title: Interfacial regulation of bacterial sphingomyelinase activity.
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 degrees 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.
authors.affiliation: Department of Biochemistry and Pharmacy, Abo Akademi University, Turku, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Jungner
authors.forename: H
authors.initials: H
authors.lastname: Ohvo
authors.forename: J P
authors.initials: JP
authors.lastname: Slotte
mesh_terms: D000818:Animals; D002518:Ceramides / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002784:Cholesterol / Q000494:pharmacology; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D008567:Membranes, Artificial; D010743:Phospholipids / Q000494:pharmacology; D010767:Phosphorylcholine; D011312:Pressure; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013109:Sphingomyelins / Q000737:chemistry / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology*; D013696:Temperature
chemical_list: D002518:Ceramides; D008567:Membranes, Artificial; D010743:Phospholipids; D013109:Sphingomyelins; D010767:Phosphorylcholine; D002784:Cholesterol; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium