Binding of adenylosuccinate synthetase to contractile proteins of muscle

1989 ◽  
Vol 257 (1) ◽  
pp. C29-C35 ◽  
Author(s):  
J. P. Manfredi ◽  
R. Marquetant ◽  
A. D. Magid ◽  
E. W. Holmes
Keyword(s):  
Ionic Strength ◽  
Dissociation Constant ◽  
Thin Filament ◽  
Contractile Proteins ◽  
Purine Nucleotide ◽  
Apparent Dissociation Constant ◽  
Thin Filaments ◽  
Adenylosuccinate Synthetase ◽  
Purine Nucleotide Cycle ◽  
Low Ionic Strength

The muscle isozyme of adenylosuccinate synthetase (AdSS), an enzyme of the purine nucleotide cycle, has previously been shown to bind to purified F-actin in buffers of low ionic strength and pH (Ogawa et al. Eur. J. Biochem. 85: 331-338, 1978). We have extended these observations by measuring the association of both crude and purified AdSS with the contractile proteins of muscle in buffers of physiological ionic strength and pH. Under these conditions, the enzyme binds to F-actin, actin-tropomyosin complexes, reconstructed thin filaments, and myofibrils but not to myosin. The apparent dissociation constant of 1.2 microM and binding maximum of 2.6 nmol enzyme/mg myofibrils indicate that binding of AdSS to myofibrils can be physiologically significant. The results suggest that AdSS in muscle may be associated with the thin filament of myofibrils.

Which value for the first dissociation constant of carbonic acid should be used in biological work?

1991 ◽  
Vol 260 (5) ◽  
pp. C1113-C1116 ◽  
Author(s):  
R. W. Putnam ◽  
A. Roos
Keyword(s):  
Ionic Strength ◽  
Activity Coefficient ◽  
Dissociation Constant ◽  
Carbonic Acid ◽  
Mass Action ◽  
Bicarbonate Concentration ◽  
Apparent Dissociation Constant ◽  
Logarithmic Form ◽  
Ion Activity ◽  
Hasselbalch Equation

The apparent first dissociation constant of carbonic acid has been defined in different ways in the literature. Harned and co-workers (8-10) have defined it in terms of molalities of the participating species, including H ions: Ks = mHmHCO3/mCO2. In contrast, Hastings and Sendroy have defined an apparent constant in which acidity is expressed as H ion activity: K'1 = aHmHCO3/mCO2. These constants differ by a factor gamma H, the activity coefficient of H ions at the prevailing ionic strength. Therefore, pK'1 is greater than pKs by an amount equal to -log gamma H, which, at mu = 0.16 M, is approximately 0.1. It is important that the correct value for the apparent dissociation constant or its logarithmic form be entered in the mass action expression or in the Henderson-Hasselbalch equation in order to prevent significant errors in the computation by means of these equations of quantities that cannot be directly measured. Specifically, for the derivation of bicarbonate concentration from PCO2 and pH (-log aH), pK'1 is to be used and not an uncorrected pKs.

AMP deamination and IMP reamination in working skeletal muscle

1980 ◽  
Vol 239 (1) ◽  
pp. C32-C38 ◽  
Author(s):  
R. A. Meyer ◽  
R. L. Terjung
Keyword(s):  
Purine Nucleotide ◽  
Threefold Increase ◽  
Adenylosuccinate Synthetase ◽  
Mild Stimulation ◽  
Fast Twitch Muscle ◽  
Important Pathway ◽  
Purine Nucleotide Cycle ◽  
Fast Twitch ◽  
Intense Stimulation

The extent of purine nucleotide cycle (PNC) turnover [i.e., the cyclic deamination of adenosine 5'-monophosphate (AMP) and reamination of inosine 5'-monophosphate (IMP)] was estimated in fast-twitch muscle of pentobarbital sodium anesthetized rats during in situ stimulation, by the accumulation of excess IMP after blocking IMP reamination with the adenylosuccinate synthetase inhibitor, hadacidin. Sodium hadacidin (100 mg/kg, iv) did not alter IMP production and was effective in blocking IMP reamination by 80%. Hadacidin had no effect on IMP levels in muscle at rest or during mild stimulation (1 Hz for 30 min). During 30 min of more intense stimulation (5 Hz), hadacidin treatment resulted in a threefold increase in IMP in the fast-twitch white gastrocnemius section, but had no effect in the fast-twitch red section. The IMP in the fast-twitch white section did not accumulate over the 30-min period, but was produced initially (within 5 min) and kept for being reaminated by hadacidin. Thus, the IMP formation and IMP reamination evident in the saline-injected animals did not occur concurrently. This suggests that the IMP reamination arm of the PNC is not an important pathway in "working" muscle.

