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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reconstituted sarcoplasmic reticulum (SR) vesicles have been prepared mixing fluorescein labelled SR, excess endogenous lipids and deoxycholate by a rapid dilution protocol and several freeze-thaw treatments. We have found that both the steady-state level and the polarization of fluorescein fluorescence of these reconstituted systems monotonically increase as a function of the lipid to protein ratio between 80 and 2000 (on a mole per mole basis). The magnitude of this increase is about 15%. Detergents, such as Triton X-100 and deoxycholate, when added to SR labelled vesicles below their critical micelle concentrations also induce similar changes in fluorescein fluorescence. We suggest that lipid dilution of protein in these reconstituted systems induce a decrease of the level of self-quenching by promoting dissociation of (Ca2+, Mg2+)-ATPase.
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PMID:Dependence of the fluorescence of fluorescein labelled (Ca2+, Mg2+)-ATPase upon the lipid to protein ratio in sarcoplasmic reticulum reconstituted systems. 293 64

Four mechanisms for the allosteric regulation of the calcium and magnesium ion activated adenosinetriphosphatase (Ca,Mg-ATPase) of sarcoplasmic reticulum were examined. Negative cooperativity in substrate binding was not supported by 3H-labeled 5'-adenylyl methylenediphosphate (AMPPCP) binding, which was best fit by a single class of sites. Although calcium had no effect on the absence of cooperativity, it did increase the affinity of the enzyme for AMPPCP. Allosteric regulation via an effector site for AMPPCP or ATP on the same ATPase chain was eliminated by the stoichiometry of ATP and AMPPCP binding, 1 mol of site per mole of enzyme. The possibility that AMPPCP acts at an effector site was eliminated by showing that it competitively inhibits the rate of phosphoenzyme formation. Allosteric regulation of kinetics via site-site interaction in an oligomer was eliminated by showing that the inhibition of ATPase activity by fluorescein isothiocyanate is linearly dependent upon its incorporation into the sarcoplasmic reticulum. The fourth mechanism considered was stimulation of ATPase activity by the binding of ATP or AMPPCP at the active site after departure of ADP but before the departure of inorganic phosphate. This hypothesis was supported by site stoichiometry and by the observation that AMPPCP or ATP stimulates v/EP, the rate of ATP hydrolysis for a given level of phosphoenzyme. Computer simulation of this branched monomeric model could duplicate all experimental observations made with AMPPCP and ATP as allosteric regulators. The condition that the affinity of ATP binding to the enzyme be reduced when it is phosphorylated, which is required by the computer model, was confirmed experimentally.
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PMID:Mechanism of allosteric regulation of the Ca,Mg-ATPase of sarcoplasmic reticulum: studies with 5'-adenylyl methylenediphosphate. 293 90

The effects of acute and long-term changes in temperature upon catalytic and calcium regulatory function of red (slow oxidative) and white (fast glycolytic) muscle from striped bass (Morone saxatilis) were determined. Acclimation to 5 degrees C or 25 degrees C had no significant effect on catalytic function (ATPase activity) or regulatory sensitivity (Ca++-activation) of myofibrils from either muscle type. Substantial differences between red and white muscle were found in the intrinsic thermal sensitivity of maximally-activated Mg++-Ca++ myofibrillar ATPase. Arrhenius plots of myofibrillar ATPase from white muscle show one significant breakpoint at 29 degrees C, with activation energies (Ea) of 2.3 and 23.4 kcal mole-1 at temperatures above and below this transition, respectively. Arrhenius plots of myofibrillar ATPase from red muscle show two transitions occurring at 22 and 9 degrees C, with Ea of 7.6 kcal mole-1 above 22 degrees C and 18.3 kcal mole-1 between 9 and 22 degrees C. Activation energies for myofibrils from red muscle increase substantially to approximately 107.3 kcal mole-1 below the 9 degrees C breakpoint. Differences in the intrinsic thermal sensitivity of red and white muscle catalytic function are apparently due to interaction of actomyosins and calcium regulatory proteins which are specific to each muscle type. The results suggest that capacity for sustained swimming in striped bass, which is powered exclusively by red muscle, will be severely impaired at cold temperature unless compensations occur above the level of contractile proteins.
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PMID:Biochemical responses to temperature in the contractile protein complex of striped bass Morone saxatilis. 294 18

