Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia)

Abstract

Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and P(i). PAPs are typically categorized into two subfamilies: Mg(2+)-dependent soluble PAP and Mg(2+)-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg(2+)-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53-60 °C and unaffected by up to 0.3 mM MgCl2. The K(m) and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na(3)VO(4), Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg(2+)-independent enzyme in plants.

Figure 1. The schematic representation of enzymatic reaction catalyzed by PAP.

The enzyme hydrolyzes the phosphoester linkage of PtdOH and generates DAG and P_i.

Figure 2. Subcellualr distribution of PAP activity in bitter melon cotyledons.

(A) bitter melon of Indian origin was used in the study. (B) Distribution of PAP activity in bitter mellow cotyledons. The cotyledon extract was successively centrifuged at 3,000 g, 18,000 g and 105,000 g. The final pellet (P3-pellet) after ultracentrifugation is generally regarded as the microsomal membranes. Following dialysis and centrifugation of the 105,000 g supernatant, the S3-pellet (contaminated microsomes) and the S3-supernatant (cytosol) were obtained. All three fractions were analyzed for PAP activity. The data presented are the mean of 2 assays for each sample.

Figure 3. Linearity of PAP assays.

PAP activity was assayed using aliquots of the proteins from the second S column fraction duing PAP purification. (A) PAP activity vs. reaction time. The assay was performed at 53°C for various times with 500 µM DPA and 0.3 mM MgCl₂. Aliquots of the enzymatic reactions were withdrawn for measurement at the indicated time points. (B) PAP activity vs. amount of enzyme. The assay was performed at 53°C using various amounts of the PAP preparation with 500 µM DPA and 0.3 mM MgCl₂. The data presented are the mean of 2 assays for each sample.

Figure 4. Effect of buffer pH and assay temperature on PAP activity.

The assay was performed at 53°C for 30 min using 5 µL of the PAP preparation with 500 µM DPA and 0.3 mM MgCl₂. (A) pH profile of PAP enzyme catalyzing PtdOH. (B) Temperature profile of PAP enzyme catalyzing PtdOH. The data presented are the mean of 2 assays for each sample.

Figure 5. Effect of Mg²⁺ on PAP activity catalyzing PtdOH.

The assay was performed at 53°C, pH 6.5 for 30 min using 5 µL of the PAP preparation with 500 µM DPA and various concentrations of MgCl₂. The data presented are the mean of 2 assays for each sample.

Figure 6. Kinetcis of PAP enzyme activity. PAP activity vs. substrate concentration.

The assay was performed at 53°C, pH 6.5 for 10 min using various concentrations of DPA. The data presented are the mean of 2 assays for each sample.

Figure 7. Effects of phosphatase inhibitors, additives and cations on PAP activity.

The assays were performed at 53°C, pH 6.5 for 10 min using 5 µL of the PAP preparation with 500 µM DPA. (A) Effect of phosphatase inhibitors on PAP activity. The phosphatase inhibitors used were NaF (1 mM), sodium tartrate (4 mM) and sodium orthovanadate (2 mM). (B) Effect of additives on PAP activity. The final concentration of each additive in the assay buffer was 100 mM, except TX-100 was used at 10% concentration. (C) Effect of cations on PAP activity. The final concentration of each cation in the assay buffer was 0.3 mM. The data presented are the mean of 2 assays for each sample.

Figure 8. SDS-PAGE and native gel analysis of protein composition and PAP activity.

(A) Denaturing gel analysis of the purified PAP fractions from Affigel Blue column by SDS-PAGE and stained by silver nitrate. Lanes 1 and 11, Bio-Rad Precision plus protein standards; lanes 2–4, 0.28, 0.55 and 0.83 µg of fraction #4; lanes 5–7, 0.19, 0.37 and 0.56 µg of fraction #5; lanes 8–10, 0.30, 0.60 and 0.90 µg of the pooled fractions 11–17. (B) PAP activity determined by in-gel phosphatase assay with DiFMUP. Lanes 1 and 8, positive control B-phycoerythrin; lanes 2–7, 0.5, 1, 3, 6, 10 and 12 µg of PAP from the pooled fraction. (C) Native gel analysis of the purified PAP fractions stained with Coomassie Blue. Lanes 1 and 8, native marker unstained protein standards; lanes 2–7, 0.5, 1, 3, 6, 10 and 12 µg of PAP from the pooled fraction.