ABSTRACT. In the presence of alcohol, phospholipase D (PLD) is known to perform transphosphatidylation activity, during which the overall reaction rate of PLD increased. To elucidate the reaction mechanism of transphosphatidylation further, we investigated rate constants of transphosphatidylation reaction of the purified α-type PLD from cabbage in the presence of various alcohols. The second-oder rate constants of PLD transphosphatidylation showed a large increase with the primary alcohols examined as expected. In the case of butanol we observed the second-oder rate constant of 33.33 ± 1.33 M -1 sec -1. This second-order rate constant of transphosphatidylation was as 400 times greater as the second-order hydrolysis rate constant of 0.078 M -1 sec -1 which was adjusted for the water concentration. A linear free energy relationship between the pKa of alcohol and transphosphatidylation rate gives a Brønsted slope of βnu = 0.12 ± 0.03. This small βnu value implicates that the transition state of break down of phosphatidyl-enzyme intermediate (E-P) is likely dissociative. Finally, a reaction mechanism of cabbage PLD is suggested on the basis of our results presented here and the histidine residue known to be located in the active site of cabbage PLD.
The study was performed to identify bioactive constituents from the inner bark of Betula schmidtii. Seven compounds were isolated through silica gel and Sephadex LH‐20 column chromatography, and identified to be betulin (1), betulinic acid (2), β‐sitosterol (3), lupeol (4), kojic acid (5), 1,7‐bis‐(ρ‐hydroxyphenyl)‐(3,4‐dihydroxy)‐hepan‐5‐one (6), and platyphyllenone (7) using EI‐MS, 1D‐, and 2D‐NMR data. Compounds 5, 6, and 7 show antioxidant effect with IC50 values of 18.23 ± 1.08, 29.37 ± 1.42, and 40.14 ± 1.75 μM in DPPH free radical scavenging activity. Compounds 4 and 5 show an antioxidant effect with IC50 values of 28.21 ± 1.80 and 37.74 ± 1.02 μM in DCHF‐DA intracellular ROS generations. Compounds 5, 6, and 7 also exhibited mushroom tyrosinase inhibitory activity with IC50 values of 17.42 ± 1.08, 47.17 ± 1.92, and 26.64 ± 1.40 μM. Compound 1 showed cell viability of 24.50 ± 6.30 and 45.60 ± 3.10% at 10 and 25 μM, while compound 6 showed 11.20 ± 1.40% at 25 μM in glutamate‐induced damaged mouse neuronal cells.
ABSTRACT. The effect of diphtheria toxin on cell membrane lipids was studied by examining the phospholipase D (PLD) activity and free fatty acids (FFA) release in HepG2 cells. The diphtheria toxin effects on lipid alteration show apparently maximal at pH 5.1, stimulating PLD activity nearly 3.5 fold and enhancing FFA release approximately 5 fold over the control. These results indicate that the membrane is perturbed and its lipid component is rearranged during the diphtheria toxin translocation. Digitonin, a random membrane perturbing detergent, exhibit about four-fold higher perturbation effect over the diphtheria toxin at neutral pH. This observation suggests that the membrane perturbation induced by diphtheria toxin appears to be rather selective. To investigate the cause of the membrane perturbation, Cibacron blue, an inhibitor of membrane pore formation, and hemagglutinin, an influenza virus with fusion peptide, were tested for their effects on diphtheria toxin action. Cibacron blue decreased the diphtheria toxin effect by almost 50%, but the lipid alteration induced by hemagglutinin was similar to the diphtheria toxin effect. These observations imply that the membrane perturbation induced by diphtheria toxin may be caused by a combination of pore formation and insertion of hydrophobic peptide of toxin to the membrane as well. Additionally, we found that the diphtheria toxin increased the HepG2 cells permeability but the cells viability was maintained at high level at the same time. DNA fragmentation which is related to apoptosis was not induced by the toxin. Under these conditions, we could demonstrate that the lipid alteration of HepG2 cells was brought about by diphtheria toxin at acidic pH.
ABSTRACT. As a model study for in vivo interplay between phospholipase D(PLD) and phospholipase A2(PLA2), effect of PLA2 on the activity of cabbage PLD was examined employing phosphatidylcholine(PC) small unilamellar vesicles(SUV). The PLD activity was inhibited in the presence of PLA2. Almost complete inhibition of PLD activity could be brought about with increasing amount of PLA2. In order to evaluate the inhibitory effect of PLA2, effects of its products LPC and oleate were examined by adding individually to the SUV. The exogenously added oleate enhanced the PLD activity, while the LPC inhibited the PLD activity. However, in combination of LPC and oleate, the PLD activity was inhibited as similariy to the PLA2 added directly to the assay system. The oleate enhancement effect was greater than the other saturated fatty acids examined. The inhibitory effect of LPC was also extended to other lysophospholipids. Among the lysophospholipids tested here, all except LPA inhibited the PLD activity. LPA showed marked activational effect. The modulational effect of the molecules produced by PLA2 on the PLD activity was further investigated by examining the stability of bilayer structure and the size of mixed vesicles in addition to carrying out the kinetics of Michaelis-Menten. The results presented here could show a possibility of in vivo clue for the modulation of PLD activity by the products of PLA2 under the certain experimental conditions.
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