At physiological pH, bovine testes calmodulin (t-CaM) upon excitation at 278 nm shows typical tyrosine fluorescence at 305 nm and a spectral band characteristic of emission from tyrosinate , at 330-350 nm. In addition, a new band at 312-320 nm appears upon excitation at 288 nm. The pH dependence of the excitation spectra demonstrates that the intense tyrosinate emission at 330-355 nm originates from direct excitation of ground-state tyrosinate . The tyrosinate emission shows complex pH dependence and reaches its highest intensities at pH 7.0 and 8.5, in both apo (Ca free) and holo (Ca saturated) t-CaM. The evidence suggests that the major contribution to the tyrosinate emission at 330-350 nm originates from Tyr-99. In holo t-CaM, the tyrosine emission at 305 nm is quenched at basic pH values and exhibits a sigmoidal pH titration curve with pK(app) 7.0. The tyrosine emission in apo t-CaM is weaker and is almost insensitive to changes in pH. The pH dependence of the emission at 316 nm is the same as the pH dependence of the tyrosine emission in both apo and holo t-CaM. The differences between the fluorescence of apo and holo t-CaM are attributed to a Ca2+-induced shift in the pKa of carboxylic side chains located in the immediate vicinity of the tyrosine residues. The enhancement of the fluorescence by Ca2+ is pH dependent and is maximal at pH 6.5. Above pH 8.0, there is almost no Ca2+ effect on the fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Large-scale growth of extreme halophilic bacteria from the Dead Sea and purification of malate dehydrogenase (and other proteins) in quantities of hundreds of milligrams makes possible a detailed study of the adaptation to high salt. Halophilic malate dehydrogenase is stable at 20 "C in NaCl solutions between 2.5 -5 M. Below 2.5 M NaCl time-dependent inactivation, paralleled by structural changes, sets in. Within the time scale of the sedimentation, diffusion and circular dichroism experiments discussed here, it was possible to analyze data corresponding to the active halophilic malate dehydrogenase between 1 M and 5 M NaCl. The striking observation was that rather minor conformational changes were observed over the whole range, yet the special properties of the halophilic enzyme seem to be related to its capacity of associating with unusually large amounts of water and of salts, quite distinct from non-halophilic counterparts. These special properties seem to be related to the intact structure of the protein. Some parallel properties of halophilic glutamate dehydrogenase are also discussed.Extreme halophilic bacteria grow best in 20 -30 % NaCl 11 -31. These obligate halophiles are red-pigmented organisms which occur naturally inwaters of high salinity such as theDead Sea, for instance. The extreme halophilic bacteria adapt themIselves to life at high salinity by having internal concentrations (of salt that equal or exceed even the high concentrations in which they grow. They lyse at low salt concentrations. For recent reviews of the unusual properties of the halobacteria, some of which bear closer resemblance to eukaryotes rather than to prokaryotes, we refer to Kushner [4] and to Bailey ,md Morton [5]. Striking compositional properties, which have also been extensively studied in our laboratory are that halophilic proteins possess a molar excess of 16-18 % negatively charged amino acids [6 -81, whereas in non-halophilic proteins the excess is only 7-9%. The content of hydrophobic amino acids is significantly low, yet the content of borderline hydrophobic amino acids (e.g. alanine and threonine) is higher than in non-halophilic counterparts.Halophilic proteins carry out identical functions to their nonhalophilic counterparts, yet in a medium in which one parameter (salt concentration) differs greatly. The halophilic character is an intrinsic property of the proteins and does not depend on additional factors [7,9,10]. The proteins are stabilized by multimolar salt concentrations and the stabilization effect of different salts follows the Hofmeister lyotropic series. At high salt concentrations (3-5 M NaCl or KC1) enzymatic activity of halophilic proteins is observed whereas non-halophilic enzymes tend to be inactivated and to precipitate [11,12]. At low salt concentrations (< 2 M NaCI) halophilic enzymes denature. Denaturation involves unfolding, dissociation and inactivation, processes which may

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The stability of halophilic malate dehydrogenase increases with increasing salt concentration and with decrease in temperature. Stabilization by various salts, at high salt concentrations, follows the Hofmeister series. The enzyme inactivation rates closely match dissociation of the dimeric enzymes into monomeric subunits and unfolding of the polypeptide chains, as followed by velocity sedimentation, light. scattering and circular dichroism measurements. The a-helix content goes to zero upon denaturation. Unusual water and salt binding properties of the native enzyme (cf. preceding paper, in this journal) are believed to be largely lost upon enzyme dissociation and unfolding. These properties thus seem to be associated with the intact structure of the enzyme.In our previous work [l] we have shown that, although malate dehydrogenase (h) becomes unstable at NaCl concentrations lower than about 2.5 M, enzyme activity and structure is maintained for short times at NaCl concentrations as low as 1 M. It was therefore possible to provide a description of the properties of the active enzyme over the NaCl concentration range 1-5 M, by performing a series of diffusion, sedimentation and circular dichroism experiments. These essentially could be considered as being performed at 'zero time', before any significant changes in enzymatic activity or conformation arises.The recent striking observation resulting from the above work is the fact that malate dehydrogenase (h), as well as glutamate dehydrogenase (h), complex in striking fashion with large amounts of both water and salt. Concepts relating to 'binding' of low-molecular-weight components to large particles in solution are largely operational and different methods may yield different results. We have used an approach based on volume exclusion, as manifested by density increments at specified compositions and thermodynamic potentials of water and salt [2,3]. This concept has previously yielded reasonable values for hydration and interaction with salts of DNA and of non-halophilic proteins [l]. The behavior of the halophilic enzymes is distinctly different from that of the non-halophilic macromolecules examined. We shall present evidence in the present work to suggest that these unusual hydration and salt binding characteristics are largely lost when malate dehydrogenase (h) dissociates and unfolds. An indication is thus provided for a mechanism of adaptation to extreme conditions of high salt concentration which is dependent on the intactness of the active enzyme structure.Ahbreviaiions. Malate dehydrogenase (h), halophilic malate dchydrogenase; glutamate dehydrogenase (h), halophilic glutamate dehydrogenase.Enzymes. Malate dehydrogenase (EC 1.1.1.37); glutamate dehydrogenase [NAD(P)+] (EC 1.4.1.3).In an earlier investigation Mevarech and Neumann [4] described the reversible inactivation of malate dehydrogenase (h) at low NaCl concentration. They suggested that inactivation of the dimeric enzyme involved dissociation into two subunits, since the kinetics of reactivation...

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