Molecular properties and kinetic studies on sphingomyelinase of Bacillus cereus.
A sphingomyelinase of Bacillus cereus was purified to a homogeneous state (512 U/mg, 2200-fold) as indicated by SDS-polyacrylamide gel electrophoresis and the molecular weight (23,300) was determined by sedimentation equilibrium. The enzyme contained loosely-bound magnesium atom. The addition of Mg2+ accelerated the enzyme reaction regardless of substrates and their physical state. The addition of Ca2+ also accelerated the enzyme reaction slightly, when water-soluble substrates, i.e., 2-hexadecanoylamino-4-nitrophenylphosphorylcholine and p-nitrophenylphosphorylcholine, were used as substrates. On the other hand, the addition of Ca2+ inhibited enzyme reaction when mixed micelles of either sphingomyelin and Triton X-100 or sodium deoxycholate were used. The surface charge on mixed micelles affected the enzyme reaction. When the mixed micelle of sphingomyelin and Triton X-100 was used as substrate, Ca2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 33 microM. On the other hand, when the mixed micelle of sphingomyelin and sodium deoxycholate was used as substrate, Ca2+ stimulated the enzyme reaction at lower concentration in the presence of a low concentration of Mg2+, although higher concentrations of Ca2+ were still inhibitory. In this case, added Ca2+ may be used as a substitute of Mg2+ to neutralize the negative charge on the mixed micelle, improving the accessibility of sphingomyelinase to the micellar substrate. A cationic detergent, cetyltrimethylammonium bromide, seemed to denature or inactivate the enzyme.
D001409:Bacillus cereus / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D003902:Detergents; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008823:Micelles; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012995:Solubility; D013108:Sphingomyelin Phosphodiesterase / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D003902:Detergents; D008823:Micelles; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium; D002118:Calcium