Interfacial regulation of bacterial sphingomyelinase activity |
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Institution: | 1. Medical Imaging Center, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China;2. Biomaterial Research Center, School of Biomedical Engineering, Southern Medical University, Guangzhou 510515, PR China |
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Abstract: | The objective of this study was to define how the quality of the buffer/membrane interface influences the activity of bacterial sphingomyelinase acting at the interface. The enzyme reaction was carried out in a zero-order trough using a surface barostat. This approach allowed for proper control of the physico-chemical properties of the substrate molecules. Since the molecular area of ceramide is smaller than that of sphingomyelin, the hydrolysis reaction could be followed `on-line' from the monolayer area decrease at constant surface pressure. The hydrolysis reaction could be divided into two separate phases, the first being the lag-phase (time between enzyme addition and commencement of the monolayer area change), and the second phase being the actual hydrolysis reaction (from which a maximal degradation rate could be determined). The activity of sphingomyelinase (Staphylococcus aureus) toward bovine brain sphingomyelin (bb-SM) was markedly enhanced by Mg2+ (maximal activation at 5 mM). Mg2+ also influenced the lag-phase of the reaction (the lag-time increased markedly when the Mg2+ concentration decreased below 1 mM). Saturated sphingomyelins (bb-SM and N-palmitoyl sphingomyelin N-P-SM]) were more slowly degraded than the mono-unsaturated N-oleoyl sphingomyelin (N-O-SM). Both bb-SM and N-P-SM monolayers underwent a phase-transition at room temperature, whereas the N-O-SM monolayer did not. The phase-transition (liquid-expanded to liquid-condensed) was observed to greatly increase the lag-time of the hydrolysis reaction. The activity of sphingomyelinase was also sensitive to the lateral surface pressure of the monolayer membrane. Maximal degradation rate was achieved at 20 mN/m (with bb-SM, 30°C); above this pressure the lag-time of the reaction increased sharply. The inclusion of 4 mol% of cholesterol into a 3H]sphingomyelin monolayer markedly increased the extent of 3H]sphingomyelin degradation, and shortened the lag-time of the reaction. The inclusion of 10 mol% of zwitterionic or negatively charged phospholipids to the 3H]sphingomyelin monolayer did not affect the sphingomyelinase reaction significantly. In conclusion, this study has demonstrated that the physico-chemical properties of the substrate molecules have a dominating influence on the activity of a bacterial sphingomyelinase acting at the buffer/membrane interface. |
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