Figure 1.
The schematic representation of enzymatic reaction catalyzed by PAP.
The enzyme hydrolyzes the phosphoester linkage of PtdOH and generates DAG and Pi.
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.
Table 1.
Purification of PAP from bitter melon cotyledons.
Figure 3.
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 Aliquots of the enzymatic reactions were withdrawn for measurement at the indicated time points.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.
Table 2.
LC/MS/MS analysis of PAP-copurified proteins from bitter melon cotyledons.