The Modification of Actomyosin ATPase Activity by Tropomyosin-Troponin and its Dependence on Ionic Strength, ATP-Concentration, and Actin-Myosin Ratio

1974 ◽  
Vol 29 (9-10) ◽  
pp. 496-505 ◽  
Author(s):  
Peter Daneker
Keyword(s):  
Ionic Strength ◽  
Atpase Activity ◽  
Actin Filaments ◽  
Regulatory Proteins ◽  
High Affinity ◽  
Low Concentrations ◽  
Dependence On Ionic Strength ◽  
High Concentrations ◽  
Low Ionic Strength ◽  
Better Than

Abstract At millimolar concentrations of ATP the ATPase activity of regulated actomyosin (which consisted of myosin and of actin containing the regulatory proteins tropomyosin and troponin) was lower than that of unregulated actomyosin (containing actin devoid of the regulatory proteins) when the ionic strength was high (> 0 .0 3 ᴍ KCl). At low ionic strength (0.03 ᴍ KCl) the ATPase activity of regulated actomyosin was similar to or even higher than that of unregulated acto­ myosin. Besides increasing ionic strength an increasing actin-myosin ratio tended to depress the ATPase activity of regulated actomyosin below that of unregulated one. At lower ATP concen­ trations (0.1 mᴍ or lower) the ATPase activity of regulated actomyosin was higher than that of unregulated actomyosin at any ionic strength and at any actin-myosin ratio. EGTA inhibited the ATPase of regulated actomyosin under any conditions at high ATP concentrations. At lower ATP concentrations EGTA inhibited either at higher ionic strength or at a higher actin-myosin ratio. The inhibition of the ATPase activity of acto-HMM by increasing ionic strength was not in­ fluenced by the regulatory proteins. - For the interpretation of these results it has been assumed that in actomyosin regulated actin can adopt three states: A low-affinity state which activates the ATPase of myosin only slightly (occurring at high ATP concentrations and in the absence of Ca2+), a high affinity state which activates the ATPase of myosin better than does unregulated actin (occurring at low concentrations of ATP and in the presence of Ca2+), and an intermediate state. This latter state (occurring at high concentrations of ATP and in the presence of Ca2+ or at low concentrations of ATP and in the absence of Ca2+) activates the ATPase of myosin less than does unregulated actin when the actin-myosin ratio is high (wide spacing of myosin on the actin filaments) but activates more (or at least not less) when the actin-myosin ratio is low (dense spacing of myosin on the actin filaments)

scholarly journals A novel procedure for the rapid purification of plastocyanin from pea (Pisum sativum) leaves

1981 ◽  
Vol 193 (1) ◽  
pp. 375-378 ◽  
Author(s):  
A R Ashton ◽  
L E Anderson
Keyword(s):  
Ionic Strength ◽  
Pisum Sativum ◽  
Deae Cellulose ◽  
High Concentrations ◽  
Rapid Purification ◽  
Low Ionic Strength

Plastocyanin is soluble at high concentrations (greater than 3 M) of (NH4)2SO4 but under these conditions will adsorb tightly to unsubstituted Sepharose beads. This observation was utilized to purify plastocyanin from pea (Pisum sativum) in two chromatographic steps. Sepharose-bound plastocyanin was eluted with low-ionic-strength buffer and subsequently purified to homogeneity by DEAE-cellulose chromatography.

scholarly journals Activation of the Adenosine Triphosphatase of Limulus polyphemus Actomyosin by Tropomyosin

1972 ◽  
Vol 59 (4) ◽  
pp. 375-387 ◽  
Author(s):  
William Lehman ◽  
Andrew G. Szent-Györgyi
Keyword(s):  
Ionic Strength ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Limulus Polyphemus ◽  
Muscle Tropomyosin ◽  
Low Ionic Strength ◽  
Full Activation

Purified actin does not stimulate the adenosine triphosphatase (ATPase) activity of Limulus myosin greatly. The ATPase activity of such reconstituted preparations is only about one-fourth the ATPase of myofibrils or of natural actomyosin. Actin preparations containing tropomyosin, however, activate Limulus myosin fully. Both the tropomyosin and the actin preparations appear to be pure when tested by different techniques. Tropomyosin combines with actin but not with myosin and full activation is reached at a tropomyosin-to-actin ratio likely to be present in muscle. Tropomyosin and actin of several different animals stimulate the ATPase of Limulus myosin. Tropomyosin, however, is not required for the ATPases of scallop and rabbit myosin which are fully activated by pure actin alone. Evidence is presented that Limulus myosin, in the presence of ATP at low ionic strength, has a higher affinity for actin modified by tropomyosin than for pure actin.