Techniques are described for using blocking agents to distinguish between enzymes which are functional monomers and oligomers. To achieve this distinction the blocking agent must react exclusively at the active site with a stoichiometry of one mole of site per mole enzyme. The effect of the blocking agent on enzymatic activity in oligomers of n = 2 and 4 are described and the optimal degree of blocking is considered for tests of enzyme activity at saturating and less than saturating substrate concentrations. For saturating concentrations and a dimer the distinction between dimer and monomer is best observed with 50 per cent of sites blocked. For a tetramer the distinction is best made at higher degrees of blockade. The use of saturating substrate concentrations is thus limited to small oligomers. If nonsaturating substrate concentrations are used and normalized double reciprocal plots of the dependence of enzyme activity on substrate concentrations are made then the distinction between monomer and oligomer can readily be made for dimers, tetramers, and higher n-mers. The principles developed to distinguished monomeric from oligomeric enzymes are applied to published data obtained with the Ca Mg-ATPase of sarcoplasmic reticulum. Fluorescein isothiocyanate is the blocking agent. Plots of the published data support both the monomeric and tetrameric models for allosteric regulation with the preponderance of the data supporting the monomeric model.
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PMID:Distinguishing between functional monomeric and oligomeric complexes of the Ca,Mg-ATPase in sarcoplasmic reticulum. 294 43

Ca2+-ATPase molecules were labeled in intact sarcoplasmic reticulum (SR) vesicles, sequentially with a donor fluorophore, fluorescein-5'-isothiocyanate (FITC), and with an acceptor fluorophore, eosin-5'-isothiocyanate (EITC), each at a mole ratio of 0.25-0.5 mol/mol of ATPase. The resonance energy transfer was determined from the effect of acceptor on the intensity and lifetime of donor fluorescence. Due to structural similarities, the two dyes compete for the same site(s) on the Ca2+-ATPase, and under optimal conditions each ATPase molecule is labeled either with donor or acceptor fluorophore, but not with both. There is only slight labeling of phospholipids and other proteins in SR, even at concentrations of FITC or EITC higher than those used in the reported experiments. Efficient energy transfer was observed from the covalently bound FITC to EITC that is assumed to reflect interaction between ATPase molecules. Protein denaturing agents (8 M urea and 4 M guanidine) or nonsolubilizing concentrations of detergents (C12E8 or lysolecithin) abolish the energy transfer. These results are consistent with earlier observations that a large portion of the Ca2+-ATPase is present in oligomeric form in the native membrane. The technique is suitable for kinetic analysis of the effect of various treatments on the monomer-oligomer equilibrium of Ca2+-ATPase. A drawback of the method is that the labeled ATPase, although it retains conformational responses, is enzymatically inactive.
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PMID:Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum. 295 Sep 38

Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of IAA per mole of ATPase was bound in 6 h without affecting the Ca2+-transport activity. [14C]IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys 674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys denotes the [14C]IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 microM IAEDANS for 5 h, at pH 7.0 and 30 degrees C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.
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PMID:Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. II. Site of labeling with iodoacetamide and its fluorescent derivative. 295 12

Myosin has two heads which can bind with F-actin and react with ATP. The skeletal muscle myosin forms each 1 mol of the myosin-phosphate-ADP complex (M-P-ADP) and the myosin-ATP complex (M-ATP). The actomyosin ATPase reaction which is coupled with muscle contraction is catalyzed only by the head which forms M-P-ADP. However, the function of M-ATP forming head in muscle contraction has not been elucidated. We studied the binding of S-1 and HMM with F-actin and the dissociation of acto-S-1 or acto-HMM by ATP or AMPPNP using the change in light-scattering and fluorescence of pyrene bound to F-actin. S-1 and HMM bound with actin at 1:1 and 1:2 molar ratio, respectively. Acto-S-1 dissociated by one mole of ATP per mole of S-1 but acto-HMM dissociated by 1 mol ATP per mol of HMM (0.5 mol/mol head). Acto-HMM dissociates by AMPPNP (or ADP) via a ternally complex. Acto-HMM bound two mole of AMPPNP, but acto-HMM dissociated by a function of (AMPPNP) but not (AMPPNP)2. These results suggested that the affinity of HMM with F-actin decreased by the binding of one mole of AMPPNP. The result presented here showed that binding of M-ATP forming head with F-actin is controlled by the ATPase reaction of the M-P-ADP forming head. It is suggested that during muscle contraction two heads react cooperatively with thin filament.
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PMID:The function of two heads of myosin in muscle contraction. 297 Feb 8

