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)

Axoplasmic vesicles that translocate on isolated microtubules in an ATP-dependent manner have an associated ATP-binding polypeptide with a previously estimated relative molecular mass of 292 kD (Gilbert, S. P., and R. D. Sloboda. 1986. J. Cell Biol. 103:947-956). Here, data are presented showing that this polypeptide (designated H1) and another high molecular mass polypeptide (H2) can be isolated in association with axoplasmic vesicles or optic lobe microtubules. The H1 and H2 polypeptides dissociate from microtubules in the presence of MgATP and can be further purified by gel filtration chromatography. The peak fraction thus obtained demonstrates MgATPase activity and promotes the translocation of salt-extracted vesicles (mean = 0.87 microns/s) and latex beads (mean = 0.92 microns/s) along isolated microtubules. The H1 polypeptide binds [alpha 32P]8-azidoATP and is thermosoluble, but the H2 polypeptide does not share these characteristics. In immunofluorescence experiments with dissociated squid axoplasm, affinity-purified H1 antibodies yield a punctate pattern that corresponds to vesicle-like particles, and these antibodies inhibit the bidirectional movement of axoplasmic vesicles. H2 is cleaved by UV irradiation in the presence of MgATP and vanadate to yield vanadate-induced peptides of 240 and 195 kD, yet H1 does not cleave under identical conditions. These experiments also demonstrate that the actual relative molecular mass of the H1 and H2 polypeptides is approximately 435 kD. On sucrose density gradients, H1 and H2 sediment at 19-20 S, and negatively stained samples reveal particles comprised of two globular heads with stems that contact each other and extend to a common base. The results demonstrate that the complex purified is a vesicle-associated ATPase whose characteristics indicate that it is a squid isoform of dynein. Furthermore, the data suggest that this vesicle-associated dynein promotes membranous organelle motility during fast axoplasmic transport.
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PMID:A squid dynein isoform promotes axoplasmic vesicle translocation. 247 67

Folding catalysts of the endoplasmic reticulum (ER), such as protein disulfide isomerase (PDI), accelerate the slow chemical steps, such as disulfide bond formation, that accompany protein folding. Molecular chaperones of the ER, notably the heavy chain-binding protein, BiP (grp78), bind and release unfolded proteins in an ATP-dependent fashion. In vitro, the fate of reduced, denatured lysozyme is dependent on whether the substrate interacts first with BiP or PDI. Depending on the ratio of PDI to substrate and order in which the components of the reaction are mixed, PDI can exhibit a foldase/chaperone activity, which increases the rate and extent of lysozyme refolding, or it can function as an anti-chaperone that promotes the formation of inactive, disulfide-linked lysozyme aggregates (Puig, A., and Gilbert, H.F. (1994) J. Biol. Chem. 269, 7764-7771). Reduced, denatured lysozyme, but not the native protein, interacts with BiP and efficiently stimulates its peptide-dependent ATPase activity. When present at substoichiometric amounts, BiP, like PDI, facilitates the formation of large, inactive lysozyme aggregates that are non-covalently associated with BiP. BiP and PDI compete for a limited number of sites in these insoluble aggregates. If BiP is present at a high molar excess, the chaperone binds unfolded lysozyme and inhibits its aggregation by maintaining it in a soluble, yet inactive, conformation, both in the presence or absence of ATP. Increasing concentrations of BiP decrease the extent, but not the initial rate, of refolding, suggesting that BiP and PDI compete for unfolded lysozyme and that the BiP-lysozyme complex is not a very good substrate for PDI either in the presence or absence of ATP. Depending on the BiP and PDI concentrations, unfolded lysozyme may either be efficiently refolded into the native conformation in a PDI-catalyzed reaction, or it may form both soluble and insoluble BiP-lysozyme complexes. In vitro, PDI- and BiP-facilitated aggregation, as well as the competition of the two proteins for substrate, reproduces many of the features of the quality control system of the ER.
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PMID:Anti-chaperone behavior of BiP during the protein disulfide isomerase-catalyzed refolding of reduced denatured lysozyme. 792 93

The pre-steady-state kinetics of the microtubule-kinesin ATPase were investigated by chemical-quench flow methods using the Drosophila kinesin motor domain (K401) expressed in Escherichia coli [Gilbert, S. P., & Johnson, K. A. (1993) Biochemistry 32, 4677-4684]. The results define a minimal mechanism: M.K + ATP in equilibrium with (M).K.ATP in equilibrium with (M).K.ADP.Pi in equilibrium with M.K.ADP + Pi in equilibrium with M.K + ADP, where M, K, and Pi represent microtubules, kinesin, and inorganic phosphate, respectively, with k+1 = 0.8-3 microM-1 s-1, k-1 = 100-300 s-1, k+2 = 70-120 s-1, k+4 = 10-20 s-1, and k+3 >> k-2 and k+3 >> k+4. Conditions were as follows: 25 degrees C, 20 mM HEPES, pH 7.2 with KOH, 5 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM potassium acetate, 1 mM DTT. The experiments presented do not determine the step in the cycle where kinesin dissociates from the microtubule or the step at which kinesin reassociates with the microtubule; therefore, the steps that may represent kinesin as the free enzyme are indicated by (M). A burst of ADP product formation was observed during the first turnover of the enzyme in the acid-quench experiments that define the ATP hydrolysis transient. The observation of the burst demonstrates that product release is rate limiting even in the presence of saturating microtubule concentrations. The pulse-chase experiments define the time course of ATP binding to the microtubule-K401 complex. At low ATP concentrations, ATP binding limits the rate of the burst. However, at high concentrations of ATP, ATP binding is faster than the rate of ATP hydrolysis with k+2 = 70-120 s-1. The amplitude of the burst of the ATP binding transient reached a maximum of 0.7 per site at saturating concentrations of ATP and microtubules. The amplitude of less than 1 is attributed to the fast k(off) for ATP (k-1 = 100-300 s-1) that leads to a partitioning of the M.K.ATP complex between ATP hydrolysis (k+2) and ATP release (k-1). These results indicate that ATP binds weakly to the M.K complex (Kd,ATP app approximately 100 microM). ADP release (k+4 = 10-20 s-1) is rate limiting during steady-state turnover, indicating that microtubules activate the kinesin ATPase by increasing k(off),ADP from 0.01 s-1 in the absence of microtubules to 10-20 s-1 at saturating microtubule concentrations.
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PMID:Pre-steady-state kinetics of the microtubule-kinesin ATPase. 811 Aug

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 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

The pathway of ATP hydrolysis by rat kinesin was established by pre-steady-state kinetic methods. A 406-residue long N-terminal fragment was shown by sedimentation equilibrium analysis to form a dimer with a K(d) of 46 nm. The pathway of ATP hydrolysis follows the Gilbert-Johnson pathway determined previously for a similarsized N-terminal fragment of Drosophila conventional kinesin. However, the rates of ADP release were at least 3-fold faster, and ATP hydrolysis was approximately 5-fold faster. Paralleling our previous mechanistic data, these results support an alternating site ATPase pathway, including a captive head state as an intermediate in the kinesin ATPase cycle. The kinetic data presented in this report once again point to the importance of the captive head state and argue against a pathway that short-circuits this key intermediate. In addition, several unique aspects of the rat kinesin kinetics reveal new aspects of the ATPase-coupling mechanism. These studies provide a baseline set of kinetic parameters against which future studies of rat kinesin mutants may be evaluated and directly correlated with the structure of the dimeric kinesin.
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PMID:Alternating site ATPase pathway of rat conventional kinesin. 1611 18