Proton nuclear-magnetic-resonance and resonance Raman studies of thermophilic cytochrome c-552 from Thermus thermophilus HB8

The pH and temperature dependences of the 270-MHz proton nuclear magnetic resonance and resonance Raman spectra of Thermus thermophilus cytochrome c-552 were studied. Observation of the NMR methyl signal of the iron-bound methionine indicates that a methionine residue is the sixth ligand of heme iron in both ferric and ferrous states, although the environment of this methionine is not similar to that in mitochondrial cytochrome c. The NMR methyl signal of the coordinated methionine in the ferrous state was observed even at 87 degrees C, indicating the retention of the methionine ligand at the sixth coordination position. None of resonance Raman lines in ferrous cytochrome c-552 at higher temperatures showed a prominant temperature-dependent frequency shift, which implies that the heme iron was still bound with strong ligands and retained the low-spin state. In either redox state overall thermal denaturation did not occur even at 87 degrees C, although the ferric form existed in thermal spin mixture of the low-spin and high-spin species at higher temperatures. The hyperfine-shifted NMR resonances of the ferric form indicated rapid exchange of the sixth ligand at alkaline pH in the process of a single-step alkaline isomerization.     

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy.

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.     

Kinetic study of isomerization of ferricytochrome c at alkaline pH

Kinetic studies of the isomerization reaction of horse heart ferricytochrome c between pH 8.5 and pH 12.1 have been carried out by using stopped-flow and rapid scanning stopped-flow techniques. Below pH 10, our results were in good agreement with the scheme proposed earlier (Davis, L. A., Schejter, A. and Hess, G. P. (1974) J. Biol. Chem. 249, 2624–2632). Above pH 10, another faster first-order process was observed, which suggested the existence of a transient species in the isomerization reaction between the species with and without a 695 nm band. The probable scheme of the isomerization reaction is considered to be

where H denotes a proton, the colored forms are the species predominant at neutral pH with a 695 nm band and the noncolored forms are the species without a 695 nm band. The transient species has a small 695 nm absorbance which suggests that the sixth ligand is still Met-80, although the protein conformation might be different from that at neutral pH.     

The distribution of 14c from [U-14c]glucose in mice using whole-body autoradiography.

Tissue distribution of radioactive carbon from [U-14C]glucose in the mouse in vivo was studied by whole-body autoradiography. The mice were frozen with Dry-Ice-acetone at 0.5, 1, 5 and 30 min, 1 and 24 hr and 1 and 3 weeks after intraperitoneal injection of [U-14C]glucose. Whole-sagittal sections of the frozen mouse, obtained by using a microtome in a cryostat, were dried in a cryostat and autoradiographed. The resulting dry autoradiographs are called untreated autoradiographs in the present work. The sections were then fixed in cold 6% (w/v) HClO4, dried at room temperature and again autoradiographed. Autoradiographs that have undergone this process are referred to as treated autoradiographs. In both untreated and treated autoradiographs, within 1 min following injection of the labeled glucose, the abdominal cavity had the highest autoradiographic density. At 1 hr, density became highest in Harder's, sublingual and duodenal glands, large intestinal mucosa and tongue, and after 3 weeks, no autoradiographic denisty was present.     

Properties of a DNA-binding protein in a culture medium of thymus cells

A DNA-binding protein, which migrated as one major protein band, with a molecular weight of 14,000, on sodium dodecylsulfate polyacrylamide gel, was purified from a culture medium of mouse thymus cells. The interaction of the isolated protein with DNA in vitro was assayed by a nitrocellulose filter binding technique. Equilibrium competition experiments demonstrated that the DNA-binding protein had the ability to differentiate among sequences of polynucleotides, indicating that the DNA-binding protein-DNA interaction was at least partially specific. This protein increased the helix melting temperature of DNA and inhibited the incorporation of [3H]dTMP into DNA by the DNA polymerase of calf thymus in vitro.     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

Dietary GABA induces endogenous synthesis of a novel imidazole peptide homocarnosine in mouse skeletal muscles

Amino Acids - Carnosine (β-alanyl-l-histidine) is an imidazole dipeptide present at high concentrations in skeletal muscles, where it plays a beneficial role. However, oral intake of carnosine...