Tyrosine and tyrosinate fluorescence of bovine testes calmodulin: calcium and pH dependence.

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)

Amino acid sequence of southern bean mosaic virus coat protein and its relation to the three-dimensional structure of the virus.

The amino acid sequence of the coat protein of southern bean mosaic virus has been determined. Chemical techniques were supplemented by a 2.8-A-resolution electron-density map which helped in assigning absolute positions for each peptide. Four amino acids and the placement of one heptapeptide near the amino terminus of the protein were interpolated by comparison with the partially known southern bean mosaic virus RNA sequence. The subunit is heavily lined with basic residues on the side facing the RNA. Subunit-subunit interactions are hydrophobic and ionic. The calcium site on the quasi-threefold axis has three glutamic residues as ligands. There is a disulfide bond, between Cys 168 and 176, within a sequence which is inserted in southern bean mosaic virus relative to the tomato bushy stunt virus structure. This portion is a helix nestling between the "beta-barrel" subunit structure and the RNA interior.

Structure and activity of malate dehydrogenase from the extreme halophilic bacteria of the Dead Sea. 1. Conformation and interaction with water and salt between 5 M and 1 M NaCl concentration.

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 degrees 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 conformation 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.

Structure and activity of malate dehydrogenase from the extreme halophilic bacteria of the Dead Sea. 2. Inactivation, dissociation and unfolding at NaCl concentrations below 2 M. Salt, salt concentration and temperature dependence of enzyme stability.

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 alpha-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. The properties thus seem to be associated with the intact structure of the enzyme.

[Diazonium derivatives of ADP for the identification of essential amino acid side chains in the active site of dehydrogenases]. / Diazoniumderivate des ADP zur Identifizierung essentieller Aminosäureseitenreste im aktiven Zentrum von Dehydrogenasen.

For affinity labeling of NAD-dependent dehydrogenases, dinucleotide analogs were prepared by connecting nitrobenzene or nitrobenzimidazole systems with adenosine diphosphate. The distance between the two parts of the molecule was varied by insertion of propyl, butyl and pentyl chains or ribose. Reduction of the nitro group with hydrazine/Raney nickel yielded the corresponding amino derivatives which were converted to the diazonium salts by nitrous acid. Due to specific linking of ADP moiety to dehydrogenases, the reactive diazonium group combines with nucleophilic amino acid side chains in the active centre of dehydrogenases, the enzymatic activity of which was protected by NAD and NADH. Fluorescence titration experiments proved a linear correlation between incorporation of nucleotide anhydride, residual activity and remaining NADH capacity of the enzymes. The different modified amino acids showed characteristic absorption bands which allowed the identification of the reacting group as well as the estimation of the stoichiometry of the reaction. The latter could be estimated by titration of the enzyme with the diazonium salt. Only in a few cases was the spectrophotometric identification of the modified amino acid side chain uncertain. This fact required enzymatic degradation of the protein followed by electrophoresis and amino acid analysis.