Since the Ca2+-regulatory mechanism for actin-myosin interaction in smooth muscle involves phosphorylation of the 20,000-Da myosin light chains, it was hypothesized that such interaction should be influenced by myosin phosphatase. Accordingly, we studied the effects of an aortic myosin light-chain phosphatase on Ca1+-dependent actin-myosin interaction in detergent-skinned porcine carotid artery and bovine aortic native actomyosin. In skinned preparations, the aortic phosphatase (16 U/ml) markedly inhibited the rate of isometric contraction in low Ca2+ (6.8 X 10(-7) M) and responsiveness to Ca2+ (force attained with 6.8 X 10(-7) Ca2+/force attained with 1.6 X 10(-6) M Ca2+), whereas relaxation was accelerated. Ca2+-dependent actomyosin ATPase activity and phosphorylation of the light chains were significantly and progressively depressed in the presence of increasing concentrations of phosphatase (0.1-0.9 U/ml). The concentration of Ca2+ (1.1 X 10(-6) M) required for half-maximal activation of either ATPase activity or light-chain phosphorylation increased by 70% in the presence of 0.1 U phosphatase/ml. Neither the maximal rate of Ca2+-sensitive ATP hydrolysis (39 +/- 0.8 nmole/min/mg actomyosin) nor the extent of phosphorylation (0.68 +/- 0.05 mole PO4/mole light chain) was altered at greater than 5 X 10(-6) M Ca2+. ATPase activity was correlated to light-chain phosphorylation under diverse conditions including the presence or absence of 1 microM calmodulin, different concentrations of phosphatase (0-0.9 U/ml), and different concentrations of Ca2+ (10(-8) to 1.25 X 10(-5) M). However, significant phosphorylation was present (20-25% of maximum) in the absence of Ca2+-dependent ATPase activity and only 15% of the maximal rate of ATP hydrolysis was expressed until phosphorylation attained 50% of its maximal value. These findings are consistent with the ordered model of myosin phosphorylation suggested by A. Persechini and D. J. Hartshorne [Science (Washington, DC), 213:1383-285, 1961] (36). They also suggest that myosin phosphatase may participate in modulating actin-myosin interactions in vascular smooth muscle.
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PMID:Phosphatase-mediated modulation of actin-myosin interaction in bovine aortic actomyosin and skinned porcine carotid artery. 298 22

When perfused cortex-free ox adrenal medulla was stimulated to secrete catecholamine by infusion of 0.1 mM acetylcholine for 4 min, the oxygen consumption increased to a value which was 0.15 +/- 0.07 mumole O2/min/g wet weight (+/- S.D., N = 12) above the pre-stimulation value of 0.49 +/- 0.15 (P less than 0.001). 1.4 +/- 0.9 (+/- S.D., N = 12) mole of catecholamine was secreted per mole of enhanced O2 consumption in the 16 min following the start of the stimulation. The rate of ATP hydrolysis by the proton-translocating Mg-ATPase of the chromaffin granule may increase on fusing with the plasma membrane of the chromaffin cell during exocytosis. However, from the amount of catecholamine secreted, this was estimated to account for less than 17% of the oxygen consumption increase. The amount of catecholamine secreted by 4 min 0.1 mM acetylcholine stimulations correlated with the enhancement of oxygen consumption (r = 0.82, P less than 0.001) but, on stimulation with 60 microM veratridine for 4 min, O2 consumption enhancement was anomalously low. This dependence on mode of stimulation suggests that ATP consumption in exocytosis itself is an inadequate explanation. Ouabain-sensitive oxygen consumption rose from undetectable levels to 18 +/- 8% (+/- S.D., N = 4) of the basal respiration during prolonged 0.1 mM acetylcholine stimulation in the absence of Ca, indicating that Na,K-ATPase was not responsible for all of the oxygen consumption enhancement.
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PMID:Enhanced oxygen consumption in adrenal medulla on stimulation with acetylcholine. 298 35

Occlusion of Rb+ by C12E8-solubilized (Na+ + K+)-ATPase from shark salt glands has been measured. The rate of de-occlusion at room temperature is about 1 s-1, which is the same as for the membrane-bound enzyme. The amount of Rb+ occluded is 3 moles Rb+ per mole membrane-bound shark enzyme, whereas only about 2 moles Rb+ are occluded by the C12E8-solubilized enzyme.
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PMID:Occlusion of Rb+ by detergent-solubilized (Na+ + K+)-ATPase from shark salt glands. 298 93


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