Computer simulations of cyclic enkephalin analogues

Molecular dynamics simulations and energy minimization studies of cyclic enkephalin analogues incorporating retro-inverso modifications have been carried out. The dynamic trajectories are analyzed in terms of the relative mobility of the 14-membered rings, conformational transitions among equilibrium states, and hydrogen-bonding patterns. The cyclization of the molecules reduces the motion of the ring structures substantially. Time-correlated conformational transitions resulting in the reorientation of peptide units are observed. Hydrogen bonds form principally C7 structures. Because of the incorporation of retro-inverso residues, C6 and C8 structures are also formed. Starting conformations for energy minimizations were obtained from the molecular dynamics simulations and from a systematic search of the conformational space available to the molecules. Several minimum energy backbone and side-chain conformations were found for each analogue. The effect of retro-inverso residues on hydrogen-bonding patterns and backbone conformations is discussed.     

Yeast metallothionein function in metal ion detoxification

A genetic approach was taken to test the function of yeast metallothionein in metal ion detoxification. A yeast strain was constructed in which the metallothionein locus was deleted (cup1 delta). The cup1 delta strain was complemented with normal or mutant metallothionein genes under normal or constitutive regulatory control on high copy episomal plasmids. Metal resistance of the cup1 delta strain with and without the metallothionein-expressing vectors was analyzed. The normally regulated metallothionein gene conferred resistance only to copper (1000-fold); constitutively expressed metallothionein conferred resistance to both copper (500-fold) and cadmium (1000-fold), but not to mercury, zinc, silver, cobalt, nickel, gold, platinum, lanthanum, uranium, or tin. Two mutant versions of the metallothionein gene were constructed and tested for their ability to confer metal resistance in the cup1 delta background. The first had a deletion of a highly conserved amino acid sequence (Lys-Lys-Ser-Cys-Cys-Ser). The second was a hybrid gene consisting of the sequences coding for the first 20 amino acids of the yeast protein fused to the monkey metallothionein gene. Expression of these genes under the CUP1 promoter provided significant protection from copper, but none of the other metals tested. These results demonstrate that there is significant flexibility in the structural requirements for metallothionein to function in copper detoxification and that yeast metallothionein is also capable of detoxifying cadmium under conditions of constitutive expression.     

Regulation of the manganese-containing superoxide dismutase is independent of the inducible DNA repair system in Escherichia coli

Studies on the induction of the manganese-containing superoxide dismutase in several strains of Escherichia coli with different mutations in recA and lexA revealed that the inductions of the Mn-isozyme and of the SOS system by oxygen free radicals are not coregulated. We also studied the synthesis of the manganese-superoxide dismutase in the temperature-dependent, protease-constitutive strain recA441(tif-1) that also contained a lac fusion in an SOS gene. A shift to the temperature at which recA441 has constitutive protease activity did not induce Mn-superoxide dismutase but did induce beta-galactosidase. The data clearly demonstrate that induction of the Mn-superoxide dismutase is independent of the SOS system.     

Complexities in the denaturation of horse metmyoglobin by guanidine hydrochloride

The denaturation of horse metmyoglobin by guanidine hydrochloride was studied at pH 6.4 and 25 degrees C. Measurements of both the peptide circular dichroism and the absorbance in the Soret region suggest that the extent of renaturation strongly depends on the time interval during which the protein is exposed to concentrated solutions of the denaturant. From the equilibrium measurements of the absorption in the Soret region, it is concluded that the unfolding of metmyoglobin is complex. This is further supported by kinetic studies of denaturation which suggest the occurrence of the least four species in the reaction.     

Utilization of carbon and nitrogen sources and acid/alkali production by cowpea rhizobia

Summary Sixteen slow-growing strains of rhizobia (15 cowpea rhizobia and oneR. japonicum) were examined to determine the effects of carbon and nitrogen sources on acid/alkali production in culture media. We found that the pH changes of the medium were more influenced by nitrogen sources than carbon sources (with the exception of ribose). When ammonium sulphate was used as a nitrogen source, all the cowpea rhizobia strains produced acid. When yeast-extract was used as a nitrogen source, however, a heterogenous pattern for acid/alkali production was found. The majority of the strains produced alkali from nitrate, glutamate and urea irrespective of carbon sources and acid from ribose irrespective of nitrogen sources.     

Leishmania mexicana: purine-metabolizing enzymes of amastigotes and promastigotes

Cultured promastigote and isolated amastigote forms of Leishmania mexicana mexicana have been surveyed for the presence of enzymes involved in purine metabolism. Quantitative but not qualitative differences between the enzymes of two forms were discovered. There were found to be significant differences between the enzyme content of L. m. mexicana and that reported for L. donovani. Extracts of both parasite forms of L. m. mexicana were found to have higher levels of adenine deaminase (EC 3.5.4.2) and guanine deaminase (EC 3.5.4.3) than adenosine deaminase (EC 3.5.4.4). There appeared to be two distinct nucleosidases (EC 3.2.2.1), one active on nucleosides, the other on deoxynucleosides. Phosphorylase (EC 2.4.2.1) could be detected only in the catabolic direction. Nucleotidases were present, but were more active on 3' (EC 3.1.3.6)- than 5' (EC 3.1.3.5)-nucleotides. Phosphoribosyltransferase (EC 2.4.2.7,.8 and .22) and nucleoside kinase (EC 2.7.1.20) activities were detected in both forms. Nucleotide-interconverting enzymes were found to be present, with IMP dehydrogenase (EC 1.2.1.14) being the most active. Cell fractionation experiments revealed that, in the promastigote, enzyme separation within the parasite may play an important part in regulating cellular purine metabolism.     

Mathematical analysis of the stimulus for the lateral line organ

Behavioral studies have shown that a blind fish is capable of detecting and recognizing stationary objects in its surroundings. It is proposed that the displacement of water caused by the fish as it moves is the basis for this detection capability. Alterations in the displacement of water around the fish, caused by the obstacle, act as stimuli for the lateral line organ. The question of how these stimuli acting on the skin of the fish, image the environment and what information is thus made available to the fish is the concern of this paper. The stimuli for the lateral line organ are derived mathematically. Two cases are treated: that of a fish gliding past an obstacle and that of one approaching an obstacle.     

Thermodynamic stability of proteins in salt solutions: A comparison of the effectiveness of protein stabilizers

The denaturation of proteins by guanidine hydrochloride was studied in the presence of different concentrations of stabilizing salts, namely potassium phosphate, ammonium sulfate, and potassium acetate. The denaturation transition was followed by observing changes in the peptide circular dichroism atpH 7.0 and 25°C. From these results the free energy of stabilization for the process native denatured was determined. It was found that the stabilizing power of the anions increased in the order acetate < sulfate < phosphate, in agreement with the anionic lyotropic series. Ribonuclease A, which is known to have a site that can bind either a phosphate or a sulfate ion, showed a larger stabilization by these anions than that for lysozyme, pepsinogen, and myoglobin.