Studies on the hydrophobic properties of sphingomyelinase.
Crude liver lysosomal sphingomyelinase (EC 3.1.4.12) displays a heterogeneous electrofocusing profile. The majority of the enzyme resolves into two major components with acidic pI values near pH 4.6 and 4.8. Several additional minor peaks of activity are seen at more basic pH values (up to pH 8.0). In the presence of 0.1% Triton X-100 (or Cutscum), the location of sphingomyelinase is shifted by about 1 pH unit to more basic pH values. Triton X-100 also increases the apparent heterogeneity of sphingomyelinase. Removal of detergent by treatment with Bio Beads SM-2 restores the acidic pI profile. This behaviour appears to be specific, since it was not shared by six glycosidases several of which hydrolyse sphingolipids. The electrofocusing profile of 3H-labelled Triton X-100 was distinct and separate from sphingomyelinase, suggesting that only a small fraction of detergent interacted directly with the enzyme. To study this behaviour in more detail we examined the effect of detergents on elution of sphingomyelinase from sphingosylphosphocholine-Sepharose. Sphingosylphosphocholine is a competitive inhibitor of sphingomyelinase (Ki 0.5 mM). Binding of enzyme was pH-dependent. Triton X-100, Cutscum and Tween 20 eluted significant amounts of enzyme at 0.01-0.02%. Total elution was achieved with up to 0.1% detergent. These data suggest that sphingomyelinase binds to neutral detergent monomers with a high degree of affinity. In excess detergent (5-7 times the critical micellar concentration) the surface charge on the protein is changed, leading to a pI shift. This behaviour probably does not occur at the active site of the enzyme, since there is no appreciable effect on substrate hydrolysis and substrate analogues were ineffective in eluting the enzyme.
D002846:Chromatography, Affinity; D006026:Glycoside Hydrolases; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D017830:Octoxynol; D010727:Phosphoric Diester Hydrolases; D010767:Phosphorylcholine / Q000031:analogs & derivatives; D011092:Polyethylene Glycols; D012685:Sepharose; D013108:Sphingomyelin Phosphodiesterase; D013110:Sphingosine / Q000031:analogs & derivatives
C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; D011092:Polyethylene Glycols; D017830:Octoxynol; D012685:Sepharose; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase; D006026:Glycoside Hydrolases; D013110:Sphingosine