acta 2_2015

Rapeseed phosphatidyl choline hydrolysis to phosphatidic acid... 337Cabbage extracts hydrolytic activityHydrolytic activities of cabbage PLD extractstowards RPC were compared to activity of standardPLD. Hundred milligrams of RPC was dissolved in10 mL of diethyl ether and added to the mixture of0.4 mg PLD st or 400 mg cabbage PLD extract, 40mM CaCl 2 , in 10 mL of acetate buffer (pH 5.6), andthen incubated at 30 O C under stirring for 30 min.Product of hydrolysis was extracted three times with40 mL of diethyl ether. Extracts were combined,dried with anhydrous sodium sulfate and evaporatedto dryness at 40 O C under vacuum. The phosphatidylcholineand PA content was analyzed byTLC and HPLC as a 1% w/v solutions in chloroform.Hydrolysis of rapeseed phosphatidylcholineHundred milligrams of RPC was dissolved in10 mL of diethyl ether and added to the mixture of200-1000 mg PLD extract from savoy cabbage, 20-160 mM CaCl 2 in 10 mL of acetate buffer of pH 5.6.The obtained mixture was then incubated at 30 O Cunder stirring for different times in the range of 15-120 min. Next, the product of hydrolysis was extractedthree times by 40 mL of diethyl ether. Extractswere combined, dried with anhydrous sodium sulfateand evaporated to dryness at 40 O C under vacuum.The PC and PA content was analyzed by TLC andHPLC as a 1% w/v solutions in chloroform.TLC analysisTLC was performed on 20 ◊ 20 cm silica gelplates. Samples of 20 µL of chloroform phospholipidssolutions were applied and the plates weredeveloped with chloroform-methanol-water (45 : 25: 0.2, v/v/v) in saturated glass chamber to 15 cmhigh. The plates were sprayed with 5% phosphoromolybdateethanol solution and dried at 105 O C.HPLC analysisFurther calculation of the products was performedby HPLC analysis using a Waters 600 unit,fitted with a UV photodiode array detector Waters996 PDA and a data processor (Millenium 32,Waters). The chromatograms were evaluated at 205nm. A 250 ◊ 5 mm column packed with Lichrospher100-10 (Knauer) was used. Column flow rate was 1mL/min and the column temperature was maintainedat 20 O C. Phospholipids were separated by isocraticelution with hexane/2-propanol/water at ratioof 1 : 4 : 1, v/v/v. Samples of 10 µL (1% w/v) wereinjected in the same solvent used for elution. Thepeaks were identified on the basis of their retentiontimes using standard phospholipids. PA concentrationwas calculated as a peak area from the standardcurve.RESULTS AND DISSCUSIONWe have characterized the hydrolytic activityof cabbage PLD extracts towards RPC. The partiallypurified enzyme extracts have been used, and theroutine purification has not been continued beyondheat precipitation step. The purified by us cabbagePLD acts readily upon PC (22, 26, 27). CabbagePLD of the highest purity proved rather unstable andthe purification was accompanied by significantreduction of enzyme activity and yield (26-28).Figure 1. Effect of reaction time on phosphatidic acids concentration (PA%) in hydrolyzed RPC(RPC 100 mg, PLD sc 400 mg, 30 O C). The calculations are given as the mean values of triplicatemeasurements