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)

Rabbit antiserum prepared against an ATPase-containing tryptic fragment of dynein by Ogawa and Mohri (J. Biol. Chem. 250: 6476-6483) specifically inhibited the ATPase activity of dynein 1 and not that of dynein 2. Varying amounts of this antidynein 1 serum were added to demembranated sperm while they were swimming in reactivating solution containing 1 mM ATP. The sperm continued to form regularly propagated flagellar bending waves, but the beat frequency decreased gradually with time, the greater part of the change occurring in the first 15 min. The beat frequency after 1 h was a function of the amount of antiserum used, and could be as low as 1 Hz. The waveforms of the treated sperm resembled those of normal reactivated sperm except that the bend angles of both the principal and reverse bends were larger in the proximal portion of flagellum. The ATPase activity and corresponding beat frequency of sperm which had been pretreated with varying amounts of antidynein 1 serum for 15 min at 0 degrees C and then diluted were both decreased as a function of the amount of antiserum added, the ATPase activity of homogenized, nonmotile sperm also decreased upon pretreatment with antiserum, but the percentage decrease was less than for motile sperm. For moderate to low concentrations of antiserum, the rates of reaction with motile and with rigor sperm were almost identical. The overall results suggest that antidynein 1 inhibits the functioning of the dynein arms, probably by blocking the ATPase sites of the dynein 1.
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PMID:The effect of antidynein 1 serum on the movement of reactivated sea urchin sperm. 13 52

Studies were carried out of temperature relationship of dansylchloride, N-3-pyrenylmaleinimide fluorescence, SR membranes, self-luminescence caused by Ca-ATPase tryptophane - provided fluorescence and of pyrene excimerization in membrane preparations of sarcoplasmic reticulum (SR) of rabbit skeletal muscles. Temperature relationship of fluorescence intensity of dansylchloride and N-3-pyrenylmaleimide in Arrhenius coordinates has bends at 15 and 35 degrees. Selffluorescence of protein samples linearly depends on temperature. Temperature relationship of the ratio between the intensities of exsimeric and monomeric forms of pyrene Fa/Fm in Arrhenius coordinates has the bend at 20-22 degrees. Hence only the latter relationship coincides with the shape of Arrhenius graph for enzymatic activity of SR Ca-ATPase.
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PMID:[Effect of temperature on fluorescence of labels and probes of various localizations incorporated into sarcoplasmic reticulum samples]. 621 13

The effects of methanol, butanol, glycerol, glucose, sucrose and inorganic anions on the activity of soluble ATPase from mouse liver mitochondria were studied. Glycerol inhibited, while methanol stimulated the enzyme activity uncompetitively with respect to ATP and competitively with respect to each other. Glycerol-induced inhibition of ATPase was competitive with respect to sulphite; methanol competed with thiocyanate for the enzyme activity. The Arrhenius plots for ATPase revealed bends at 20 degrees and 30 degrees C in the presence of sulphite, chlorine, thiocyanate, glycerol and methanol. It was assumed that all the compounds tested influenced soluble ATPase by changing the nucleophilic activity of H2O.
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PMID:[Mechanism of glycerol and methanol action on soluble ATPase of mitochondria]. 622 18

Electron microscope (EM) studies of the eukaryotic flagellum reveal that the organelle contains a 9 + 2 arrangement of microtubules, the axoneme, with nine doublets surrounding two singlets enveloped by a membrane which is continuous with that of the cell; various linkages and projections are associated with the microtubules. Strong experimental evidence supports the idea that the forces required for bend formation on eukaryotic flagella are derived from active relative sliding of the peripheral doublets. Dynein arms, which project from each peripheral microtubule and possess ATPase activity, interact with a neighbouring doublet and undergo conformational changes which induce sliding. To form and propagate coordinated bends along a flagellum the sliding must be resisted in a controlled manner by structures within the axoneme. The regulatory mechanism responsible for the control of inter-doublet sliding is not known in detail, but ultrastructural studies suggest that interactions between the radial spokes attached to each doublet and the central complex of the axoneme may be involved. We report here the treatment of flagella with a 9 + 2 microtubular structure from the trypanosomid flagellate Crithidia oncopelti to produce motile axonemes with only one central microtubule. We conclude that the complete central complex is not involved in the conversion of microtubule sliding into axonemal bending, but may be both associated with the control of wave propagation and essential for bend initiation.
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PMID:Motile flagellar axonemes with a 9 + 1 microtubule configuration. 644 61

