Distribution of charged residues stabilizes individual helices in myoglobins

The effect of the distribution of charged residues on stability of alpha helices in isolated peptides and in globular proteins exemplified by myoglobins from 62 different species is discussed. A highly simplified set of rules is used to account for the interaction of charged groups with the dipole of an alpha helix. Only the position and sign of a charge with respect to the center of the helix and its ability to participate in intrahelical salt bridges determine its effect. These rules lead to a linear correlation between the helicity in variant C-peptide helices from RNAse and the extent to which the charge distribution opposes the helix dipole. Of the sample of 496 helices in the myoglobins studied, 456 exhibit arrangements of charges which oppose the effective dipole moment of the helix according to this calculation. A number of variants occur which leave the backbone moment of helices A-D unchanged, or even add to it. However no such variants exist in the sequences of helices E-H. We suggest that the E, F, G and H helices in myoglobins which show the strongest reversal of the helix dipole participate in the structures of early intermediates in folding of the chain. Stable helix structures should be more likely to occur in these isolated sequences also, and introduction of charge alterations in helices E to H should affect the initial refolding rate of mutant myoglobins.     

Search for the optimal sequence of the ribosome binding site by random oligonucleotide-directed mutagenesis.

Synthetic DNA duplexes corresponding to the ribosome binding site (RBS) were synthesized through the phosphite method on solid support. The synthetic RBS DNA with partial random sequences was inserted into an appropriate site between the lpp-lac promoter and the beta-galactosidase structural gene in plasmid pMKT2. The level of beta-galactosidase expression was correlated with the color intensity of the recombinant colonies on X-gal plates. The bluest colonies were isolated and characterized with respect to beta-galactosidase enzyme activity and RBS sequence. There was good correlation between color intensity and the level of the enzyme activity, and this provided a reliable phenotypic screening method in the search for the optimal regulatory sequences. Novel RBS sequences obtained here show not only the unique nucleotide distribution, but also strong complemetarity to the 3' end region of 16S rRNA, from which could be deduced a generalized RBS sequence, the position of the SD region, and the 16S rRNA position mediated during translation initiation.     

Respiration-driven proton translocation in Thiobacillus versutus and the role of the periplasmic thiosulphate-oxidizing enzyme system

The periplasmic location of enzymes A and B of the thiosulphate-oxidizing multienzyme system of Thiobacillus versutus has been further confirmed by differential radiolabelling of periplasmic and cytoplasmic proteins. The stoichiometries of respiration-driven proton translocation in T. versutus were determined using the oxygen pulse and the initial rate methods. A value for the H⁺/O quotient (number of protons translocated per oxygen atom reduced) of about 2.8 was found for the oxidation of thiosulphate, and of about 2.5 for sulphite. The H⁺/O quotient for endogenous respiration was about 5.7. The data are shown to be in good agreement with the scheme proposed previously for thiosulphate oxidation by this organism. Proton generation during the oxidation of thiosulphate or sulphite is indicated to occur in the periplasm rather than by pumping across the cytoplasmic membrane. The results also suggest that a H⁺/O quotient of six occurs during NADH oxidation (from endogenous metabolism measurements) and that the terminal cytochrome oxidase, aa₃, does not function as a proton pump.Abbreviations DCCD dicyclohexyl carbodiimide - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - IEF isoelectric focusing - HIC hydrophobic interaction chromatography - EAI ethyl acetimidate hydrochloride - IAI isethionyl acetimidate     

Distribution of Clostridium botulinum in Japan and in Shinkiang district of China

Soil specimens obtained from several areas of Japan, which are closely located to or facing the Continental land of China, were examined for the distribution of Clostridium botulinum, especially pertaining to types A and B. A total of 266 specimens of Japan, when cultured, showed no type A or B toxicity, although 30 (11.3%), 4 (1.5%), and 10 (3.8%) of the specimens showed C1, C2, and type E toxicities, respectively. On the contrary, types A and/or B toxicities were shown, by the same method, in 14 of 20 specimens of Shinkiang district, China. The highest number of C. botulinum cells found in one gram of soil specimen was 25 for type A and 10 for type B.     

