Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100.
We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U
D000818:Animals; D002794:Choline / Q000031:analogs & derivatives*; D004355:Drug Stability; D005260:Female; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives*; D007700:Kinetics; D008823:Micelles; D009596:Nitrophenols / Q000378:metabolism*; D017830:Octoxynol; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D011092:Polyethylene Glycols / Q000494:pharmacology*; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism; D014468:Umbelliferones / Q000378:metabolism*
D008823:Micelles; D009596:Nitrophenols; D013109:Sphingomyelins; D014468:Umbelliferones; D010767:Phosphorylcholine; D006923:Hymecromone; D011092:Polyethylene Glycols; C013847:bis-(4-methylumbelliferyl)phosphate; C010733:2-hexadecanoylamino-4-nitrophenylphosphorylcholine; D017830:Octoxynol; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase; D002794:Choline