The 8-anilino-1-naphthalenesulfonate (Ans) fluorescence in nonenergized and energized mitochondria was measured at various concentrations of Ans and KCl. Under the same experimental conditions, the zeta potential was determined from measurements of the electrophoretic mobility of mitochondria. The fluorescence intensity under various conditions was represented quantitatively in terms of the Langmuir adsorption isotherm where the electrostatic interaction acting between Ans and mitochondria was properly taken into account. The values of qN (q) proportionally constant related to quantum yield of Ans: N, the maximum number of the adsorption site) and deltaG (nonelectrical part of the free-energy change due to the binding of Ans to mitochondria) were constant irrespective of difference in energy state and in ionic strength in media. It was concluded that changes in the Ans fluorescence in mitochondria are mainly attributed to changes in the surface potential of mitochondria.
Vinyl acetate (VA) is a potential and practical acetylating reagent for the optical resolution of enantiomers in the racemate by lipase (triacylglycerol hydrolase, EC 3.1.1.3) catalyzed transesterification in organic solvent: VA irreversibly reacts with the active serine (Ser) residue in lipase to give the acetyl-enzyme intermediate [Enz-Ac] because vinyl alcohol is immediately converted to acetaldehyde by keto-enol tautomerization. This irreversibility is considered to accelerate the reaction rate. Such a reaction is called "irreversible transesterification" (1) and applied to the optical resolution of numerous secondary alcohols (1-5). On the other hand, the analyses of optical resolution data are complicated for lipase catalyzed transesterification with VA. It is reported that the enantioselectivity [E: the enantiomeric ratio for the homocompetitive reaction (6-9), i.e., as the reaction is irreversible] at low conversion is greater than that at high conversion (>50%) for Pseudomonas fluorescens lipase (PFL) catalyzed transesterification with racemic b-methyl-(2-643
Pharaonis phoborhodopsin (ppR, also called Natronobacterium pharaonis sensory rhodopsin II) and its transducer protein, pharaonis halobacterial transducer of ppR (pHtrII), form a signaling complex, and light signals are transmitted from the sensor to the transducer by the protein–protein interaction. A truncated pHtrII(1–159) consisting of intramembrane helices (expressing amino acid residues from the first to the 159th position) and ppR form the complex in a solution containing 0.1%n‐dodecyl‐β‐d‐maltoside. At 75–85°C, the time‐dependent color loss of ppR was caused by denaturation. We found that pHtrII(1–159) retarded the denaturation rate of ppR. This increase in the thermal stability was used as a probe for the binding ability in the dark. Tyr199 of ppR and Asn74 of pHtrII(1–114) were proposed as amino acid residues interacting with each other through hydrogen bonding. Then, ppR and pHtrII(1–159) mutants at these positions were prepared to examine the effect on the binding in the dark. The wild‐type and Y199F mutant can bind pHtrII(1–159), suggesting that the hydrogen bonding between these specific amino acid residues may not be the only cause of the binding, but the hydrophobic interaction via phenyl ring of ppR may contribute dominantly.
The mechanism by which UV‐C irradiation inactivates M13 bacteriophage was studied by analyzing the M13 genome using agarose gel electrophoresis and South‐Western blotting for pyrimidine dimers. The involvement of singlet oxygen (1O2) was also investigated using azide and deuterium oxide and under deoxygenated conditions. With a decrease in M13 infectivity on irradiation, single‐stranded circular genomic DNA (sc‐DNA) was converted to Form I and Form II, which had an electrophoretic mobility between that of sc‐DNA and linear‐form DNA. However, the amount of sc‐DNA remaining was not correlated with the survival of M13. The formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts ((6–4)PP) increased as a function of irradiation dose. The decrease in M13 infectivity was highly correlated with the increase in CPD and (6–4)PP, whereas no change was seen in M13 coat protein on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 8‐Oxo‐7,8‐dihydro‐2′‐deoxyguanosine did not form in the M13 genome after UV‐C irradiation. Inactivation of M13 was neither enhanced by deuterium oxide nor inhibited by azide. Deoxygenation of the M13 suspension did not affect the inactivation, indicating that 1O2 did not participate in the inactivation of M13 by UV‐C irradiation under these conditions. These results indicated that UV‐C irradiation induced not only CPD and (6–4)PP formation but also additional tertiary structural change in DNA inside the M13 virions, resulting in primary damage and a loss of infectivity. The indirect effect of UV‐C irradiation such as 1O2 production followed by oxidative damage to nucleic acids and proteins might have contributed less, if at all, to the inactivation of M13 than the direct effect of UV‐C.
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