We determined the crystal structure of the motor domain of the fast fungal kinesin from Neurospora crassa (NcKin). The structure has several unique features. (i) Loop 11 in the switch 2 region is ordered and enables one to describe the complete nucleotide-binding pocket, including three inter-switch salt bridges between switch 1 and 2. (ii) Loop 9 in the switch 1 region bends outwards, making the nucleotide-binding pocket very wide. The displacement in switch 1 resembles that of the G-protein ras complexed with its guanosine nucleotide exchange factor. (iii) Loop 5 in the entrance to the nucleotide-binding pocket is remarkably long and interacts with the ribose of ATP. (iv) The linker and neck region is not well defined, indicating that it is mobile. (v) Image reconstructions of ice-embedded microtubules decorated with NcKin show that it interacts with several tubulin subunits, including a central beta-tubulin monomer and the two flanking alpha-tubulin monomers within the microtubule protofilament. Comparison of NcKin with other kinesins, myosin and G-proteins suggests that the rate-limiting step of ADP release is accelerated in the fungal kinesin and accounts for the unusually high velocity and ATPase activity.
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PMID:Structure of a fast kinesin: implications for ATPase mechanism and interactions with microtubules. 1170 93

The molecular chaperone machine composed of Escherichia coli Hsp70/DnaK and Hsp40/DnaJ binds and releases client proteins in cycles of ATP-dependent protein folding, membrane translocation, disassembly, and degradation. The J-domain of DnaJ simultaneously stimulates ATP hydrolysis in the ATPase domain and capture of the client protein in the peptide-binding domain of DnaK. ATP-dependent binding of DnaJ to DnaK mimics DnaJ-dependent capture of a client protein. The dnaJ mutation that replaces aspartate-35 with asparagine (D35N) in the J-domain causes a defect in binding of DnaJ to DnaK. The dnaK mutation that replaces arginine-167 with alanine (R167A) in the ATPase domain of DnaK(R167A) restores binding of DnaJ(D35N). This genetic interaction was said to be allele-specific because wild-type DnaJ does not bind to DnaK(R167A). The J-domain of DnaJ binds to the ATPase domain of DnaK in its capacity as modulator of DnaK ATPase activity and conformational behavior. Surprisingly, the mutations affect the domainwise interaction in an almost opposite manner. D35N increases the affinity of the J-domain for the ATPase domain. R167A has no affect on the affinity of the ATPase domain for the D35N mutant J-domain, but it reduces the affinity for the wild-type J-domain. Previous amide ((1)H, (15)N) NMR chemical shift perturbation mapping in the J-domain suggested that the ATPase domain binds to J-domain helix II and the flanking loops. In the D35N mutant J-domain, chemical shift perturbations include additional effects at amides in the flexible loop II-III and helix III, which have been proposed to undergo an induced fit conformational change upon binding to DnaK. The integrated magnitudes of chemical shift perturbations for the various J-domain and ATPase domain pairs correlate with the free energies of binding. Thus, the J-domain structure can be described as a dynamic ensemble of conformations that is constrained by binding to the ATPase domain. J-domain helix II bends upon binding to the ATPase domain. D35N increases helix II bending, but less so in combination with R167A in the ATPase domain. Taken together, the results suggest that D35N overstabilizes an induced fit conformational change in loop II-III and helix III that is necessary for the J-domain to couple ATP hydrolysis with a conformational change in DnaK, and R167A destabilizes the induced conformation. Conclusions from this work have implications for understanding mechanisms of protein-protein interaction that are involved in allosteric regulation and genetic suppression.
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PMID:Structure and energetics of an allele-specific genetic interaction between dnaJ and dnaK: correlation of nuclear magnetic resonance chemical shift perturbations in the J-domain of Hsp40/DnaJ with binding affinity for the ATPase domain of Hsp70/DnaK. 1271 34

