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The enzymatic activity and structural stability of the extremely halophilic enzyme malate dehydrogenase (EC 1 . I . I .37) isolated from Dead Sea halobacteria depend in a different way on the concentration of inorganic salts. At low salt concentration (<2.0 M NaCI) the enzyme is inactivated in a first-order reaction. When the salt concentration is increased the inactivated enzyme is reactivated in a second-order reaction. The inactivation at low salt concentration is ascribed to dissociation of the dimeric enzyme to inactive subunits and the reactivation at high salt concentration is determined by the rate of reassociation of the subunits to dimers. At low salt concentrations another process participates which causes an apparently irreversible (first order) denaturation. The rate E x t r e m e l y halophilic bacteria are organisms which grow in saturated NaCl solutions and thus constitute a fascinating example of biological adaptation. It was reported that the internal salt concentration in bacteria of the genus Halobacterium is about 4 M KCI and 1.5 M NaCl (Ginzburg et al., 1971; Christian and Waltho, 1962). Besides the tolerance of high salt levels, halobacteria vary the intracellular salt content in response to changes in the environmental conditions. The halobacteria of the Dead Sea change the NaCl content between 3 M during the growing phase and 0.5 M under starvation conditions (Ginzburg et al., 1971). Obviously, the biochemical machinery of these bacteria including the enzymes is adapted not only to extremely high salt concentration but also to appreciable changes in the internal salt content. As a correlate, the catalytic activity of the isolated enzymes depends strongly on the salt concentration (Baxter and Gibbons, 1956; Holmes and Halvorson, 1965). At salt concentrations lower than 2.0 M most of the halophilic enzymes studied are not stable and spontaneously inactivate (Lanyi, 1974). Whereas some quantitative studies have been performed on the inactivation of halophilic enzymes (Hochstein and Dalton, 1973; Holmes and Halvorson, 1969, reactivation studies have remained on a qualitative level (Hubbard and Miller, 1970; Keradjopoulos and Wulff, 1974). One of the reasons for the small number of detailed biochemical and biophysical investigations on halophilic enzymes is encountered in the difficulty of purifying these enzymes. Recently, an efficient procedure has been developed for the purification to homogeneity of considerable amounts of the halophilic enzyme malate dehydrogenase of Dead Sea halobacteria (Mevarech et al., 1977). Initial physicochemical studies showed that the molecular weight of the active enzyme + From the Polymer Department, Weizmann Institute of Science, Rehovot, Israel (M.M.), and Max-Planck-lnstitut fur Biochemie, DX033 Martinsried bei Munchen, West Germany (E.N.). Receioed Jonuary 20, 1977. This work was supported by a grant from the Stiftung Vol kswagenwcrk. constants of the inactivation and reactivation processes depend i n a characteristic manner on salt concentration and salt type (NaCI, KCI, NH4CI, and (NH4)$304), and on temperature. The thermodynamic analysis suggests that a t concentrations below 0.1 5 M the salt mainly screens the fixed charges of the subunits, whereas at concentrations higher than 0.8 M the salt dominantly stabilizes hydrophobic interactions between the enzyme subunits. I f the NaCI concentration is varied in a cyclic manner, the enzymatic activity changes along a hysteresis loop. This observation indicates relatively long-lived metastable states, reflected in the extremely small values of the rate constants of both the inactivation and reactivation process in the range of NaCl concentration where hysteresis exists. is 84 000 and that the enzyme is composed of two subunits. The amino acid composition indicated a very high negative net charge. In the present study a method is proposed to separate the effect of salt concentration and salt type on the catalytic activity from that on the structural stability of the enzyme. As a peculiar result, we note that in a certain concentration range the enzymatic activity displays relatively long-lived metastable states that result in a hysteresis loop upon cyclic variation of the salt concentration. Materials and Methods Chemicals. For the enzyme assay, oxaloacetic acid and NADH' (Sigma) were used. All salts employed were of analytical grade. Solutions. All solutions contain, in addition to the desired amounts of salt, 10 mM sodium phosphate. The standard buffer solution contains 4.26 M NaCI, 0.01 M sodium phosphate. The pH of all solutions was adjusted to 7.3 (Metrohm pH meter, Model E-300B, Metrohm EA 120U combined glass electrode). All solutions were filtered through 0.45-pm Millipore f i l ters. Enzyme Preparation and Assay. The purification of the halophilic enzyme malate dehydrogenase of Dead Sea halobacteria has been previously described (Mevarech et al., 1977). Enzymatic activity was measured in 1 mL of the standard buffer containing, additionally, 0.1 mM NADH and 0.25 mM oxaloacetate. The oxidation of NADH was followed at 340 nm with a Zeiss PMQ 11 spectrophotometer equipped wi th a linear-to-log converter and a recorder. Enzymatic activity is expressed in international units (IU). Fluorescence Intensity Measurements. The changes in the intrinsic fluorescence of the enzyme were determined by using an Aminco Bowman spectrofluorometer. The excitation and I Abbreviation used: N A D H , reduced nicotinamide adenine dinucleotide. 3786 B I O C H E M I S T R Y , V O L . 1 6 , N O . 1 7 , 1 9 7 7 E F F E C T O F S A L T O N T H E H A L O P H I L I C M A L A T E D E H Y D R O G E N A S E

Malate dehydrogenase isolated from extremely halophilic bacteria of the Dead Sea. 2. Effect of salt on the catalytic activity and structure