The data demonstrate that rPlp is a relatively themostable phospholipase. Figure 4 Effects of chemical and physical conditions on rPlp activity. (A) Effect of rPlp concentration on enzymatic activity. (B) The effect of temperature on rPlp activity. (C) The effect of pH on rPlp activity. (D) The effect of EGTA rPlp activity. The effect of pH on enzyme activity was Adriamycin chemical structure determined for pH values ranging from 2 to 12. The data showed that rPlp had a broad pH optimum from pH 5.3 to pH 8.7 with activity dropping off rapidly at pH values above and below the optimum (Figure 4C). rPlp activity was not affected by treatment with the chelating reagents EGTA (Figure 4D) or EDTA (data not shown) at concentrations

up to 100 mM. Additionally, treatment with divalent metal ions, such as calcium or magnesium had no effect on activity (data MI-503 purchase not shown). Plp is a secreted protein in V. anguillarum Subcellular fractions from V. anguillarum strains M93Sm and S262 (plp) were prepared and phospholipase A2 activity examined using BPC and TLC. Initial studies revealed that at 37°C phospholipase A2 activity was detected in all cell fractions, including the culture supernatant, periplasm, cytoplasm, cytoplasmic membrane, and outer membrane, from both M93Sm and S262 (Figure 5A). However, when the assay was performed at 64°C

(to inactivate heat labile phospholipases), phospholipase A2 activity in S262 was significantly decreased in all fractions including the supernatant (Figure 5B). Additionally, when the assay was performed at 64°C for M93Sm subcellular fractions, only the culture supernatant exhibited phospholipase activity against BPC (about 100-fold higher activity compared to the phospholipase activity of the S262 supernatant). The data demonstrated that Plp was secreted into the culture supernatant of V. anguillarum, which corresponds with in silico analysis of the deduced Plp amino acid sequence (Accession number DQ008059) by SignalP that Plp has a signal peptide [18]. TLC results

also revealed that there was at least one other protein in V. anguillarum M93Sm exhibiting phospholipase A2 activity besides the secreted, heat stable Plp protein. This was a themolabile PLA2 activity inactivated at 64°C. Figure 5 The phospholipase activity assays detected by TLC of Ribonuclease T1 various cell fractions prepared from wild type (wt) strain M93sm and plp mutant strain S262 (plp-) were performed at 37 ° C (A) and 64 ° C (B). PBS buffer, LB20, and PBS buffer + 1% sarcosylate were served as negative controls. The refolded rPlp protein (PLP +) served as positive control. The top spots on each chromatogram are the BPC substrate and the bottom spots are the BLPC reaction product. The proteins from the same cell fractionation preparations were analyzed by SDS-PAGE and Western blot analysis (C) as described in the Methods. The refolded rPlp protein was served as positive control.