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)

We have used electron paramagnetic resonance to study the orientation of myosin heads in the presence of nucleotides and nucleotide analogs, to induce equilibrium states that mimic intermediates in the actomyosin ATPase cycle. We obtained electron paramagnetic resonance spectra of an indane dione spin label (InVSL) bound to Cys 707 (SH1) of the myosin head, in skinned rabbit psoas muscle fibers. This probe is rigidly immobilized on the catalytic domain of the head, and the principal axis of the probe is aligned nearly parallel to the fiber axis in rigor (no nucleotide), making it directly sensitive to axial rotation of the head. On ADP addition, all of the heads remained strongly bound to actin, but the spectral hyperfine splitting increased by 0.55 +/- 0.02 G, corresponding to a small but significant axial rotation of 7 degrees. Adenosine 5'-(adenylylim-idodiphosphate) (AMPPNP) or pyrophosphate reduced the actomyosin affinity and introduced a highly disordered population of heads similar to that observed in relaxation. For the remaining oriented population, pyrophosphate induced no significant change relative to rigor, but AMPPNP induced a slight but probably significant rotation (2.2 degrees +/- 1.6 degrees), in the direction opposite that induced by ADP. Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) relaxed the muscle fiber, completely dissociated the heads from actin, and produced disorder similar to that in relaxation by ATP. ATP gamma S plus Ca induced a weak-binding state with most of the actin-bound heads disordered. Vanadate had negligible effect in the presence of ADP, but in isometric contraction vanadate substantially reduced both force and the fraction of oriented heads. These results are consistent with a model in which myosin heads are disordered early in the power stroke (weak-binding states) and rigidly oriented later in the power stroke (strong-binding states), whereas transitions among the strong-binding states induce only slight changes in the axial orientation of the catalytic domain.
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PMID:Orientation of intermediate nucleotide states of indane dione spin-labeled myosin heads in muscle fibers. 874 17

When active insect fibrillar flight muscle is stretched, its ATPase rate increases and it develops "negative viscosity," which allows it to perform oscillatory work. We use a six-state model for the cross-bridge cycle to show that such "stretch activation" may arise naturally as a nonlinear property of a cross-bridge interacting with a single attachment site on a thin filament. Attachment is treated as a thermally activated process in which elastic energy must be supplied to stretch or compress the cross-bridge spring. We find that stretch activation occurs at filament displacements where, before the power stroke, the spring is initially in compression rather than in tension. In that case, pulling the filaments relieves the initial compression and reduces the elastic energy required for attachment. The result is that the attachment rate is enhanced by stretching. The model also displays the "delayed tension" effect observed in length-step experiments. When the muscle is stretched suddenly, the power stroke responds very quickly, but there is a time lag before dissociation at the end of the cycle catches up with the increased attachment rate. This lag is responsible for the delayed tension and hence also for the negative viscosity.
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PMID:Stretch activation and nonlinear elasticity of muscle cross-bridges. 874 18

We have developed a new technique for measurements of piconewton forces and nanometer displacements in the millisecond time range caused by actin-myosin interaction in vitro by manipulating single actin filaments with a glass microneedle. Here, we describe in full the details of this method. Using this method, the elementary events in energy transduction by the actomyosin motor, driven by ATP hydrolysis, were directly recorded from multiple and single molecules. We found that not only the velocity but also the force greatly depended on the orientations of myosin relative to the actin filament axis. Therefore, to avoid the effects of random orientation of myosin and association of myosin with an artificial substrate in the surface motility assay, we measured forces and displacements by myosin molecules correctly oriented in single synthetic myosin rod cofilaments. At a high myosin-to-rod ratio, large force fluctuations were observed when the actin filament interacted in the correct orientation with a cofilament. The noise analysis of the force fluctuations caused by a small number of heads showed that the myosin head generated a force of 5.9 +/- 0.8 pN at peak and 2.1 +/- 0.4 pN on average over the whole ATPase cycle. The rate constants for transitions into (k+) and out of (k-) the force generation state and the duty ratio were 12 +/- 2 s-1, and 22 +/- 4 s-1, and 0.36 +/- 0.07, respectively. The stiffness was 0.14 pN nm-1 head-1 for slow length change (100 Hz), which would be approximately 0.28 pN nm-1 head-1 for rapid length change or in rigor. At a very low myosin-to-rod ratio, distinct actomyosin attachment, force generation (the power stroke), and detachment events were directly detected. At high load, one power stroke generated a force spike with a peak value of 5-6 pN and a duration of 50 ms (k(-)-1), which were compatible with those of individual myosin heads deduced from the force fluctuations. As the load was reduced, the force of the power stroke decreased and the needle displacement increased. At near zero load, the mean size of single displacement spikes, i.e., the unitary steps caused by correctly oriented myosin, which were corrected for the stiffness of the needle-to-myosin linkage and the randomizing effect by the thermal vibration of the needle, was approximately 20 nm.
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PMID:Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle: unitary steps and forces. 877 Feb 15

