Mechanism of action of troponin . tropomyosin. Inhibition of actomyosin ATPase activity without inhibition of myosin binding to actin - PubMed (original) (raw)

. 1981 Jan 25;256(2):575-8.

Mechanism of action of troponin . tropomyosin. Inhibition of actomyosin ATPase activity without inhibition of myosin binding to actin

J M Chalovich et al. J Biol Chem. 1981.

Abstract

The regulation of vertebrate skeletal muscle contraction by the troponin . tropomyosin complex is generally thought to be the result of tropomyosin physically blocking the myosin binding site of actin in the absence of Ca2+. This mechanism was tested during steady state ATP hydrolysis by comparing the degree of association of myosin subfragment 1 (S-1) with the actin . troponin . tropomyosin complex in the absence and presence of Ca2+. Binding in the presence of ATP was determined by stopped flow absorbance measurements at 25 degrees C. Although the steady state ATPase rate was reduced 96% in the absence of Ca2+, the association constant of S-1 with regulated actin was virtually the same in the absence of Ca2+ (1.3 X 10(4) M-1) as in the presence of Ca2+ (2.3 X 10(4) M-1). The association constant of S-1 to regulated actin in the presence of Ca2+ was similar to the association constant of S-1 to unregulated actin. These results suggest that the troponin . tropomyosin complex does not inhibit the actin-activated ATPase activity by preventing the binding of S-1 . ATP or S-1 . ADP . Pi to actin; rather, it may act by blocking the release of Pi from the acto-S-1 . ADP . Pi complex.

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Figures

Fig. 1

Fig. 1. Final absorbance of the complex formed upon binding of S-1 to actin or regulated actin as a function of S-1 concentration

The final absorbance was measured after completion of ATP hydrolysis. Conditions: 78 μ

m

actin, 17 μ

m

native tropomyosin (where applicable), 1.0 m

m

ATP, 3.0 m

m

MgCl2, 1 m

m

EGTA, 10 m

m

imidazole, pH 7.0, 25°C. •, ▪, stopped flow measurement; ○, standard spectrophotometer measurement.

Fig. 2

Fig. 2. Double reciprocal plots of the fraction of S-1 bound, in the presence of ATP, as a function of free actin concentration

The conditions are the same as in Fig. 1 with 20 μ

m

S-1, except where noted. A, binding in the presence of calcium. EGTA was replaced with 0.5 m

m

CaCl. Binding to regulated (closed symbols) and unregulated actin (open symbols) are shown. B, binding in the absence of calcium. S-1 concentration was varied from 10 μ

m

(▴) to 40 μ

m

(▵). In both A and B, different symbols represent different protein preparations. The solid lines are least squares fits to the data (see “Materials and Methods”).

Fig. 3

Fig. 3. Time course of the fraction of S-1 bound to actin or regulated actin during ATP hydrolysis

Conditions are the same as in Fig. 1 with 20 μ

m

S-1 and free actin concentrations of 90 μ

m

(regulated + Ca2+), 100 μ

m

(unregulated), and 130 μ

m

(regulated + EGTA).

Fig. 4

Fig. 4. Time course of the transmitted light intensity after mixing regulated actin with either S-1 (upper curve) or buffer (lower curve) in the presence of ATP and EGTA

The lower curve has been moved down about 1 V for comparison. Conditions are the same as in Fig. 1 with the following final protein concentrations: S-1, 20 μ

m

; actin, 60 μ

m

; native tropomyosin, 13 μ

m

. 35% of the S-1 was bound to actin during steady state ATP hydrolysis in this experiment.

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References

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