Harvey M. Levy

Effect of temperature on the rate of hydrolysis of adenosine triphosphate and inosine triphosphate by myosin with and without modifiers. Evidence for a change in protein conformation

Abstract The temperature dependence of the myosin-catalyzed hydrolysis of inosine triphosphate gives a biphasic Arrhenius plot curving sharply near 16°C. In contrast, the Arrhenius plot with adenosine triphosphate as substrate is linear. When enzyme modifiers such as 2,4-dinitrophenol or p -chloromercuribenzoate are added to the system, the temperature dependence of ATP hydrolysis becomes very similar to that for ITP with native enzyme. Using 32 P-labeled ATP, it has been shown that ATP and ITP compete for the same site and that even in the presence of dinitrophenol, ATP is bound to the enzyme far more tightly than ITP. These data can be explained by assuming a temperature-induced change in the conformation of the enzyme-substrate complex. A consistent framework to explain the complex relationship between temperature, substrate, metal ion, and modifier can be obtained using this concept of a flexible active site.

Studies on the superprecipitation of actomyosin suspensions as measured by the change in turbidity I. Effects of adenosine triphosphate concentration and temperature

Abstract Superprecipitation of actomyosin induced by ATP or ITP was studied by measuring the increase in turbidity which occurs during the process. The maximum rate of superprecipitation was markedly slowed by lowering temperature. Below 20°, the apparent energy of activation of the process is greater than 50 000 cal. However, the apparent binding of ATP (ITP) was increased by lowering temperature, as was the efficiency (change in turbidity per unit of ATP hydrolyzed). Efficiency was also increased by increasing ATP concentration. It was inferred from these results that superprecipitation is not a simple stoichiometric function of nucleotide hydrolysis, but requires the concerted action of two or more molecules of ATP (ITP) operating simultaneously or sequentially at two or more sites in the protein complex.

Studies on the superprecipitation of actomyosin suspensions as measured by the change in turbidity II. The relationship to hydrolysis of adenosine and inosine triphosphate

Abstract Using a turbidity method, we have directly compared ATP- and ITP-induced superprecipitation with the hydrolysis of these nucleotides by actomyosin. Studies on the concentration dependence of the two processes indicate that superprecipitation requires the binding of nucleotide at two or more sites on the protein complex. The operation of ATP or ITP at the hydrolytic site may be one of these requirements, but it is not sufficient. The involvement of two different sites in superprecipitation was supported by the observation that ATP and ITP, together, can operate synergistically. When ITP modifies the protein, then ATP hydrolysis and ATP-induced superprecipitation appear to be stoichiometrically related.

Heat Inactivation of the Relaxing Site of Actomyosin: Prevention and Reversal with Dithiothreitol

Adenosine triphosphate and magnesium (MgATP) inhibit contraction by binding to a specific relaxing site on natural actomyosin gel. This inhibitory control site is distinct from the active sites where MgATP causes contraction.In high concentrations of MgATP, calcium triggers contraction by releasing the protein from substrate inhibition, allowing the contractile reactions to occur. Heating the protein for 5 minutes at 43�C selectively inactivates the relaxing site. After this treatment, actomyosin with MgATP contracts as well without calcium as with it. That this effect of heat is prevented and reversed by dithiothreitol (an agent that reduces disulfide bonds) indicates that the structure of the relaxing site depends on certain labile sulfhydryl groups, which may be those of tropomyosin. When these are oxidized to disulfide bonds, the site loses its activity; when the disulfide bonds are reduced, the site regains its activity.