scholarly journals Effects of deletion of tropomyosin overlap on regulated actomyosin subfragment 1 ATPase

1989 ◽  
Vol 258 (3) ◽  
pp. 831-836 ◽  
Author(s):  
D H Heeley ◽  
L B Smillie ◽  
E M Lohmeier-Vogel
Keyword(s):  
Ionic Strength ◽  
Binding Model ◽  
Thin Filaments ◽  
System A ◽  
Atpase Assay ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Low Ionic Strength ◽  
Atpase Inhibition

The role of the overlap region at the ends of tropomyosin molecules in the properties of regulated thin filaments has been investigated by substituting nonpolymerizable tropomyosin for tropomyosin in a reconstituted troponin-tropomyosin-actomyosin subfragment 1 ATPase assay system. A previous study [Heeley, Golosinka & Smillie (1987) J. Biol. Chem. 262, 9971-9978] has shown that at an ionic strength of 70 mM, troponin will induce full binding of nonpolymerizable tropomyosin to F-actin both in the presence and absence of calcium. At a myosin subfragment 1-to-actin ratio of 2:1 ([actin] = 4 microM) and an ionic strength of 50 mM, comparable levels of ATPase inhibition were observed with increasing levels of tropomyosin or the truncated derivative in the presence of troponin (-Ca2+). Large differences were noted, however, in the activation by Ca2+. Significantly lower ATPase activities were observed with nonpolymerizable tropomyosin and troponin (+Ca2+) over a range of subfragment 1-to-actin ratios from 0.25 to 2.5. The concentration of subfragment 1 required to generate ATPase activities exceeding those seen with actomyosin subfragment 1 alone under these conditions was 3-4-fold greater when nonpolymerizable tropomyosin was used. Similar effects were seen at the much lower ionic strength of 13 mM and are consistent with the reduced ATPase activity with nonpolymerizable tropomyosin observed previously [Walsh, Trueblood, Evans & Weber (1985) J. Mol. Biol. 182, 265-269] at low ionic strength and a subfragment 1-to-actin ratio of 1:100. Little cooperativity in activity as a function of subfragment 1 concentration with either intact tropomyosin or its truncated derivative was observed under the present conditions. Further studies are directed towards an understanding of these effects in terms of the two-state binding model for the attachment of myosin heads to regulated thin filaments.

Extractability and properties of the contractile proteins of vertebrate smooth muscle

1973 ◽  
Vol 265 (867) ◽  
pp. 169-181 ◽  
Keyword(s):  
Smooth Muscle ◽  
Ionic Strength ◽  
Striated Muscle ◽  
Smooth Muscles ◽  
Contractile Proteins ◽  
Biosynthetic Activity ◽  
Salting Out ◽  
Low Ionic Strength ◽  
The Comparative Study ◽  
Muscle Myosin

The vertebrate smooth muscles differ from the striated ones by their larger extracellular space, the smaller size of their cells and their high content in extracellular components. Furthermore, the smooth muscle cell is a bifunctional biological unit able to carry on also an important biosynthetic activity. The contractile proteins of vertebrate smooth muscle are extractable at low ionic strength contrarily to those of striated muscle. The partition of the salt extractable nitrogen between the low and high ionic strength extracts is very different in these two cases. Acidification of low ionic strength extracts of vertebrate smooth muscle at pH 5 allows precipitation of the contractile proteins quantitatively together with a large amount of contaminants typical of the smooth muscles. Comparison of the contractile proteins of vertebrate smooth muscle with their striated counterparts shows that actin is a very constant component of the contractile machinery, that tropomyosin holds an intermediate position, while myosin is the most variable. The smooth muscle myosin differs not only by some general properties as salting-out range and thermostability, but also by the behaviour of various parts of the molecule. The globular head has a different ATPase activity and is responsible for the very peculiar immunological behaviour of this myosin. The point along the myosin rod which is attacked by trypsin is much more resistant to proteolysis. The light meromyosin is more soluble and differs very much in amino acid composition. The comparative study of myosin reveals only minor variations from one species to the other but more or less wide ones within each species according to the type of muscle examined.

