• KSBB Journal
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• v.5 no.2
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• pp.119-124
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• 1990

Phospholipase D catalyzes the phosphatidohydrolysis and transphosphatidylation of phospholipid in the biological systems. In this study we were partially purified phospholipase D from Chinese cabbage and the characterization of the enzyme was carried out in a multistirring batch system bioreactor. The enzyme showed optimum activity at pH ,5.6, highest activity at 37$^{\circ}C$ and Ca2+ is important for the enzyme activity. Optimum concentrations of Ca2+ for phosphatidohydrolysis was 20 mM and for transphosphatidylation was 40 mM, respectively. Some organic solvents such as diethylether, isopropylether and butylacetate were activated the enzyme activity. On the other hand, EDTA, Ba2+, Mn2+ and Zn2+ showed inhibitory effect on the enzyme activity. The base acceptors in transphosphatidylation by the Chinese cabbage phospholipase D were tested. Various poly-and monohydroxy alcohols were found to be active.

• Journal of the Korean Chemical Society
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• v.52 no.5
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• pp.488-494
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• 2008

• Journal of the Korean Chemical Society
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• v.47 no.5
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• pp.466-471
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• 2003

The effect of polyamines on the cabbage phospholipase D(PLD) activity was investigated. The PLD activity was determined by pH-stat titration of phosphatidic acid, one of the enzymatic reaction product, using phosphatidyl choline small unilamellar vesicles as a substrate. The cabbage PLD was activated approximately 4 fold by spermine at 1 mM concentration. This spermine effect appears to be similar to the previous report on the PLD activation of rat brain mitochondrial fraction. It was also found that cationic polypetides such as polylysine and polyhistidine exerted a marked enhancement effect on the cabbage PLD. Particularly polyhistidine exerted approximately 5.5 fold enhancement effect at 0.062 mM concentration. The polyamine effect on the cabbage PLD was reexamined in the phosphatidylcholine/sodium dodecyl sulfate mixed micellar system. The relevance of polyamine effect on PLD activity is discussed in relation to the active site of PLD.

• Bulletin of the Korean Chemical Society
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• v.17 no.10
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• pp.905-908
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• 1996

A substrate specificity of cabbage phospholipase D (PLD) was studied using the synthetic phospholipids having different head groups. The phospholipids were synthesized from phosphatidylcholine and appropriate bases by transphosphatidylation of PLD. The bases used were ethanolamine, serine, ethanol and γ-hydroxybutyric acid. The phosphatidic acid, the product of PLD, was separated in TLC and measured densitometrically. The kinetic parameters were estimated for each substrate and the effects of pH, SDS, Ca2+ and other metal ions were examined. Vmax values found were 3.75, 2.36, 5.59, 1.63, 2.30 nmol/min/μg protein for phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylethanol, and phosphatidylburytic acid, respectively. These results indicate a broad specificity of cabbage PLD toward phospholipids with different head groups. Particularly phosphatidylserine was most easily hydrolyzed by PLD and its activity did not depend on Ca2+.

• KSBB Journal
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• v.6 no.3
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• pp.271-279
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• 1991

Phosphatidylglycerol(PG) and two unnatural phospholipids, phosphatidylethyleneglycol (PEG) and phosphatidylpropyleneglycol(PPG), were synthesized from ovolecithin using cabbage phospholipase D(PLD) in a emulsion system. Optimum pH and temperature for the enzymatic synthesis of PG, PEG and PPG in the emulsion system was 5.0-5.6 and 37$^{\circ}C$, respectively. The maximum activity for transphosphatidylation was obtained with 30-80 mM Ca++. Addition of 25% glycerol was required to convert completely ovolecithin to PG, whereas 16% glycerol was sufficient to attain the highest rate of conversion for both PEG and PPG syntheses, the highest conversion rate was obtained with addition of either 10% ethyleneglycol or propyleneglycol. However, the concentration of alcoholic acceptor should be increased up to 20% to improve selectivity up to 100% for PEG or PPG synthesis. Identification of PEG and PPG was made by analyzing the polyvalent alcohols released after their hydrolysis by HCl or PLD.

• Journal of the Korean Chemical Society
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• v.54 no.2
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• pp.208-214
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• 2010

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 ${\alpha}$-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 $33.33{\pm}1.33M^{-1}sec^{-1}$. This second-order rate constant of transphosphatidylation was as 400 times greater as the second-order hydrolysis rate constant of $0.078M^{-1}sec^{-1}$ which was adjusted for the water concentration. A linear free energy relationship between the $pK_a$ of alcohol and transphosphatidylation rate gives a Br${\o}$nsted slope of ${\beta}_{nu}$ = 0.12 ${\pm}$ 0.03. This small ${\beta}_{nu}$ 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.

