Cabbage Phospholipase D Research Articles

포스포리파제 D 촉매반응에서 포스파티딜 전달반응 활성에 미치는 알코올의 영향

포스포리파제 D(PLD)는 알코올 존재 하에서 포스파티딜 전달반응이 일어나며 동시에 PLD의 전체 반응속도도 증가하는 것으로 알려져 있다. 이 포스파티딜 전달반응을 자세히 구명하기위해 본 실험에서는 양배추에서 정제한 <TEX>${\alpha}$</TEX>-type PLD를 가지고 여러 종류의 알코올 존재 하에서 포스파티딜 전달반응의 반응속도를 검토하였다. 실험 결과 검토한 1차 알코올들에 의해 PLD의 포스파티딜 전달반응 속도는 기대했던 대로 크게 증가하였다. 부탄올의 경우 2차 반응속도는 <TEX>$33.33{\pm}1.33M^{-1}sec^{-1}$</TEX>로 얻어졌으며, 이 전달반응 속도를 물의 농도를 고려한 가수분해 속도(<TEX>$0.078M^{-1}sec^{-1}$</TEX>)와 비교하면 무려 400배 이상의 격차를 보여주었다. 알코올의 <TEX>$pK_a$</TEX>과 포스파티딜 전달반응 속도와의 자유에너지 선형 관계로부터 브뢴스테드 <TEX>${\beta}_{nu}$</TEX> 값 <TEX>$0.12{\pm}0.03$</TEX>을 얻었다. 비교적 적은 <TEX>${\beta}_{nu}$</TEX> 값으로 미루어보아 끊어지는 포스파티딜-효소 중간체(E-P)의 전이상태는 상당히 해리된 상태일 것으로 추정된다. 이들 결과와 양배추 PLD의 활성부위의 히스티딘 잔기를 참작하여 양배추 PLD의 반응메커니즘을 제안하였다. 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 <TEX>${\alpha}$</TEX>-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 <TEX>$33.33{\pm}1.33M^{-1}sec^{-1}$</TEX>. This second-order rate constant of transphosphatidylation was as 400 times greater as the second-order hydrolysis rate constant of <TEX>$0.078M^{-1}sec^{-1}$</TEX> which was adjusted for the water concentration. A linear free energy relationship between the <TEX>$pK_a$</TEX> of alcohol and transphosphatidylation rate gives a Br<TEX>${\o}$</TEX>nsted slope of <TEX>${\beta}_{nu}$</TEX> = 0.12 <TEX>${\pm}$</TEX> 0.03. This small <TEX>${\beta}_{nu}$</TEX> 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.

Conversion of Phosphatidylcholine to Phosphatidylglycerol with Phospholipase D and Glycerol

AbstractWhen phosphatidylcholine (PtdCho) is acted upon by the enzyme phospholipase D (PLD) in the presence of glycerol and water, at least two products can arise: phosphatidic acid (PtdOH) from hydrolysis and PtdGly from transphosphatidylation. Commercial PLD preparations from Streptomyces chromofuscus, Streptomyces sp., and cabbage were examined for their ability to selectively promote PtdGly formation in a two phase aqueous‐organic solvent system. Factors examined were enzyme amount, pH, glycerol concentration, and the type of organic solvent. The reaction of PtdCho to give products such as PtdGly was followed by HPLC using a stationary phase consisting of a polymerized poly (vinyl alcohol) on silica gel with ELSD. The identities of all products were confirmed by retention times and HPLC‐MS analyses. Under all tested conditions PLD from S. chromofuscus gave at most a 15% yield of PtdGly. Higher amounts of added glycerol inhibited this PLD. Nearly quantitative conversion to PtdGly was obtained with cabbage PLD when the mol ratio of glycerol to PtdCho was at least 64 (mol water/glycerol = 105). With PLD from Streptomyces sp. a nearly quantitative yield of PtdGly was obtained when the mol ratio of glycerol to PtdCho was at least 5.3 (mol water/glycerol = 1,266), demonstrating that this PLD had a higher selectivity for glycerol than cabbage PLD. When the glycerol concentration was very low, the level of PtdOH increased, and cardiolipin (diphosphatidylglycerol) was generated. The highest mol ratio PtdGly to PtdOH was observed when the solvents isopropyl ether or dichloromethane were used.