四川宝兴雉类生态和垂直分布的调查研究

宝兴位于四川盆地西缘山地,境内纵贯世界闻名的大雪山——即夹金山。 自19世纪30年代至20世纪30年代,先后有Verreaux(1838—1871),Geoffroy(1866),David(1871),Seinhoe(1871),Sharpe(1875),David et Oustalet(1877),Oustaler (1891—92),Styan(1899),Laubmann(1920),La Touch(1925—30),Smith(1931—34),Bangs(1932),Seys(1934)等对宝兴进行过调查和采集,而我     

Properties of a small basic Peptide from pumpkin seeds

A small basic peptide with an unusual amino acid composition has been isolated from the seeds of pumpkin, Cucurbita maxima. Amino acid analysis and sequence data show the protein to be about 36 residues in length, with an approximate composition Lys₁, Arg₁₄, Asp₃, (Glu + Gln)₁₅, Gly₁, Pro₁, Trp₁. On the basis of composition, the molecular weight is approximately 5000 daltons and the nitrogen content by weight is 20.4%. Twelve amino acids are entirely lacking. The peptide is slightly toxic to mouse B-16 melanoma cells, but its in vivo function is unknown. It does not appear to be derived from cucurbitin, the pumpkin storage globulin; however, it could be a storage peptide involved in nitrogen mobilization during the early stages of germination.     

Genetic organization and nucleotide sequence of the stability locus of IncFII plasmid NR1

The stability (stb) locus of IncFII plasmid NR1 was mapped to a 1700 base-pair NaeI-TaqI restriction fragment. A series of unstable plasmids that contained insertion, deletion, and point mutations that inactivated the stability function was isolated. The unstable point mutants examined were all stabilized (complemented) in trans by a copy of the wild-type stb locus, suggesting that the mutations had inactivated diffusible gene products. The nucleotide sequence of the stb locus contained two tandem open reading frames, designated stbA and stbB, that encoded essential trans-acting protein products with predicted sizes of 36,000 Mr and 13,000 Mr, respectively. A third open reading frame, stbC, that could encode a peptide of 8000 Mr was contained within stbB in the complementary DNA strand. Plasmid-encoded proteins of 36,000 Mr and 13,000 Mr were identified in minicell experiments as the products of stbA and stbB, respectively. Unstable deletion mutants that retained the promoter proximal region of the stb locus upstream from stbA but had deleted both stbA and stbB were stabilized in trans by plasmids that could supply StbA and StbB. In contrast, deletion mutants that had lost the stbAB promoter region were not complemented in trans, indicating that this region contained an essential cis-acting site (or sites). Unlike some other loci that mediate stable plasmid inheritance, cloned copies of the wild-type stb locus of NR1 did not exert strong incompatibility (i.e. trans destabilization) against other stb+ derivatives of plasmid NR1 present in the same cell.     

Transforming activities of sodium fluoride in cultured Syrian hamster embryo and BALB/3T3 cells

The transforming activity of sodium fluoride was studied in the SHE and the BALBl3T3 cell culture systems. Initiating and promoting activities were then investigated by means of the orthogonal methodology. Sodium fluoride was found to induce morphological transformation of SHE cells seeded on a feeder layer of X-irradiated cells at high concentrations (75–125 g/ ml). When the cells were seeded in the absence of a feeder-layer, the transformation frequencies increased in a dose-dependent manner with the concentrations of sodium fluoride ranging from 0 to the highly toxic concentration of 200 g/ml. In the BALBl3T3 cell system, sodium fluoride was negative in the standard Kakunaga procedure, while through the experiment designed by table L₈ (2⁷) of the orthogonal method, an initiating-like effect and a weak promoting activity were detected within the concentrations ranging from a 25 g/ ml to a 50 g/ ml concentration which is highly toxic for BALBl3T3 cells. From these results, it is suggested that, besides a genetic mode of action, sodium fluoride could possibly act through a non-genotoxic mechanism.Abbreviations CE cloning efficiency - NaF sodium fluoride - SHE Syrian hamster embryo - TF transformation frequency