1. The effects of cyclopiazonic acid (CPA) and thapsigargin (TG), both of which are known to inhibit sarcoplasmic reticular Ca(2+)-ATPase, on the mechanical activities, intracellular Ca2+ level and electrical activities of smooth muscle of the carotid artery of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY) were compared. 2. Both CPA and TG induced elevation of tension of the smooth muscle, which was composed of a phasic and a tonic component. The level of tension attained, especially the tonic component, was greater in the preparation from SHRSP. 3. The elevation of tension was associated with an increased intracellular Ca2+ level. Both the elevation of tension and the increase in intracellular Ca2+ were diminished by the removal of extracellular Ca2+ or by the application of verapamil. 4. The resting membrane potential of the preparations from SHRSP were depolarized to a greater extent than those from WKY.CPA depolarized the smooth muscle from both SHRSP and WKY, and the final level was also more depolarized in the preparation from SHRSP. 5. These results indicate that the elevation of tension induced by these drugs is mainly due to increased Ca2+ influx through voltage-dependent Ca2+ channels, and the difference in the action between the preparation from SHRSP and that from WKY can be explained mainly by the changes in the channels. 6. Thus, differences in the action of these drugs on the tension of smooth muscle between preparations from WKY and SHRSP can mainly be explained by the difference in the membrane potential which is related to the difference in voltage-dependent Ca2+ influx.
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PMID:Effects of cyclopiazonic acid and thapsigargin on electromechanical activities and intracellular Ca2+ in smooth muscle of carotid artery of hypertensive rats. 879 54

We have reported that aldosterone is synthesized and cytochrome P450aldo mRNA exists in the vasculature. To clarify the pathophysiological role of vascular aldosterone in hypertension, we compared aldosterone production in the mesenteric arteries of stroke-prone spontaneously hypertensive rats (SHRSP) with that in Wistar-Kyoto rats (WKY). The expressions of mRNA of cytochrome P450aldo, mineralocorticoid receptor, and alpha 1, Na,K-ATPase in the mesenteric arteries were compared between the two groups. Aldosterone concentration in the perfusate of the vasculature was measured by radioimmunoassay after purification with high-performance liquid chromatography. Cytochrome P450aldo and mineralocorticoid receptor mRNA levels were quantified by Southern blot analysis of the products of reverse-transcribed polymerase chain reaction. Levels of alpha 1 Na,K-ATPase mRNA were measured by Northern blot analysis. Vascular aldosterone and cytochrome P450aldo mRNA levels of 2-week-old SHRSP were significantly increased compared with those of age-matched WKY. However, vascular aldosterone in 4- and 9-week-old SHRSP did not differ from that in age-matched WKY. Expression levels of mineralocorticoid receptor mRNA in the vasculature of 4- and 9-week-old SHRSP were significantly increased compared with those in age-matched WKY. Concentrations of vascular alpha 1 Na,K-ATPase mRNA of 2-, 4-, and 9-week-old SHRSP also were significantly higher than those in age-matched WKY. These results suggest that vascular aldosterone contributes to the pathophysiology of hypertension in SHRSP in the early stage.
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PMID:Vascular aldosterone in genetically hypertensive rats. 903 78