Asymmetric in vitro synthesis of diastereomeric phosphatidylglycerols from phosphatidylcholine and glycerol by bacterial phospholipase D

Using chiral-phase HPLC, we determined the stereochemical configuration of the phosphatidylglycerols (PtdGro) synthesized in vitro from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho, R configuration) or 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PtdEtn, R configuration) and glycerol by transphosphatidylation with bacterial phospholipase D (PLD). The results obtained with PLD preparations from three Streptomyces strains (S. septatus TH-2, S. halstedii K5, and S. halstedii subsp. scabies K6) and one Actinomadura species were compared with those obtained using cabbage and peanut PLD. The reaction was carried out at 30 degrees C in a biphasic system consisting of diethyl ether and acetate buffer. The resulting PtdGro were then converted into bis(3,5-dinitrophenylurethane) derivatives, which were separated on an (R)-1-(1-naphthyl)ethylamine polymer. In contrast to the cabbage and peanut PLD, which gave equimolar mixtures of the R,S and R,R diastereomers, as previously established, the bacterial PLD yielded diastereomixtures of 30-40% 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol (R,S configuration) and 60-70% 1,2-diacyl-sn-glycero-3-phospho-3'-sn-glycerol (R,R configuration). The highest disproportionation was found for the Streptomyces K6 species. The present study demonstrates that bacterial PLD-catalyzed transphosphatidylation proceeds to a considerable extent stereoselectively to produce PtdGro from PtdCho or PtdEtn and prochiral glycerol, indicating a preference for the sn-3' position of the glycerol molecule.

Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol

In this study, the effect of temperature on the stereoselectivity of phospholipase D (PLD) toward the two primary hydroxyl groups of glycerol in the transphosphatidylation reaction of phosphatidylcholine to phosphatidylglycerol (PtdGro) was investigated. For this purpose, PLD from bacteria (Streptomyces septatus TH-2, S. halstedii subsp. scabies K6, and Actinomadura sp.) and cabbage were tested. At the reaction temperatures employed (0-60 degrees C), the proportions of the two PtdGro diastereomers, namely, 1,2-dioleoyl-sn-glycero-3-phospho-3'-sn-glycerol (R,R configuration) and 1 ,2-dioleoyl-sn-glycero-3-phospho-1'-sn-glycerol (R,S configuration), which were produced with PLD from Streptomyces TH-2 and Actinomadura sp., changed gradually from 50% R,R and 50% R,S at 50-60 degrees C to 70% R,R and 30% R,S at 0 degrees C. These alterations suggested that the stereoselectivity of the bacterial PLD toward the two primary hydroxyl groups of prochiral glycerol was significantly influenced by reaction temperature. PLD from Streptomyces K6 showed relatively little effect of temperature on stereoselectivity, giving 65-69% R,R in the temperature range of 60-10 degrees C examined. The plots of In ([R,R]/[R,S]) vs. 1/T gave good linear fits for these three bacterial PLD. No temperature effect was observed for cabbage PLD, which gave an almost equimolar mixture of the R,R and R,S diastereomers in the range from 0 to 40 degrees C. The temperature-dependent change in enantiomeric selectivity of the bacterial PLD promises potentially profitable commercial exploitation.

폴리아민에 의한 양배추 포스포리파제 D의 활성화

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 phos- phatidyl choline small unilamellar vesicles as a substrate. The cabbage PLD was activated approximately 4 fold by sper- mine 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 enhance- ment effect at 0.062 mM concentration. The polyamine effect on the cabbage PLD was reexamined in the phos- phatidylcholine/sodium dodecyl sulfate mixed micellar system. The relevance of polyamine effect on PLD activity is discussed in relation to the active site of PLD.

Using O-( n-alkyl)- N-( N, N′-dimethylethyl)phosphoramidates to investigate the role of Ca 2+ and interfacial binding in a bacterial phospholipase D

