Evaluation of in vitro conditions influencing Phosphatidylinositol-specific Phospholipase C production by Staphylococcus aureus

The aim of this study was to evaluate the growth conditions affecting production of hosphatidylinositol-specific phospholipase-C (PIPLC) by Staphylococcus aureus ATCC 9144. A simple colorimetric method for direct estimation of phospholipids was adapted for rapid assaying of PIPLC. First phase of the study involved preliminary screening of nutritional and physical parameters. In the second phase, Plackett-Burman statistical design was employed to identify the most critical factors. Of the eleven variables studied, peptone, sodium carbonate (buffer), and rotational speed exhibited significant positive influence while glucose showed a negative influence on PIPLC activity. Thus, low C/N ratio, aeration, and buffering agent were identified as important factors controlling production of PIPLC. These results contribute to the existing knowledge on PIPLC regulation in Staph. aureus and would help optimize production of this enzyme for its successful application in biochemical research and industry. KEY WORDS: Phosphatidylinositol-specific phospholipase C Staphylococcus aureus Enzyme production Plackett-Burman design Colorimetric assa

Increased activity of goat liver plasma membrane alkaline phosphatase upon release by phosphatidylinositol-specific phospholipase C

263-270Mammalian alkaline phosphatase (ALP) is attached to the plasma membrane by a unique glycosylphosphatidylinositol (GPI) anchor. The influence of such a complex anchoring device on the enzyme function is not fully understood. Here, we report the effect of cleavage of the GPI anchor on the activity of goat liver plasma membrane ALP (GLPM-ALP). Phosphatidylinositol-specific phospholipase C (PI-PLC) purified from Bacillus cereus was used for the cleavage of the GPI anchor (delipidation) and hence for release of ALP from the membrane. Detergents — octyl-β-D-glucopyranoside (OG) and triton X100 (TX100) were also used for solubilization of ALP from the membrane. Resistance to solubilization by TX100 suggested the association of GPI-ALP with lipid rafts. Solubilization of GLPM-ALP with OG had no effect on the enzyme activity; however, delipidation with PI-PLC resulted in enhanced ALP activity. Kinetic analysis showed catalytic activation of PI-PLC-treated GLPM-ALP with an increase in Vmax (35%) without a significant change in Km. Moreover, this change in Vmax was observed to be independent of pH and buffer. The results suggested the implication of GPI anchor in modulating the catalytic property of GLPM-ALP, thus indicating the role of this special anchoring structure in the enzyme regulation