Tsie Joursal of Biological Ceiewstry

Halobacterium cutirubrum extracts exhibit no menadione reductase activity in the absence of salt; activity increases with salt concentration to a maximum at 2 M or higher concentrations of sodium chloride. The enzyme activity lost at low salt concentrations can be restored by adding 2 M NaCl. In addition to this reversible loss of activity, the enzyme also exhibits a time-dependent irreversible inactivation at salt concentrations below 2 M. The effects of salt on the initial enzyme activity and on the irreversible inactivation have a reciprocal relationship to one another. A mechanism for the salt effect is proposed, on the basis of kinetic data, whereby the active, stable form of the enzyme is in a salt-dependent equilibrium with an inactive, unstable form. The effect of NaCl, according to this model, is not to prevent the irreversible denaturation of the enzyme but rather to shift the equilibrium in favor of the active, stable form. The pH dependence of the inactivation rate constant and the reversibility of the pH effect indicate that there is an acid-base equilibrium, with pK = 9.3, between a less and a more stable intermediate form of the enzyme. Low concentrations of MgC12, CaCl*, and some polyamines also have activating and protecting effects on menadione reductase. The mode of action of these substances is shown to be unlike that of NaCl but to consist in the alteration of the intermediate form of the enzyme in such a way as to make it more stable and partially active.