O-( n-alkyl)- N-( N, N′-dimethylethyl)phosphoramidates ( n=6, 8, and 10; C n PNC) were synthesized and characterized as inhibitors of phospholipase D (PLD) activity toward phosphatidylcholine presented as monomers, micelles, and bilayers. Detailed studies with recombinant Streptomyces chromofuscus PLD, a Ca 2+-activated enzyme that does not show large changes in catalytic activity toward the same substrate as a monomer or micelle, showed that the longer the inhibitor chain length, the more potent C n PNC is as a competitive inhibitor toward all the substrates. However, the physical state of the inhibitor did affect the maximum inhibition attainable. For a fixed concentration of diC 4PC (monomer substrate), C n PNC inhibition reached a maximum around the CMC of the inhibitor; the inhibition was reduced at higher inhibitor concentrations, in part caused by the lower solubility of the aggregated inhibitor. With diC 4PC as the substrate and using concentrations of C 10PNC that were below its CMC, the K i for C 10PNC was 0.030±0.003 mM, ∼13-fold less than the K m for substrate. Aggregated substrates showed significant inhibition of PLD by C n PNC, although as the substrate chain length increased, inhibition by a given C n PNC was diminished. With POPC vesicles, the apparent K i for C 10PNC was 0.030 of the apparent K m. The availability of these inhibitors allowed us to show that PC analogues can bind to the active site of S. chromofuscus PLD in the absence of Ca 2+. Once bound at the active site, the inhibitor does not significantly affect the divalent ion-dependent partitioning of the enzyme to PC surfaces. Of the two other PLD enzymes examined, cabbage PLD, but not Streptomyces sp. PMF, was able to catalyze the cleavage of the PN bond. Differential susceptibility of PLDs to these phosphoramidates may eventually be useful in studying PLD isozymes in cells.

Effect of Cabbage Phospholipase D Treatment on the Oxidative Stability of Beef Homogenate and Egg Yolk Phosphatidylcholine Liposomes

ABSTRACT:The possibility that enzymatic hydrolysis of phospholipids increases the oxidative stability of muscle food was examined using cabbage phospholipase D (PLD). As judged by the amounts of thiobarbituric acid‐reactive substances and carbonyl content, treatment of meat homogenate with 500 ppm (840 units) PLD increased its oxidative stability during storage at 4°C for 7 d, as compared to the nontreated ones. The thin‐layer chromatographic analysis confirmed the formation of phosphatidic acid (PA) in the 500 ppm (840 units) cabbage PLD‐treated samples. In a nonheme and heme iron‐induced lipid oxidation of egg yolk phosphatidylcholine liposomes, cabbage PLD treatment also improved their oxidative stability. Cabbage PLD treatment is, therefore, likely to improve the oxidative stability of meat homogenate via PA generation.

Inhibition of Streptomyces chromofuscus Phospholipase D Activity by Dichloro-(2,2′:6′,2′-terpyridine)-platinum (II) Dihydrate

To determine the catalytic site of Streptomyces chromofuscus phospholipase D (PLD), which lacks an HKD motif, we examined the effects of inhibitors on the hydrolytic activity of the PLD by comparing it with cabbage and Streptomyces PLDs, which have two HKD motifs. We showed that dichloro-(2,2′:6′,2"-terpyridine)-platinum (II) dihydrate, a His- and Cys-directed chemical modifier, had inhibitory effects on the activities of all types of PLD examined. On the other hand, N -ethylmaleimide, a thiol-directed modifier had no such effects on PLD activity. These results suggest that the His residue plays an important role in the activity of Streptomyces chromofuscus PLD.

Purification and Characterization of Phospholipase D from Cabbage Leaves.

Phospholipase D (PLD) was purified from cabbage leaves. The molecular weight of purified PLD was estimated as approximately 73 and 87 kDa by gel filtration using Superdex 200 HR column and, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. The transphosphatidylation capacity for phosphatidylcholine of this enzyme reached over 90% , and the enzyme's hydrolysis efficiency for phosphatidylcholine was five-fold higher than that for phosphatidylglycerol. These findings indicated that the cabbage PLD efficiently transferred phosphatidylcholine to phosphatidylglycerol. On N-terminal amino acid sequence analysis of the band separated by SDSPAGE, two sequences with differing N-terminus were detected. This N-terminal difference may have been generated by processing during maturation of PLD.

