A low viscosity G-actin preparation

Grant, Robert Jason; Cohen, Lawrence B.; Clark, Eloise E.; Hayashi, Teru

doi:10.1016/0006-291x(64)90032-4

Cited by 15 publications

(3 citation statements)

References 8 publications

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“…If the model of actin denaturation described in eq 1 is correct then the reduction of the rate of actin inactivation could be ascribed to one of three modes of action: (A) the cation could induce aggregation of G-actin; (B) the cation could shift the equilibrium to the left, i.e., increase the affinity of G-actin for ADP; and (C) the cation could increase the stability of G-actin containing no nucleotide and thereby decrease the a of eq 3. Grant et al (1964) found that ADP-G-actin does not aggregate when low concentrations of MgCl2 are added at 0°, and no significant change in viscosity was demonstrated in this study upon addition of 0. to ADP-G-actin solutions. Our data indicate that G-actin had the same affinity for ADP in the presence or absence of added MgCl2 (Table I).…”

Section: Discussioncontrasting

confidence: 52%

Binding of adenosine diphosphate to G-actin

West¹

1970

Biochemistry

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The binding of adenosine diphosphate (ADP) by G-actin was studied under various conditions. Binding constants were determined by measuring either the rates of denaturation of G-actin solutions in the presence of varying nucleotide concentrations, or the concentration of free nucleotide in G-actin solutions at equilibrium. The binding constant of G-actin for ADP at 0" was 2-5 X lo6 M -~; a t 25" it was 4 X l o 4 M -~, whereas at 0" ATP bound to G-actin with an affinity constant of 2.4 X IO7 M -~, Changing the I n view of the possible role of actin-bound nucleotide in energy-transfer processes leading to muscle contraction, studies of the properties of actin-bound nucleotide are important. Several recent publications have presented conflicting evidence on the extent to which ADP-G-actin dissociates in solution to form ADP and native G-actin. Higashi and Oosawa (1965) derived a binding constant of G-actin for ADP of 1.3 X lo4 M -~ from studies of changes in absorbance at 230 m p after addition of ADP to ADP-G-actin solutions. Thus, according to these data, at 1.5 mg/ml of ADP-G-actin in the absence of added ADP only 20% of the nucleotide was actually bound to the protein. Seidel et al. (1967) determined the amount of native actin containing no bound nucleotide and estimated that the binding constant of G-actin for ADP a t 4" was 4 X IO6 M -~. I n contrast, Hayashi and Rosenbluth (1964) inferred that less than 5 % of the total nucleotide present was free after studies of removal of free nucleotide by Dowex 1 from 1 mgjml of ADP-G-actin solutions at 0". West et al. (1967a) studied the kinetics of ADP hydrolysis by apyrase and found that only 7 of the nucleotide was dissociated in 1.5 mg/ml of ADP-G-actin solutions at 0". If the free nucleotide in these studies was equivalent to that found at equilibrium these results imply a binding constant greater than 7 X 106 M -~, a value 500-fold greater than that calculated by Higashi and Oosawa (1965) and 20-fold greater than that determined by Seidel et al. BBCB and a Veterans Administration Hospital Clinical Investigatorship. actin-bound cation from magnesium to calcium did not appear to alter ADP binding. Free magnesium at a concentration of 0.2 mM reduced the rate of G-actin denaturation by onethird but there was no change in the affinity of G-actin for ADP.It was concluded that the mechanism by which free magnesium stabilizes G-actin depends on a prolonged lifetime of nucleotide-free G-actin molecules without change in the properties of G-actin containing bound nucleotide. excess of magnesium at 0" retarded thermal denaturation of ADP-G-actin. It seemed possible that such a stabilizing effect of free MgCI2 could have been mediated by an increased affinity of G-actin for ADP.Our results show tighter ADP binding to G-actin at 0" than found in previous studies. A marked temperature dependence for ADP binding to G-actin was found. Nucleotide binding was not altered if calcium was substituted for actin-bound magnesium, nor did addition of free magnesium alter nucleotide b...

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Section: Discussioncontrasting

confidence: 52%

Binding of adenosine diphosphate to G-actin

West¹

1970

Biochemistry

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“…Pro teinases have been isolated from muscle of various species such as cattle (Balls, 1938), rat (Koszalka and Miller,i960), and Albacore (Groninger, 1964). The activities of several of these enzymes have been determined on various natural and synthetic substrates (Landmann, 1963;Bodwell and Pearson, 1964 (Mihashi, 1964;Grant, 1964). While there is a possibility that this subfraction is an Integral part of the molecule, Takahashi et.…”

Section: A Biochemistry Of Rigor Mortismentioning

confidence: 99%

Post‐Rigor Changes in Selected Physicochemical Properties of Myosin B Fraction of Turkey Muscle

Scharpf

Marion

Forsythe

1966

Journal of Food Science

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SUMMARY Changes in selected physicochemical properties of the myosin B fraction of turkey muscle were followed during a 44‐hr post‐rigor aging period. Sedimentation patterns of myosin B fractions prepared 48 hr after death indicated a decrease in a high‐molecular‐weight component thought to he myosin B gel. It is concluded that myosin B fractions lose gel‐forming ability with resolution of rigor. Density‐gradient centrifugal patterns showed small increases in slowly sedimenting components of myosin B fractions with aging. These components were. high in ATP‐ase activity in the presence of calcium ions and showed no reduction of viscosity with addition of ATP. Some evidence favors the concept of dissociation of myosin B into myosin A and actin with resolution of rigor. This dissociation, however, may also represent disruption of myosin R gel formed during and after extraction from muscle.

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“…1963). More recent values range from 56,000 to 63,000 in which the Archibald technique was used (Mihashi, 1964;Grant, 1964).…”

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confidence: 99%

Post-rigor changes in selected physicochemical properties of myosin B fraction of turkey muscle

Scharpf

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Density gradient centrifugation 9. Viscosity measurements 52 10. Electrophoresis 54 11. Ultraviolet spectra P. Experimental Design 55 IV. EXPERIMENTAL RESULTS A. Myosin 3 Adenosine Triphosphatase Activity 57 B. Ion Exchange Chromatography of Myosin B Fraction C. Gel Filtration of Myosin B Fraction 64 D. Ultracentrifugal Analyses of Myosin B Fraction E. Centrifugal Analyses of Myosin 3 Gel F. Density Gradient Centrifugation of Myosin B Fraction 88 G. Electrophoresis of Myosin B Fraction 92 V. DISCUSSION A. Adenosine Triphosphatase Activity of Myosin 3 Fraction B. Gel Filtration of Myosin B Fraction 93 C. Ultracentrifugal Analyses of Myosin B Fraction D. Centrifugal Analyses of Myosin B Gel E. Density Gradient Centrifugation of Myosin B Fraction 108 F. Electrophoresis of Myosin B Fraction

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A low viscosity G-actin preparation

Cited by 15 publications

References 8 publications

Binding of adenosine diphosphate to G-actin

Binding of adenosine diphosphate to G-actin

Post‐Rigor Changes in Selected Physicochemical Properties of Myosin B Fraction of Turkey Muscle

Post-rigor changes in selected physicochemical properties of myosin B fraction of turkey muscle

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