scholarly journals Evidence from Insect Fibrillar Muscle about the Elementary Contractile Process

1967 ◽  
Vol 50 (6) ◽  
pp. 139-156 ◽  
Author(s):  
J. W. S. Pringle
Keyword(s):  
Ionic Strength ◽  
Atpase Activity ◽  
Striated Muscle ◽  
Contractile Activity ◽  
High Elasticity ◽  
Flight Muscles ◽  
The Cross ◽  
Cross Bridges ◽  
Low Ionic Strength ◽  
Water Bugs

Bundles of myofibrils prepared from the dorsal longitudinal flight muscles of giant water bugs show oscillatory contractile activity in solutions of low ionic strength containing ATP and 10-8-10-7 M Ca2+. This is due to delay between changes of length and changes of tension under activating conditions. The peculiarities of insect fibrillar muscle which give rise to this behavior are (1) the high elasticity of relaxed myofibrils, (2) a smaller degree of Ca2+ activation of ATPase activity in unstretched myofibrils and extracted actomyosin, and (3) a direct effect of stretch on ATPase activity. It is shown that the cross-bridges of striated muscle are probably formed from the heads of three myosin molecules and that in insect fibrillar muscle the cycles of mechanochemical energy conversion in the cross-bridges can be synchronized by imposed changes of length. This material is more suitable than vertebrate striated muscle for a study of the nature of the elementary contractile process.

scholarly journals A 72,000-mol-wt protein from tomato inhibits rabbit acto-S-1 ATPase activity.

1983 ◽  
Vol 96 (6) ◽  
pp. 1761-1765 ◽  
Author(s):  
M Vahey
Keyword(s):  
Skeletal Muscle ◽  
Ionic Strength ◽  
Atpase Activity ◽  
Rabbit Skeletal Muscle ◽  
Ion Concentration ◽  
Actin Binding ◽  
Molar Ratio ◽  
Reversible Inhibitor ◽  
Skeletal Muscle Myosin ◽  
Low Ionic Strength

Tomato activation inhibiting protein (AIP) is a molecule of an apparent molecular weight of 72,000 that co-purifies with tomato actin. In an assay system containing rabbit skeletal muscle F-actin and rabbit skeletal muscle myosin subfragment-1 (myosin S-1), tomato AIP dissociated the acto-S-1 complex in the absence of Mg+2ATP and inhibited the ability of F-actin to activate the low ionic strength Mg+2ATPase activity of myosin S-1. At a molar ratio of 5 actin to 1 AIP, a 50% inhibition of the actin-activated Mg+2ATPase activity of myosin S-1 was observed. The inhibition can be reversed by raising the calcium ion concentration to 1 X 10(-5) M. The AIP had no effect on the basal low ionic strength Mg+2ATPase activity of myosin S-1 in the absence of actin. The protein did not bind directly to actin nor did it cause depolymerization or aggregation of F-actin but appeared, instead, to interact with the actin binding site on myosin S-1. Since AIP is a potent, reversible inhibitor of the rabbit acto-S-1 ATPase activity, it is postulated that it may be responsible for the low levels of actin activation exhibited by tomato F-actin fractions containing the AIP.

scholarly journals Affinity chromatography of immobilized actin and myosin

1975 ◽  
Vol 149 (2) ◽  
pp. 365-379 ◽  
Author(s):  
R C Bottomley ◽  
I P Trayer
Keyword(s):  
Ionic Strength ◽  
Salt Concentration ◽  
Free Solution ◽  
Heavy Meromyosin ◽  
Chemical Modifications ◽  
Low Concentrations ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
One Step ◽  
Low Ionic Strength

Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder.

Actomyosin-like protein of arterial wall

1963 ◽  
Vol 205 (6) ◽  
pp. 1247-1252 ◽  
Author(s):  
Ronald S. Filo ◽  
J. Caspar Ruegg ◽  
David F. Bohr
Keyword(s):  
Ionic Strength ◽  
Atpase Activity ◽  
Arterial Wall ◽  
Structural Protein ◽  
High Solubility ◽  
Viscosity Change ◽  
Relaxing Factor ◽  
Contractile System ◽  
Crude Preparation ◽  
Low Ionic Strength

