Neutral magnesium-dependent sphingomyelinase from liver plasma membrane: purification and inhibition by ubiquinol.
Plasma membranes isolated from pig liver contained almost no acid sphingomyelinase but significant neutral magnesium-dependent sphingomyelinase that was activated by phosphatidylserine. We report here the purification to apparent homogeneity of neutral sphingomyelinase of about 87 kDa from liver plasma membranes. The purified enzyme strictly required magnesium and had a neutral optimal pH. In contrast with neutral sphingomyelinase purified from other sources (such as brain), the enzyme purified from from liver plasma membrane was not inhibited by GSH and, strikingly, it was not activated by phosphatidylserine. Liver sphingomyelinase was inhibited by several lipophilic antioxidants in a dose-dependent way. Ubiquinol-10 was more effective than alpha-tocopherol, alpha-tocopherylquinone, alpha-tocopherylquinone, and ubiquinone-10, and inhibition was noncompetitive. Differential inhibition of neutral sphingomyelinase by antioxidants did not correlate with different levels of protection against lipid peroxidation. The purified sphingomyelinase was not inhibited significantly by ubiquinone-10 and ubiquinol- 10, but ubiquinol-0 and ubiquinone-0 inhibited by 30 and 60% respectively. Our results demonstrate a direct inhibitory effect of ubiquinol on the plasma membrane n-SMase and support the participation of this molecule in the regulation of ceramide-mediated signaling.
Martín
S F
SF
Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Universidad de Córdoba, Spain.
Navarro
F
F
Forthoffer
N
N
Navas
P
P
Villalba
J M
JM
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D015398:Signal Transduction; D012995:Solubility; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification*; D013552:Swine; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000494:pharmacology*
D000975:Antioxidants; D004791:Enzyme Inhibitors; D014451:Ubiquinone; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium; C003741:ubiquinol