Relationship between the ATPase Activity and the ATP-Induced Fluorescence Enhancement of SH-Modified Heavy Meromyosin during Its Fractional Inactivation by Vanadate plus ADP: Evidence for Heterogeneity in the Active Sites1

Kawamura, Takanori; Higuchi, Wakako; Emoto, Y.; Tawada, Katsuhisa

doi:10.1093/oxfordjournals.jbchem.a135215

Cited by 2 publications

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“…[ ,] )4 + " 1 + *4[ ,] °} If K4 is known for two pH values it is possible, given Á)a;P and ATa.v, to calculate values for K¡ and A)a;PV of eq 14. The various equilibria of eq 14 are described by eq 18-21 and the conservation equations, eq [22][23][24]. Equation 16 in combination with these…”

Section: Resultsmentioning

confidence: 99%

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Vanadium(V) oxyanions: the interaction of vanadate with pyrophosphate, phosphate, and arsenate

Gresser¹,

Tracey²,

Parkinson³

1986

J. Am. Chem. Soc.

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Section: Resultsmentioning

confidence: 99%

“…The formation of mixed phosphate/vanadate anhydrides is also relevant to the inhibition of myosin ATPase by adenosine diphosphate plus vanadate. [20][21][22]…”

Section: [Hasv073"][h30+]mentioning

confidence: 99%

Vanadium(V) oxyanions: the interaction of vanadate with pyrophosphate, phosphate, and arsenate

Gresser¹,

Tracey²,

Parkinson³

1986

J. Am. Chem. Soc.

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Active-site titration of enzymes at high concentration. Application to myosin ATPase

1986

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The number of active sites of soluble and filamentous myosin and of its subfragments, heavy meromyosin and subfragment-1, has been determined. The titration involves steady-state kinetic measurements at a high enzyme concentration and varying substrate concentrations (or vice versa), in the presence of a substrateregenerating system. Some practical and theoretical conditions for its execution are given, and, in particular, the effect of a possible heterogeneity of the active sites on the titration curves is analysed. Under the experimental conditions of the study, the number of active sites is close to that of myosin heads, and the heads seem to be functionally identical ; the catalytic constants k,,, and K, characterizing each active site are similar within some limits (1 -2 for the ratio of k,,, values; 1 -5 for that of K , values).The determination of the absolute concentration of enzymes requires the titration of their active sites [l]. Ths is usually possible by combined steady-and presteady-state kinetic measurements, if an enzyme-bound intermediate accumulates during the reaction of the enzyme with its substrate and if a reaction product is concomitantly released. In the case where no intermediate accumulates, this active-site titration is not applicable.An alternative method involving steady-state kinetic measurements may be used if the apparent affinity of the enzyme for its substrate is high. In this case, the rate of the reaction at high enzyme concentration is maximum when the substrate concentration becomes equal to that of the concentration of enzyme active sites. Although this possibility has been known for a long time [2], it seems to have been hardly used, to our knowledge, except for a recent application to the active-site titration of soluble myosin and of its subfragments [3-61. This last study was simplified particularly by the regeneration of the substrate during the course of experiments due to an efficient substrate-regenerating system. In the present paper, we develop some theoretical and practical conditions for this titration as well as some pitfalls which may arise when it is carried out.Muscle myosins are double molecules made up of one pair of heavy chains and two pairs of light chains, the so-called alkali and regulatory subunits. In the case of fast-twitch muscle myosins, there are two different alkali subunits, the A1 and A2 chains, which have however no distinct effect on of two different heavy chains in an approximate ratio of 60:40. At the present time, the number and the identity of active sites of the two-headed myosin are still under debate [3,9 -121 and the question of the functional homogeneity or heterogeneity of these sites is not yet resolved. These points will be discussed from the results of active-site titrations of myosin and of its subfragments. THEORYSeveral authors have discussed steady-state enzyme kinetics with high concentrations of substrate and enzyme [2, 13 -161. For the following general enzyme reaction mechanism : E + S e E S .+ E + P , the initial rate ...

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Relationship between the ATPase Activity and the ATP-Induced Fluorescence Enhancement of SH-Modified Heavy Meromyosin during Its Fractional Inactivation by Vanadate plus ADP: Evidence for Heterogeneity in the Active Sites1

Cited by 2 publications

References 0 publications

Vanadium(V) oxyanions: the interaction of vanadate with pyrophosphate, phosphate, and arsenate

Vanadium(V) oxyanions: the interaction of vanadate with pyrophosphate, phosphate, and arsenate

Active-site titration of enzymes at high concentration. Application to myosin ATPase

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