Experimental31p nmr study of the influence of ionic strength on the apparent dissociation constant of mgatp

1994 ◽  
Vol 2 (2) ◽  
pp. 101-107 ◽  
Author(s):  
I. Mottet ◽  
R. Demeure ◽  
B. Gallez ◽  
C. Grandin ◽  
B. E. Van Beers ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Dissociation Constant ◽  
Apparent Dissociation Constant ◽  
Nmr Study

scholarly journals Does actin bind to the ends of thin filaments in skeletal muscle?

1985 ◽  
Vol 100 (1) ◽  
pp. 282-291 ◽  
Author(s):  
S Ishiwata ◽  
T Funatsu
Keyword(s):  
Ionic Strength ◽  
High Ionic Strength ◽  
Structure Population ◽  
Electron Microscopic ◽  
Thin Filaments ◽  
Thick Filaments ◽  
Low Ionic Strength ◽  
Organizational Mechanisms ◽  
Actin Concentration

We examined whether or not purified actin binds to the ends of thin filaments in rabbit skeletal myofibrils. Phase-contrast, fluorescence, and electron microscopic observations revealed that actin does not bind to the ends of thin filaments of intact myofibrils. However, in I-Z-I brushes prepared by dissolving thick filaments at high ionic strength, marked binding of actin to the free ends, i.e., the pointed ends, of thin filaments was observed when actin was added at an early phase of polymerization. As the polymerization of actin proceeded, the binding efficiency decreased. The critical actin concentration for this binding was higher than that for polymerization in solution. The binding of G-actin was not observed at low ionic strength. On the basis of these results, we suggest that a particular structure suppressing the binding of actin is present at the free ends of thin filaments in intact myofibrils and that a part of the end structure population is eliminated or modified at high ionic strength so that further binding of actin becomes possible. The myofibril and I-Z-I brush appear to be useful systems for studies aimed at elucidating the organizational mechanisms of actin filaments in vivo.

Conformational Transitions in Escherichia coli Ribosomal RNAs as Visualized by Dedicated STEM

1988 ◽  
Vol 46 ◽  
pp. 176-177
Author(s):  
J.S. Wall ◽  
V. Maridiyan ◽  
S. Tumminia ◽  
J. Hairifeld ◽  
M. Boublik
Keyword(s):  
Ionic Strength ◽  
Three Dimensional ◽  
Conformational Transitions ◽  
Dark Field ◽  
Dimensional Structure ◽  
Ribosomal Rnas ◽  
Field Mode ◽  
Freeze Dried ◽  
High Resolution Images ◽  
Low Ionic Strength

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).

An Evaluation of a Plasma Fractionation System

1960 ◽  
Vol 4 (01) ◽  
pp. 031-044
Author(s):  
George Y. Shinowara ◽  
E. Mary Ruth
Keyword(s):  
Biological Activity ◽  
Ionic Strength ◽  
Plasma Proteins ◽  
High Concentration ◽  
Significant Evidence ◽  
Native Proteins ◽  
Mobility Data ◽  
Fractionation Procedure ◽  
The Individual ◽  
Low Ionic Strength

SummaryFour primary fractions comprising at least 97 per cent of the plasma proteins have been critically appraised for evidence of denaturation arising from a low temperature—low ionic strength fractionation system. The results in addition to those referable to the recovery of mass and biological activity include the following: The high solubilities of these fractions at pH 7.3 and low ionic strengths; the compatibility of the electrophoretic and ultracentrifugal data of the individual fractions with those of the original plasma; and the recovery of hemoglobin, not hematin, in fraction III obtained from specimens contaminated with this pigment. However, the most significant evidence for minimum alterations of native proteins was that the S20, w and the electrophoretic mobility data on the physically recombined fractions were identical to those found on whole plasma.The fractionation procedure examined here quantitatively isolates fibrinogen, prothrombin and antithrombin in primary fractions. Results have been obtained demonstrating its significance in other biological systems. These include the following: The finding of 5 S20, w classes in the 4 primary fractions; the occurrence of more than 90 per cent of the plasma gamma globulins in fraction III; the 98 per cent pure albumin in fraction IV; and, finally, the high concentration of beta lipoproteins in fraction II.

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