A structural protein is extractable from hog carotids in solutions of low ionic strength (0.05 m KCl + 0.02 m histidine buffer). The following procedures cause precipitation of this protein: 1) standing 12 hr at 2–4 C, 2) dialysis against 0.05 m KCl, or 3) addition of 10 mm CaCl2. The crude preparation obtained by any of these precipitation procedures fails to show superprecipitation on the addition of ATP, but when dissolved in 0.6 m KCl it does demonstrate a viscosity change on the addition of ATP and is capable of ATPase activity. If this crude preparation is purified by repeated calcium precipitation and dialysis against 0.05 m KCl, it then shows the following characteristics typical of actomyosin: 1) superprecipitation, 2) reversible viscosity change on addition of ATP, and 3) ATPase activity characteristically influenced by calcium, or magnesium, or ionic strength. We conclude that this structural protein from hog carotid is an actomyosin-like protein involved in a standard contractile system, and its initial high solubility at low ionic strength may be due either to a reduction in bound calcium or to the presence of an unusually effective solubilizing factor which, like relaxing factor, can be inhibited by calcium.

scholarly journals Inhibition of fly head acetylcholinesterase by bis-[(m-hydroxyphenyl)trimethylammonium iodide] esters of polymethylenedicarbamic acids

1970 ◽  
Vol 117 (2) ◽  
pp. 221-230 ◽  
Author(s):  
J. H. Davies ◽  
W. R. Campbell ◽  
C. W. Kearns
Keyword(s):  
Functional Group ◽  
Straight Chain ◽  
Experimental Conditions ◽  
Maximum Slope ◽  
Large Molecules ◽  
High Affinity ◽  
Affinity Constants ◽  
Low Concentrations ◽  
High Concentrations ◽  
Pass Through

A series of bis-[(m-hydroxyphenyl)trimethylammonium iodide] esters of polymethylenedicarbamic acids and a number of (m-hydroxyphenyl)trimethylammonium iodide esters of straight-chain N-alkylcarbamic acids have been examined as inhibitors of acetylcholinesterase from fly head. Evidence is presented suggesting that inhibition of acetylcholinesterase by the bis-carbamates is due to carbamoylation of the enzyme, as is generally thought to be the case with esters of N-alkylcarbamic acids. Inhibition is irreversible. The (m-hydroxyphenyl)trimethylammonium iodide ester of N-hexylcarbamic acid also inhibits fly head acetylcholinesterase irreversibly. There is therefore no need to implicate a second functional group in bis-carbamate esters to explain the irreversible inhibition of the enzyme. An unusual feature of the inhibition is that inhibition lines do not pass through 100% enzyme activity at t=0, except for rather low concentrations of inhibitor (<10μm for the octamethylene compound). Also, inhibition lines tend towards a maximum slope as inhibitor concentration is increased. The first observation indicates complex-formation, even in the presence of high concentrations of substrate, and by using measurements of inhibition at relatively high inhibitor concentrations, affinity constants K′a have been calculated. K′a varies from 0.1μm for the dodecamethylene compound to 10μm for the tetramethylene compound, in the presence of 3.75mm-acetylthiocholine, indicating high affinity for the enzyme. The second observation shows that, owing to this high affinity, the enzyme becomes saturated with inhibitor under the experimental conditions employed, and from the limiting slope values of the carbamoylation rate constant (k2) have been calculated. k2 varies from 0.15min−1 for the tetramethylene compound to 1min−1 for the decamethylene compound. Variations of potency in this series are therefore mainly due to changes in affinity (100-fold) rather than in carbamoylation rate (sevenfold). The observation that large molecules may acylate the enzyme raises certain problems, which are discussed.

Correlation between Ca2+-ATPase activity of rat islet cells and insulin secretion

1992 ◽  
Vol 134 (2) ◽  
pp. 221-225 ◽  
Author(s):  
C. M. Gronda ◽  
G. B. Diaz ◽  
J. P. F. C. Rossi ◽  
J. J. Gagliardino
Keyword(s):  
Enzyme Activity ◽  
Insulin Secretion ◽  
Ionic Strength ◽  
Atpase Activity ◽  
Islet Cells ◽  
Experimental Conditions ◽  
Physiological Regulation ◽  
Enhancing Effect ◽  
Cellular Targets ◽  
Low Ionic Strength

