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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Kinesin is a microtubule-based motor protein that contains two identical force-generating subunits. The kinesin binding sites along the microtubule lie 8 nm apart (the dimension of the tubulin dimer), which implies that kinesin must translocate a minimum distance of 8 nm per hydrolysis cycle. Measurements of kinesin's microtubule-stimulated ATPase activity (approximately 20 ATP per sec) and velocity of transport (approximately 0.6 micron/sec), however, suggest that the net distance moved per ATP (approximately 30 nm) may be greater than one tubulin dimer under zero load conditions. To explore how kinesin translocates during its ATPase cycle, we constructed a microscope capable of tracking movement with 1-nm resolution at a bandwidth of 200 Hz and used this device to examine microtubule movement driven by a single kinesin motor. Regular stepwise movements were not observed in displacement traces of moving microtubules, although Brownian forces acting on elastic elements within the kinesin motor precluded detection of steps that were < 12 nm. Though individual steps of approximately 16 nm were occasionally observed, their infrequent occurrence suggests that kinesin rarely moves abruptly by distances of two or more tubulin subunits during its ATP hydrolysis cycle. Instead it is more likely that kinesin moves forward by the distance of only a single tubulin subunit under zero load conditions.
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PMID:High-resolution tracking of microtubule motility driven by a single kinesin motor. 818 52

Kinesin is an ubiquitous heterotetrameric microtubule-based motor which translocates membrane-bound organelles. Since organelle motility and motor protein function can be regulated by components of signaling pathways, the ability of purified bovine brain kinesin (kinesin) to be phosphorylated and to recognize calmodulin (CaM) was tested. Extensively purified "kinesin" was found to consist of several forms of both heavy (KHC) and light (KLC) chains. Phosphorylation of kinesin by a variety of protein kinases was examined; cAMP-dependent protein kinase (cAMP-PK) was the most active enzyme leading to the incorporation of up to 8 mol P/mol kinesin. Phosphorylation occurred predominantly on the KLCs and led to substantial acidic pI shifts. Peptide maps indicated that multiple phosphorylation sites exist on each KLC. Incubation of kinesin in vitro with protein kinase C (PKC) led to the phosphorylation of both KHCs and KLCs. In vivo phosphorylation of KHC and KLCs was demonstrated by immunoprecipitation of [32P]-labeled kinesin from cultured rat hippocampal pyramidal neurons; kinesin phosphorylation was stimulated by 8-chlorophenyl-thio-cAMP or 12-O-tetradecanoylphorbol-13-acetate. Native bovine brain kinesin was shown to bind 125I-CaM by nucleotide-dependent pelleting with stable microtubules. Specific calcium-dependent binding of 125I-CaM to KLCs but not KHC was found using a ligand blotting assay. cAMP-PK phosphorylated kinesin bound 125I-CaM less well than untreated protein in both ligand blotting and microtubule-pelleting paradigms. Calcium-dependent binding of CaM to kinesin inhibited the ATPase activity of native kinesin but not of cAMP-PK phosphorylated kinesin. These results suggest that the KLCs have a regulatory function and integrate information coming from diverse signaling pathways to modulate the activity and function of kinesin.
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PMID:Calmodulin binding to and cAMP-dependent phosphorylation of kinesin light chains modulate kinesin ATPase activity. 838 85

Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy-chain phosphorylation, and (d) ADP release is fast. Thus, during steady-state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily, other than myosin-II, providing the opportunity to obtain insights about the evolution of all myosin isoforms.
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PMID:Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase. 860 84

Kinesin and kinesin-like proteins (KLPs) are microtubule-based motor proteins that play important roles in organelle transport. Based on the homology to these proteins, a katD cDNA has now been isolated from a library prepared from flowers of Arabidopsis thaliana ecotype Columbia. Sequence analysis of the katD cDNA revealed an open reading frame of 2691bp [corrected], encoding a protein of 987 amino acids. Comparison of the nucleotide sequences of katD genomic and cDNA clones revealed the presence of 18 introns, 17 of which conform to the GU-AG rule. The central region of the KatD polypeptide exhibits substantial amino acid sequence homology to the motor domain of kinesin heavy chains, although the motor domain of KatD appears to be phylogenetically distant from those of other KLPs in plants. The amino-terminal region of KatD shares marked sequence similarity with the calponin homology domain, whereas the approximately 240-residue carboxyl-terminal region shows no significant homology to other known proteins. The predicted secondary structure of KatD revealed the lack of an alpha-helical coiled coil structure typical of kinesin heavy chains, suggesting that KatD may function as a monomeric motor. A recombinant truncated KatD protein containing the putative motor domain was shown both to bind to mammalian microtubules in a manner dependent on a non-hydrolyzable ATP analog, and to possess microtubule-dependent ATPase activity. Immunoblot and Northern blot analyses showed that both KatD protein and mRNA are expressed specifically in floral tissues. These results suggest that the structurally distinct KatD protein functions as a floral tissue-specific motor protein.
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PMID:Characterization of katD, a kinesin-like protein gene specifically expressed in floral tissues of Arabidopsis thaliana. 1019 70

Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr(291) of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr(291) results in complete loss of function in vivo. In vitro, the T291M mutation disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514). The pre-steady-state kinetic analysis presented here shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the gamma-phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin.
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PMID:A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor. 1076 90

Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar approximately 3-fold reduction in both microtubule gliding velocity and microtubule-stimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.
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PMID:ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility. 1081 95

Kinesin, a microtubule-based motor, and myosin, an actin-based motor, share a similar core structure, indicating that they arose from a common ancestor. However, kinesin lacks the long lever-arm domain that is believed to drive the myosin power stroke. Here, we present evidence that a much smaller region of ca. 10-40 amino acids serves as a mechanical element for kinesin motor proteins. These 'neck regions' are class conserved and have distinct structures in plus-end and minus-end-directed kinesin motors. Mutagenesis studies also indicate that the neck regions are involved in coupling ATP hydrolysis and energy into directional motion along the microtubule. We suggest that the kinesin necks drive motion by undergoing a conformational change in which they detach and re-dock onto the catalytic core during the ATPase cycle. Thus, kinesin and myosin have evolved unique mechanical elements that amplify small, nucleotide-dependent conformational changes that occur in their similar catalytic cores.
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PMID:Searching for kinesin's mechanical amplifier. 1083 98

The microtubule-based motor molecule cytoplasmic dynein has been proposed to be regulated by a variety of mechanisms, including phosphorylation and specific interaction with the organelle-associated complex, dynactin. In this study, we examined whether the intermediate chain subunits of cytoplasmic dynein are involved in modulation of ATP hydrolysis, and thereby affect motility. Treatment of testis cytoplasmic dynein under hypertonic salt conditions resulted in separation of the intermediate chains from the remainder of the dynein molecule, and led to a 4-fold enhancement of ATP hydrolysis. This result suggests that the accessory subunits act as negative regulators of dynein heavy chain activity. Comparison of ATPase activities of dyneins with differing intermediate chain isoforms showed significant differences in basal ATP hydrolysis rates, with testis dynein 7-fold more active than dynein from brain. Removal of the intermediate chain subunits led to an equalization of ATPase activity between brain and testis dyneins, suggesting that the accessory subunits are responsible for the observed differences in tissue activity. Finally, our preparative procedures have allowed for the identification and purification of a 1:1 complex of dynein with dynactin. As this interaction is presumed to be mediated by the dynein intermediate chain subunits, we now have defined experimental conditions for further exploration of dynein enzymatic and motility regulation.
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PMID:Modulation of cytoplasmic dynein ATPase activity by the accessory subunits. 1112 10

The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro-polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear evidence that the movement of pollen tube organelles is not just actin based; rather, they show a microtubule-based motion as well. This unexpected finding suggests new insights into the use of pollen tube microtubules, which could be used for short-range transport, as actin filaments are in animal cells.
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PMID:In vitro assays demonstrate that pollen tube organelles use kinesin-related motor proteins to move along microtubules. 1250 35

Conventional kinesin is a highly processive, microtubule-based motor protein that drives the movement of membranous organelles in neurons. Using in vivo genetics in Drosophila melanogaster, Glu164 was identified as an amino acid critical for kinesin function [Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514]. Glu164 is located at the beta-strand 5a/loop 8b junction of the catalytic core and projects toward the microtubule binding face in close proximity to key residues on beta-tubulin helix alpha12. Substitution of Glu(164) with alanine (E164A) results in a dimeric kinesin with a dramatic reduction in the microtubule-activated steady-state ATPase (5 s(-1) per site versus 22 s(-1) per site for wild-type). Our analysis shows that E164A binds ATP and microtubules with a higher affinity than wild-type kinesin. The rapid quench and stopped-flow results provide evidence that ATP hydrolysis is significantly faster and the precise coordination between the motor domains is disrupted. The data reveal an E164A intermediate that is stalled on the microtubule and cannot bind and hydrolyze ATP at the second head.
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PMID:Motor domain mutation traps kinesin as a microtubule rigor complex. 1261 54


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