The importance of Gly-93 and Gly-94 in transmembrane segment M1 of the Na+,K+-ATPase for interaction with Na+ and K+ was demonstrated by functional analysis of mutants Gly-93-Ala and Gly-94-Ala. In the crystal structures of the Ca2+-ATPase, the corresponding residues, Asp-59 and Leu-60, are located exactly where M1 bends. Rapid kinetic measurements of K+-induced dephosphorylation allowed determination of the affinity of the E2P phosphoenzyme intermediate for K+. In Gly-94-Ala, the K+ affinity was reduced 9-fold, i.e., to the same extent as seen for mutation of the cation-binding residue Glu-329. Furthermore, Gly-94-Ala showed strongly reduced sensitivity of the E1P-E2P equilibrium to Na+, with accumulation of E2P even at 600 mM Na+, indicating that interaction of E2P with extracellular Na+ is impaired. On the contrary, in Gly-93-Ala, the affinity for K+ was slightly increased, and the E1P-E2P equilibrium was displaced in favor of E1P. In both mutants, the affinity of the cytoplasmically facing sites of E1 for Na+ was reduced, but this effect was relatively small compared with the effects seen for E2P in Gly-94-Ala. Comparison with Ca2+-ATPase mutagenesis data suggests that the role of M1 in binding of the transported ions is universal among P-type ATPases, despite the low sequence homology in this region. Structural modeling of Na+,K+-ATPase mutant Gly-94-Ala on the basis of the Ca2+-ATPase crystal structures indicates that the alanine side chain comes close to Ile-287 of M3, particularly in E2P, thus resulting in a steric clash that may explain the present observations.
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PMID:Mutation of Gly-94 in transmembrane segment M1 of Na+,K+-ATPase interferes with Na+ and K+ binding in E2P conformation. 1604

In the presence of ATP, unphosphorylated smooth muscle myosin can form a catalytically inactive monomer that sediments at 10 Svedbergs (10 S). The tail of 10 S bends into thirds and interacts with the regulatory domain. ADP-P(i) is "trapped" at the active site, and consequently the ATPase activity is extremely low. We are interested in the structural basis for maintenance of this off state. Our prior photocross-linking work with 10 S showed that tail residues 1554-1583 are proximal to position 108 in the C-terminal lobe of one of the two regulatory light chains ( Olney, J. J., Sellers, J. R., and Cremo, C. R. (1996) J. Biol. Chem. 271, 20375-20384 ). These data suggested that the tail interacts with only one of the two regulatory light chains. Here we present data, using a photocross-linker on position 59 on the N-terminal lobe of the regulatory light chain (RLC), demonstrating that both regulatory light chains of a single molecule can cross-link to the light meromyosin portion of the tail. Mass spectrometric data show four specific cross-linked regions spanning residues 1428-1571 in the light meromyosin portion of the tail, consistent with cross-linking two RLC to one light meromyosin. In addition, we find that position 59 can cross-link internally to residues 42-45 within the same RLC subunit. The internal cross-link only forms in 10 S and not in unphosphorylated heavy meromyosin (lacking the light meromyosin), suggesting a structural rearrangement within the RLC attributed to the interaction of the tail with the head.
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PMID:The N-terminal lobes of both regulatory light chains interact with the tail domain in the 10 S-inhibited conformation of smooth muscle myosin. 1701 38

Outer and inner dynein arms generate force for the flagellar/ciliary bending motion. Although nucleotide-induced structural change of dynein heavy chains (the ATP-driven motor) was proven in vitro, our lack of knowledge in situ has precluded an understanding of the bending mechanism. Here we reveal nucleotide-induced global structural changes of the outer and inner dynein arms of Chlamydomonas reinhardtii flagella in situ using electron cryotomography. The ATPase domains of the dynein heavy chains move toward the distal end, and the N-terminal tail bends sharply during product release. This motion could drive the adjacent microtubule to cause a sliding motion. In contrast to in vitro results, in the presence of nucleotides, outer dynein arms coexist as clusters of apo or nucleotide-bound forms in situ. This implies a cooperative switching, which may be related to the mechanism of bending.
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PMID:Nucleotide-induced global conformational changes of flagellar dynein arms revealed by in situ analysis. 2052 Jun 59

For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)(2)FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)(2)FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)(2)FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism.
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PMID:Structural kinetics of myosin by transient time-resolved FRET. 2124 57


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