ABSTRACT Using medium with a low ionic strength, a low concentration of Ca2+ and Mg2+ and devoid of K+, we have measured Ca2+-ATPase activity in the homogenates of rat islets preincubated for 3 min with several hormones in the presence of 3·3 mmol glucose/l. Insulin secretion was also measured in islets incubated for 5 min under identical experimental conditions. Islets preincubated with glucose (3·3 mmol/l) and glucagon (1·4 μmol/l) plus theophylline (10 mmol/l), ACTH (0·11 nmol/l), bovine GH (0·46 μmol/l), prolactin (0·2 μmol/l) or tri-iodothyronine (1·0 nmol/l) have significantly lower Ca2+-ATPase activity than those preincubated with only 3·3 mmol glucose/l. All these hormones increased the release of insulin significantly. Dexamethasone (0·1 μmol/l) and somatostatin (1·2 μmol/l) enhanced the Ca2+-ATPase activity while adrenaline (10 μmol/l) did not produce any significant effect on the activity of the enzyme. These hormones decreased the release of insulin significantly. These results demonstrated that islet Ca2+-ATPase activity was modulated by the hormones tested. Their inhibitory or enhancing effect seemed to be related to their effect on insulin secretion; i.e. those which stimulated the secretion of insulin inhibited the activity of the enzyme and vice versa. Hence, their effect on insulin secretion may be due, in part, to their effect on enzyme activity and consequently on the concentration of cytosolic Ca2+. These results reinforce the assumption that Ca2+-ATPase activity participates in the physiological regulation of insulin secretion, being one of the cellular targets for several agents which affect this process. Journal of Endocrinology (1992) 134, 221–225

scholarly journals Concentration-dependent effects of endogenous S-nitrosoglutathione on gene regulation by specificity proteins Sp3 and Sp1

2004 ◽  
Vol 380 (1) ◽  
pp. 67-74 ◽  
Author(s):  
Khalequz ZAMAN ◽  
Lisa A. PALMER ◽  
Allan DOCTOR ◽  
John F. HUNT ◽  
Benjamin GASTON
Keyword(s):  
Cystic Fibrosis ◽  
Nitrosative Stress ◽  
Regulatory Gene ◽  
Housekeeping Genes ◽  
Regulatory Proteins ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Low Concentrations ◽  
High Concentrations ◽  
Cystic Fibrosis Transmembrane

The activities of certain nuclear regulatory proteins are modified by high concentrations of S-nitrosothiols associated with nitrosative stress. In the present study, we have studied the effect of physiological (low µM) concentrations of the endogenous S-nitrosothiol, GSNO (S-nitrosoglutathione), on the activities of nuclear regulatory proteins Sp3 and Sp1 (specificity proteins 3 and 1). Low concentrations of GSNO increased Sp3 binding, as well as Sp3-dependent transcription of the cystic fibrosis transmembrane conductance regulatory gene, cftr. However, higher GSNO levels prevented Sp3 binding, augmented Sp1 binding and prevented both cftr transcription and CFTR (cystic fibrosis transmembrane conductance regulator) expression. We conclude that low concentrations of GSNO favour Sp3 binding to ‘housekeeping’ genes such as cftr, whereas nitrosative stress-associated GSNO concentrations shut off Sp3-dependent transcription, possibly to redirect cellular resources. Since low micromolar concentrations of GSNO also increase the maturation and activity of a clinically common CFTR mutant, whereas higher concentrations have the opposite effect, these observations may have implications for dosing of S-nitrosylating agents used in cystic fibrosis clinical trials.

Rhodobacter sphaeroides haem protein: a novel cytochrome with nitric oxide dioxygenase activity

2008 ◽  
Vol 36 (5) ◽  
pp. 992-995 ◽  
Author(s):  
Bor-Ran Li ◽  
J.L. Ross Anderson ◽  
Christopher G. Mowat ◽  
Caroline S. Miles ◽  
Graeme A. Reid ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Ionic Strength ◽  
Rhodobacter Sphaeroides ◽  
Protein A ◽  
Reduced Form ◽  
Gene Encoding ◽  
High Affinity ◽  
Haem Protein ◽  
Low Ionic Strength ◽  
Dioxygenase Activity

Rhodobacter sphaeroides produces a novel cytochrome, designated as SHP (sphaeroides haem protein), that is unusual in having asparagine as a redox-labile haem ligand. The gene encoding SHP is contained within an operon that also encodes a DHC (dihaem cytochrome c) and a membrane-associated cytochrome b. DHC and SHP have been shown to have high affinity for each other at low ionic strength (Kd=0.2 μM), and DHC is able to reduce SHP very rapidly. The reduced form of the protein, SHP2+ (reduced or ferrous SHP), has high affinity for both oxygen and nitric oxide (NO). It has been shown that the oxyferrous form, SHP2+–O2 (oxygen-bound form of SHP), reacts rapidly with NO to produce nitrate, whereas SHP2+–NO (the NO-bound form of SHP) will react with superoxide with the same product formed. It is therefore possible that SHP functions physiologically as a nitric oxide dioxygenase, protecting the organism against NO poisoning, and we propose a possible mechanism for this process.

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