Properties of a Purified Halophilic Malic Dehydrogenase

Abstract

, AND H. ORIN HALVORSON. Properties of a purified halophilic malic dehydrogenase. J. Bacteriol. 90:316-326. 1965.-The malic dehydrogenase (MDH) from Halobacterium salinarium required high concentrations of monovalent ions for stability and activity. Studies of inactivation rates at different salt concentrations suggested that approximately 25% NaCl (w/v) is required to stabilize MDH. From 50 to 100% reactivation, depending on the salt concentration present during inactivation, could occur in 2.5 to 5 M NaCl or KCI. The optimal salt concentration for activity of MDH was a function of the pH, and ranged from 1 to 3 M NaCl or KCl. The effect of salt concentration on the pH-activity curves occurred chiefly below pH 7.0. Inactivation of MDH with heat or thiol reagents showed that the enzyme was more labile in the state induced by absence of salt. The activation of MDH by salts was attributed to a decreased rate of dissociation of MDH and reduced nicotinamide adenine dinucleotide (NADH2). The inactivation of the enzyme in the absence of salt could be largely prevented by the presence of NADH2. The S20,. of MDH decreased threefold at low salt concentrations. The enzyme was assumed to be in its native compact configuration only in the presence of a high concentration of salt.

Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum polynucleotide phosphorylase

“…1) than for maximum activity (Fig. 2) and is similar in this respect to H. salinarium malate dehydrogenase (Holmes & Halvorson, 1965b). This difference may be due to the presence of substrate in the assay medium, since it is well known (Dixon & Webb, 1964) that an enzyme can be protected from denaturation by its substrate.…”

Salt-dependent properties of proteins from extremely halophilic bacteria.

“…It may be assumed that the polypeptide chain in these enzymes is capable of a reversible, tighter folding at the higher salt concentrations, which increases the activation energy of unfolding. Unlike the case of menadione reductase, lowering the NaCl concentration did not result in inactive states for these enzymes, but under these conditions the structures were unstable, and both isocitrate dehydrogenase (62) and malic dehydrogenase (58,60) were inactivated in a time-dependent manner. Both of these enzymes, as well as several others (58), could be reactivated by dialysis against high concentrations of NaCl, although not by directly adding salt.…”

“…Finally, some enzymes from halophilic bacteria do not require salt or are inhibited by low concentrations, such as the fatty acid synthetase (100) and RNA-dependent RNA polymerase 87 Although the salt requirement of enzyme activity is variable in halophilic systems, all enzymes investigated have shown a timedependent inactivation at lower salt concentrations and especially in the absence of salt. In many cases the inactivation followed first-order kinetics (3,5,60,75,76,84,96). Lanyi found (76) that apparent first-order inactivation rate constants could be calculated at various salt concentrations for menadione reductase, an NADH dehydrogenase linked to the membranebound respiratory chain (28,29,74,75) of H. cutirubrum.…”

“…If the effect of salts on the activity of extremely halophilic enzymes is interpreted in terms of binding sites, as it has been done by several authors (3,5,60), the kinetics yield dissociation constants in the neighborhood of molar concentrations of salt and up to 4 to 5 binding sites. Dissociation constants of ion pairs, such as K+Cl-, formed by random encounter of a monovalent ion with another, are also in the order of 1 M (45,68).…”

Methanogens and the diversity of archaebacteria.

“…The malate dehydrogenase from several halobacteria and a methanogen have been characterized (174,175,319,320,439,452). The molecular weights and subunit compositions of the archaebacterial enzymes are very similar to the eubacterial and eucaryotic enzymes.…”