The conformation of calmodulin: a substantial environmentally sensitive helical transition in Ca4-calmodulin with potential mechanistic function

The conformation of Ca4-calmodulin in solution, as assessed by far-UV peptide circular dichroism, contains significantly less alpha-helix than the proposed X-ray crystal structure. We now show that Ca4-calmodulin adopts significant additional helical structure in solution in the presence of a helicogenic solvent (50%, v/v, aqueous 2,2,2-trifluoroethanol or 50%, v/v, methylpentane-5,5-diol). We suggest that the long continuous helix (residues 66-92 of the crystal structure) is not necessarily a normal feature of the calmodulin structure in solution, and may be due in part to the conditions of crystallisation. This result is supported by time-resolved tyrosine fluorescence anisotropy studies indicating that Ca4-calmodulin in solution is an essentially compact globular structure which undergoes isotropic rotational motion. We conclude that, under appropriate ionic and apolar environmental conditions, Ca4-calmodulin undergoes a substantial helical transition, which may involve residues in the central region of the molecule. Such a transition could have an important function in determining specificity and affinity in interactions of calmodulin with different target sequences of Ca2+-dependent regulatory enzymes.     

Hydrolysis of dansyl-peptide substrates by leucine aminopeptidase: origin of dansyl fluorescence changes during hydrolysis

The origin of the fluorescence changes observed in stopped-flow experiments of the hydrolysis of three 5-(dimethylamino)naphthalene-1-sulfonyl-(dansyl) peptide substrates by porcine kidney cytosol leucine aminopeptidase has been investigated. The substrates used all have the potential to accept energy from aromatic residues of the enzyme via resonance energy transfer when they are bound as enzyme-substrate complexes, indicating that fluorescence changes due to the buildup and decay of such intermediates are possible. However, the fluorescence of these substrates differs from that of the products, and direct excitation of their dansyl groups during hydrolysis can also be responsible for the observed fluorescence changes due to changes in the concentrations of free substrate and product. The dansyl fluorescence changes observed with excitation wavelengths near 280 nm are not accompanied by quenching of the enzyme fluorescence, as would be expected if there were enzyme-to-substrate energy transfer. The magnitude of the maximal fluorescence change at a fixed concentration of substrate is also independent of the enzyme concentration. Furthermore, the excitation profile for the fluorescence changes shows that they arise from direct excitation of the dansyl group. Thus, there is no energy transfer in these reactions, and the fluorescence changes observed arise from direct excitation of the dansyl group and reflect the instantaneous concentration of substrate. This behavior contrasts sharply with that for the reaction of carboxypeptidase A with dansyl-Gly-Tyr, which has been studied as a positive control for an energy-transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Phosphate on the Corrosion of Carbon Steel and on the Composition of Corrosion Products in Two-Stage Continuous Cultures of Desulfovibrio desulfuricans

A field isolate of Desulfovibrio desulfuricans was grown in defined medium in a two-stage continuous culture apparatus with different concentrations of phosphate in the feed medium. The first state (V1) was operated as a conventional chemostat (D = 0.045 h⁻¹) that was limited in energy source (lactate) or phosphate. The second stage (V2) received effluent from V1 but no additional nutrients, and contained a healthy population of transiently starved or resting cells. An increase in the concentration of phosphate in the medium fed to V1 resulted in increased corrosion rates of carbon steel in both V1 and V2. Despite the more rapid corrosion observed in growing cultures relative to that in resting cultures, corrosion products that were isolated under strictly anaerobic conditions from the two culture modes had similar bulk compositions which varied with the phosphate content of the medium. Crystalline mackinawite (Fe₉S₈), vivianite [Fe₃(PO₄)₂ · 8H₂O], and goethite [FeO(OH)] were detected in amounts which varied with the culture conditions. Chemical analyses indicated that the S in the corrosion product was almost exclusively in the form of sulfides, while the P was present both as phosphate and as unidentified components, possibly reduced P species. Some differential localization of S and P was observed in intact corrosion products. Cells from lactate-limited, but not from phosphate-limited, cultures contained intracellular granules that were enriched in P and Fe. The results are discussed in terms of several proposed mechanisms of microbiologically influenced corrosion.     

The destruction of spores of Bacillus subtilis by the combined effects of hydrogen peroxide and ultraviolet light

Ultraviolet light irradiation of bacterial spores in the presence of hydrogen peroxide has been shown to produce synergistic kills when compared with ultraviolet light (u.v.) and hydrogen peroxide used sequentially. This use in combination has been patented for the commercial sterilization of packaging before filling with UHT-processed products. Previous results have shown that lamps producing u.v. light with a maximum output at about 254 nm were extremely effective. Results obtained using a Synchrotron radiation source to produce a narrow band of irradiation now shows that the greatest kill of spores of Bacillus subtilis in the presence of hydrogen peroxide is obtained with radiation at ˜270 nm. Such results suggest that the action of the u.v. light is not directly on the spore DNA but may be related to the production of free hydroxyl radicals from hydrogen peroxide.     

The effect of sodium chlorate and nitrapyrin on the nitrification mediated nitrosation process in soils

Summary The influence of total nitrification to nitrate or partial nitrification to nitrite on the soil organic nitrogen status was examined. NH ₄ ⁺ –¹⁵N was added to the soil in the absence and the presence of NaClO₃, respectively nitrapyrin. The first chemical inhibits only nitrate formation, the second inhibits total nitrification. The accumulation of nitrite nitrogen in the soil at levels up to 5 mg kg^–1 increased the loss of nitrogen. Yet, it did not increase the binding of mineral nitrogen into soil organic matter, relative to the control soil. The data suggest that the biochemistry of the nitrite formation process, rather than the levels of nitrite ions formed, are of primary importance in the role of nitrification mediated nitrosation of soil organic matter.