nitrophenols and 2-hexadecanoylamino-4-nitrophenylphosphorylcholine

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

Topics: Animals; Choline; Drug Stability; Female; Hydrolysis; Hymecromone; Kinetics; Micelles; Nitrophenols; Octoxynol; Phosphoric Diester Hydrolases; Phosphorylcholine; Placenta; Polyethylene Glycols; Sphingomyelin Phosphodiesterase; Sphingomyelins; Umbelliferones

Topics: Choline; Genetic Carrier Screening; Humans; Leukocytes; Niemann-Pick Diseases; Nitrophenols; Phosphorylcholine

Topics: Adolescent; Child; Child, Preschool; Choline; Female; Fibroblasts; Humans; Male; Methods; Niemann-Pick Diseases; Nitrophenols; Phosphoric Diester Hydrolases; Phosphorylcholine; Sphingomyelin Phosphodiesterase

Niemann-Pick disease is caused by a deficiency of sphingomyelinase in organs and tissues. Determinations of sphingomyelinase activity had required the use of sphingomyelin labeled with radiocarbon or radiohydrogen. These materials are expensive, and their use is restricted to laboratories with radioactive counting facilities. An analogue of sphingomyelin, 2-hexadecanoylamino-4-nitrophenylphosphorylcholine, was synthesized. This substance is hydrolyzed by highly purified sphingomyelinase, and by sphingomyelinease in extracts of human liver tissue, cultured skin fibroblasts, cultured amniotic cells and washed leukocyte preparations. Extracts of tissues and cells from patients with Niemann-Pick disease Type A do not hydrolyze this compound, whereas heterozygotes and patients with Niemann-Pick disease Type C have an intermediate level of hydrolytic activity. Thus, the analogue is a reliable chromogenic reagent for the diagnosis of patients with Niemann-Pick disease and the detection of heterozygous carriers of the Niemann-Pick trait.

Topics: Amniotic Fluid; Cells, Cultured; Cerebrosides; Choline; Clinical Enzyme Tests; Fibroblasts; Glucosylceramidase; Heterozygote; Homozygote; Humans; Hydrogen-Ion Concentration; Hydrolysis; Leukocytes; Liver; Niemann-Pick Diseases; Nitrophenols; Phosphoric Diester Hydrolases; Phosphorylcholine; Proteins; Skin; Sphingomyelin Phosphodiesterase; Sphingomyelins