The interaction of myosin with actin, coupled with hydrolysis of ATP, is the molecular basis of muscle contraction. The head segment of myosin, called S1, contains the distinct binding sites for ATP and actin and is responsible for the ATPase activity. The myosin-catalyzed ATP hydrolysis consists of several intermediate steps and each step is accompanied by conformational changes in the S1 segment. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1 x ADP x Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate, beryllium fluoride (BeFx) or aluminum fluoride (AlF4-), yields stable complexes which mimic the intermediates of the ATP hydrolysis. In this work, tertiary structure changes in S1 in the vicinity of aromatic residues was studied by comparing near-UV circular dichroism (CD) spectra from S1-nucleotide-phosphate analog complexes in the presence of Mg2+ and other cations. A significant difference between the MgATP and MgADP spectra indicated notable tertiary structural changes accompanying the M**ADP x Pi --> M*ADP transition. The spectra of the S1 x MgADP x BeFx and S1 x MgADP x AlF4- complexes resemble to those obtained upon addition of MgATPgammaS and MgATP to S1, and correspond to the M* x ATP and M** x ADP x Pi intermediates, respectively. We have found recently that the presence of divalent metal cations (Me2+) is essential for the formation of stable S1 x MeADP x PA complexes. Moreover, the nature of the metal cations strongly influences the stability of these complexes [Peyser, Y. M., et al. (1996) Biochemistry 35, 4409-4416]. In the present work we studied the effect of Mg2+, Mn2+, Ca2+, Ni2+, Co2+, and Fe2+ on the near-UV CD spectrum of the ATP, ADP, ADP x BeFx, and ADP x AlF4- containing S complexes. The CD spectra obtained with ADP, ATP ADP x BeFx and ADP x AlF4- were essentially identical in the presence of Co2+ and rather similar in the case of Ca2+, while they were partially different in other cases. An interesting correlation was found between actin activation and ATP versus ADP difference spectra in the presence of various metal ions. The distribution of the fractional concentration of the intermediates of ATP hydrolysis was estimated in the presence of each cation from the CD spectra with phosphate analogs. In the presence of Mg2+ the predominant intermediate is the M** x ADP x Pi state, which is in accordance with the kinetic studies. On the other hand with non-native cations the predominant intermediate is the M* x ADP state and the release of ADP is the rate limiting step in the myosin-catalyzed ATP hydrolysis. According to the results, the near-UV CD spectrum originating from aromatic residues in S1 not only can distinguish identifiable states in the ATP hydrolysis cycle but can also pinpoint to changes in the tertiary structure caused by complex formation with nucleotide or nucleotide analog and various divalent metal cations. These findings, that are correlative with actin activation, and thus with the power stroke, suggest new strategies for perturbing S1 structure in the continuous efforts directed toward the elucidation of the mechanism of muscle contraction.
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PMID:Effect of metal cations on the conformation of myosin subfragment-1-ADP-phosphate analog complexes: a near-UV circular dichroism study. 913 78