Aluminum fluoride inhibition of cabbage phospholipase D by a phosphate-mimicking mechanism

Aluminum fluoride (AlF 4 −) inhibited phospholipase D (PLD) purified from cabbage in both PIP 2-dependent and PIP 2-independent assays, consistent with its previously observed effect on mammalian PLD. The possibility that AlF 4 − may exert this effect through its known phosphate-mimicking property was examined. Inorganic phosphate, as well as two phosphate analogs, beryllium fluoride and orthovanadate, also inhibited cabbage PLD. Enzyme kinetic studies confirmed that PLD followed Hill kinetics, characteristic for allosteric enzymes, with an apparent Hill coefficient ( n app) of 3.8, indicating positive cooperativity among multiple substrate-binding sites and suggesting possible functional oligomerization of the enzyme. AlF 4 − modification of PLD kinetics was consistent with a competitive mode of enzyme inhibition. It is therefore proposed that AlF 4 −, and other phosphate analogs, inhibits plant PLD by competing with a substrate phosphate group for a substrate-binding site, thereby preventing the formation of an enzyme-phosphatidyl intermediate. This may be a conserved feature of PLD superfamily enzymes.

Transphosphatidylation capacity of phospholipase D from cabbage (Brassica oleracea L. var. capitata L.) leaves and Streptomyces chromofuscus

The phospholipase D (PLD) was purified from leaves of cabbage (Brassica oleracea L. var. capitata L.). The molecular weight of the enzyme was estimated as 87 and 73.5-kDa on SDS-PAGE and gel filtration using Superdex 200 HR 10/30 column. The transphosphatidylation capacity of cabbage PLD was shown to be higher than that of Streptomyces chromofuscus PLD. Although cabbage PLD showed a single band, Streptomyces chromofuscus PLD gave several bands on nondenaturing and denaturing PAGE. These results indicated that the cabbage PLD was a monomer protein, while the Streptomyces chromofuscus PLD was hetero oligomer, having several types with different molecular weights. Furthermore, the cabbage PLD included the duplicated HxKxxxxD catalytic motifs in the molecule, but the Streptomyces chromofuscus PLD did not. Previously, YAMANE et al. reported that the transphosphatidylation capacity of Streptomyces sp. PLD was higher than that of Streptomyces chromofuscus. These results indicated that the high degree of the transphosphatidylation capacity of cabbage PLD was due to the existence of HxKxxxxD catalytic motifs.

Cyclosporin A inhibits oxidant and calcium stimulated phospholipase D activity in the rat intestinal mitochondria

Mitochondrial swelling and calcium cycling occurs during oxidative stress and can be prevented by cyclosporin A (CysA). Our earlier work has shown that enterocyte mitochondria contains a phospholipase D (PLD) which can be activated by superoxide or calcium. In this study, we have shown that enterocyte mitochondrial PLD activated by these agents can be inhibited by cyclosporin A. This was clearly shown by the absence of phosphatidic acid (PA) formation and phosphatidylethanolamine (PE) degradation. Since this PLD specifically utilizes PE as substrate, PLD activity was also assessed by ethanolamine formation which was inhibited by CysA. CysA also inhibited the cabbage PLD activity as judged by phosphatidylethanol formation. These results suggest that cyclosporin A is an inhibitor of PLD and this may be one of the mechanism by which CysA protects enterocyte mitochondria from oxidative stress.

Inhibition of phospholipase D by lysophosphatidylethanolamine, a lipid-derived senescence retardant.

Phospholipid signaling mediated by lipid-derived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, flowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dose-dependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.

Hydrolytic activity of phospholipase D from plants and Streptomyces spp. against phosphatidylcholine monolayers at the polarized oil/water interface

A previously proposed method of measuring phospholipase D (PLD) activity using the change in electrical capacitance of a phospholipid monolayer at the polarized oil/water interface has been applied, with some modifications, to a comparative study of the hydrolytic activity of four PLDs frequently used in transphosphatidylation. Distinctive differences in the activities of PLDs from Streptomyces spp. and PLDs from plants were revealed. The two PLDs from Streptomyces spp. hydrolyzed the monolayer completely, whereas peanut and cabbage PLDs hydrolyzed up to 75% and 60% of the monolayer respectively. Peanut PLD did not show an absolute requirement for Ca 2+, whereas the hydrolytic activity of S. chromofuscus PLD was appreciably enhanced at 50 mM Ca 2+. The rate of hydrolysis for PLDs from plants showed little dependence on interfacial potential, while the rate for PLDs from Streptomyces spp. was highest when the potential of the aqueous phase was −90 mV relative to that of the oil phase. The Frumkin two-parallel-plate condenser model for the adsorption of PLD provided a good description of the observed potential-dependent activity of PLD.