Molecular properties and kinetic studies on sphingomyelinase of Bacillus cereus

Abstract

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 (23300) was determined by sedimentation equilibrium. The enzyme contained loosely-bound magnesium atom. The addition of Mg 2+ accelerated the enzyme reaction regardless of substrates and their physical states. The addition of Ca 2+ also accelerated the enzyme reaction slightly, when water-soluble substrates, i.e., 2-hexadecanoylamino-4nitrophenylphosphorylcholine and p-nitrophenylphosphorylcholine, were used as substrates. On the other hand, the addition of Ca 2+ 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, Ca 2+ proved to be a competitive inhibitor against Mg 2+, with a K 1 value of 33 μ M. On the other hand, when the mixed micelle of sphingomyelin and sodium deoxycholate was used as substrate, Ca 2+ stimulated the enzyme reaction at lower concentration in the presence of a low concentration of Mg 2+, although higher concentrations of Ca 2+ were still inhibitory. In this case, added Ca 2+ may be used as a substitute of Mg 2+ 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.