Rats are generally believed to be insensitive for cardiac glycosides. However, like in humans, the hemodynamic effects may be related to the pathophysiological condition. Since the hemodynamic effects of cardiac glycosides have never been investigated in rats with heart failure, the aim of the present experiments was to investigate the role of the pathophysiological condition in the rat. Therefore, hemodynamic and cardiac effects of ouabain were investigated both in normal rats and rats with heart failure due to myocardial infarction (MI). Since the effects of ouabain may also depend on the treatment scheme, rats were treated either for a short-term period or a long-term period. Three weeks after sham surgery or ligation of the left coronary artery (MI), Wistar rats were treated for two weeks with ouabain (14.4 mg/kg.d s.c.), either continuously (osmotic minipumps) or intermittently (once daily). A separate group of rats was treated for 45-60 min (1-100 microg/kg.min ouabain; i.v. infusion 5 weeks after MI). Hemodynamic measurements were performed at rest and after volume loading in conscious rats, chronically instrumented with an electromagnetic flow probe and catheters. Induction of MI resulted in a significant increase in total peripheral resistance (TPR), and a significant decrease in basal and maximal cardiac output following volume loading (basal CO: sham, 92 +/- 5; MI, 74 +/- 5 ml/min; maximal CO: sham, 152 +/- 4; MI, 105 +/- 7 ml/min; n = 7-11). Chronic intermittent ouabain treatment further increased TPR in MI rats. In contrast, continuous ouabain treatment normalized TPR in rats. Only in continuously treated MI rats, basal and maximal CO improved significantly (basal: 83 +/- 4; maximal: 134 +/- 7 ml/min; n = 7). Acute treatment dose-dependently worsened the hemodynamic conditions of MI rats, since TPR and MAP increased and CO and stroke volume decreased significantly. These experiments demonstrate that ouabain can improve cardiac function in rats, although only in MI rats and strongly depending on the delivery regimen. Thus, in contrast to the general belief, the presently used rat model is suitable for investigation of cardiac glycosides in heart failure. The preferential improvement of cardiac function in MI rats continuously treated with ouabain may depend upon changes in Na+,K+-ATPase or altered neurohumoral conditions due to MI and chronic treatment.
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PMID:Ouabain improves cardiac function in vivo in rats with heart failure after chronic but not acute treatment. 927 26

Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed us to identify separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. The G691C mutant, on the other hand, exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle.
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PMID:Cold-sensitive mutants G680V and G691C of Dictyostelium myosin II confer dramatically different biochemical defects. 934 98

Synapse loss, deposits of amyloid beta-peptide (Abeta), impaired energy metabolism, and cognitive deficits are defining features of Alzheimer's disease (AD). Estrogen replacement therapy reduces the risk of developing AD in postmenopausal women. Because synapses are likely sites for initiation of neurodegenerative cascades in AD, we tested the hypothesis that estrogens act directly on synapses to suppress oxidative impairment of membrane transport systems. Exposure of rat cortical synaptosomes to Abeta25-35 (Abeta) and FeSO4 induced membrane lipid peroxidation and impaired the function of the plasma membrane Na+/K+-ATPase, glutamate transporter, and glucose transporter. Pretreatment of synaptosomes with 17beta-estradiol or estriol largely prevented impairment of Na+/K+-ATPase activity, glutamate transport, and glucose transport; other steroids were relatively ineffective. 17Beta-estradiol suppressed membrane lipid peroxidation induced by Abeta and FeSO4, but did not prevent impairment of membrane transport systems by 4-hydroxynonenal (a toxic lipid peroxidation product), suggesting that an antioxidant property of 17beta-estradiol was responsible for its protective effects. By suppressing membrane lipid peroxidation in synaptic membranes, estrogens may prevent impairment of transport systems that maintain ion homeostasis and energy metabolism, and thereby forestall excitotoxic synaptic degeneration and neuronal loss in disorders such as AD and ischemic stroke.
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PMID:17Beta-estradiol attenuates oxidative impairment of synaptic Na+/K+-ATPase activity, glucose transport, and glutamate transport induced by amyloid beta-peptide and iron. 940 14

Glutamate (Glu) plays an important role in the early development of brain injuries caused by ischemia, i.e. stroke, or brain trauma. Glu induces a rapid astroglial swelling which, in turn, deranges the composition of neuroactive substances in the extracellular space. We report that Glu can induce astroglial cell swelling by interaction with metabotropic Glu receptors (mGluRs). Furthermore, the Na(+)-K(+)-2Cl- cotransporter, a Na(+)-K(+) ATPase, and the Na(+)-dependent electrogenic Glu carrier seem to be involved in this Glu-induced astroglial cell swelling. Two methods for studying cell swelling arc described. One is based on variations in the signal emitted by the fluorescent probe fura-2/AM when excited at its isosbestic point. These variations were shown to be directly proportional to variations in intracellular volume. Relative changes in cell volume and intracellular calcium concentration could be detected simultaneously in single astroglial cells. The other method used permits the cell volume to be calculated in relative terms with the aid of image processing techniques.
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PMID:Glutamate induced astroglial swelling--methods and mechanisms. 941 5


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