself has motor activity in vitro, by making a complex of wild-type KIF3B and a chimeric motor protein (KIF3B head and KIF3A rod tail). Subcellular fractionation of mouse brain homogenates showed a considerable amount of the native KIF3 complex to be associated with membrane fractions other than synaptic vesicles. Immunoprecipitation by anti-KIF3B antibody-conjugated beads and its electron microscopic study also revealed that KIF3 is associated with membranous organelles. Moreover, we found that the composition of KAP3 is different in the brain and testis. Our findings suggest that KIF3B forms a heterodimer with KIF3A and functions as a new microtubule-based anterograde translocator for membranous organelles, and that KAP3 may determine functional diversity of the KIF3 complex in various kinds of cells in vivo.
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PMID:KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for membrane organelle transport. 755 60


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