• Bulletin of the Korean Chemical Society
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• v.13 no.4
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• pp.381-384
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• 1992

The hydrolytic susceptibility of large unilamellar vesicle (LUV) toward cabbage phospholipase D (PLD) was studied. The activity of PLD was determined by pH stat titration method. Using phosphatidylcholine LUV as substrate a pH optimum of 6.96 was observed. For maximal activity the optimal temperature of $31^{\circ}C$ and 10 mM of Ca2+ were required. The apparent Km value estimated was 2.5 mM. The hydrolytic activity of PLD toward PC LUV was somewhat high despite the absence of activator in assay system and this high susceptibility of PC LUV may be attributed to the structural properties of LUV. The effect of amphiphatic substances such as dicetyl phosphate and phosphatidic acid on the enzyme activity were also examined in mixed LUVs.

• Animal cells and systems
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• v.7 no.3
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• pp.213-219
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• 2003

The response of plants to pathogens and wounding is dependent upon very sensitive perception mechanisms. Although genetic approaches have revealed a variety of resistance genes that activate common defense responses, defense-related proteins are not well characterized in plants. Therefore, we used a proteomic approach to determine which defense-related proteins are induced by salicylic acid (SA) and wounding in Chinese cabbage. We found that SA and wounding induce pathogenesis-related protein 1a (PR1a) at both protein and mRNA levels using proteomics and Northern blot analysis, respectively. This indicates that our proteomic approach is useful for identifying defense-related proteins. We also identified several other proteins that are induced by SA or wounding. Among the seven SA-induced proteins identified, four may be defense-related, including defense-related protein, phospholipase D (PLD), resistance protein RPS2 homolog, and L-ascorbate peroxidase. Out of the six wounding-induced proteins identified, three may be defense-related: heat shock cognate protein 70 (HSC70), polygalacturonase, and peroxidase P7. The precise functions of these proteins in plant defense responses await further study. However, identification of the defense-related proteins described in this study should allow us to better understand the mechanisms and signal transduction pathways involved in defense responses in Chinese cabbage.

• Journal of the Korean Chemical Society
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• v.40 no.9
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• pp.630-634
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• 1996

In order to explore a substrate specificity for cabbage phospholipase D, we examined the PLD reactivity toward the phosphatidylcholines with different chain length of acyl groups. The selected acyl chains were the saturated fatty acid of $C_8:0,\;C_{12}:0,\;C_{16}:0,\;C_{20}:0$. The reactivity of these phospholipids were dependent largely on the ratio of PC : SDS. The PC : SDS ratio showing the optimal PLD activity were found to be 1:1.4, 1:2.2, 1:2.5, and 1:3.6 respectively as the increase of the acyl chain length. Likewise the optimum temperature for the maximal PLD activity were altered markedly to 25$^{\circ}C$, 30$^{\circ}C$, 35$^{\circ}C$, 45$^{\circ}C$ when the length of acyl chains increased. On the contrary the pH and concentration of $Ca^{2+}$ necessary for the optimum PLD activity were not altered significantly. The kinetic parameter $V_{max}$ for short acyl chain substrate was greater than the values for the longer acyl chain, which indicates the fastest rate of hydrolysis. By the same token, the reactivity of longer chain substrate became slower for the hydrolysis activity.

• Journal of the Korean Chemical Society
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• v.50 no.5
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• pp.362-368
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• 2006

SH group modifying chemicals were used to characterize the eight cysteine residues of cabbage PLD. 5,5-dithiobis(2-nitrobenzoate)(DTNB) was used to titrate the SH group of cysteine residues . Based on the optical density at 412nm due to the reduced DTNB, 4 SH groups are found to be present in a native PLD while 8 SH groups in the denatured PLD whose tertiary structure was perturbed by 8M urea. The results imply that among the 8 cysteine residues of PLD, the half(4) are exposed on the surface whereas the other half are present at the interior of the enzyme tertiary structure. The PLD was inactivated by SH modifying reagents such as p-chloromercuribenzoate(PCMB), iodoacetate, iodoacetamide, and N-ethylmaleimide. At the addition of dithiothreitol(DTT) only the PCMB inhibited PLD activity was recovered reversibly. The micro-environment of the exposed SH group of cysteine residues was examined with various disulfide compounds with different functional groups and we found that anionic or neutral disulfides appear to be more effective than the positively charged cystamine for inactivating the PLD activity. The effect of redox state of cysteine residues on the PLD activity was further explored with H2O2. The oxidation of SH groups by H2O2 inhibited the PLD activity more than 70%, which was mostly recovered by DTT. From these results, we could confirm chemically that all the cysteine residues of PLD are present as in their reduced SH forms and the 4 SH groups exposed on the surface of the enzyme may play important roles in the regulation of PLD activity.