Characterization of the neutral pH-optimum sphingomyelinase from rat brain: inhibition by copper II and ganglioside GM3.
A neutral pH-optimum sphingomyelinase (N-SMase), solubilized from rat brain membranes, was characterized with respect to metal and membrane lipid effects. Chromatofocusing chromatography, which separates proteins according to pI, showed two N-SMase activities. One eluted at pH 4.7 and the other required 0.4 M NaCl before elution. Kinetically, the two preparations appeared similar. The N-SMase eluting at pH 4.7 was most extensively studied here. Of the phospholipids studied, only phosphatidylserine showed any influence on N-SMase and that was to increase its activity by as much as 50%. Neither serine nor phosphatidic acid had any effect. Of the cations tested, none was able to replace Mg2+ as a required activator. However, it was found that several metals were inhibitory, with Cu2+ being most effective (IC50 = 5 microM). Gangliosides, particularly the monosialoganglioside, GM3 (IC50 approximately 50 microM), inhibited N-SMase. Other glycolipids showed little effect on activity, even the immediate precursor to GM3 - lactosylceramide. The ganglioside sugar, N-acetylneuraminic acid, also had no effect on N-SMase activity. None of these inhibitors affected the acidic pH-optimum sphingomyelinase. Other sphingolipid compounds such as ceramide - the enzymatic product - and sphingosylphosphorylcholine (lysosphingomyelin) showed no capacity to inhibit N-Smase, implying that the enzyme may have a selective substrate-binding site.
Lister
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MD
Sphinx Pharmaceuticals Corporation, Biomolecular Research, Durham, NC 27717.
Crawford-Redick
C L
CL
Loomis
C R
CR
D000535:Aluminum / Q000494:pharmacology; D000077410:Aluminum Chloride; D017607:Aluminum Compounds; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D002412:Cations; D002712:Chlorides / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology*; D005290:Ferric Compounds / Q000494:pharmacology; D005679:G(M3) Ganglioside / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010743:Phospholipids / Q000494:pharmacology; D051381:Rats; D013107:Sphingolipids / Q000494:pharmacology; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D015032:Zinc / Q000494:pharmacology; D017967:Zinc Compounds
D017607:Aluminum Compounds; D002412:Cations; D002712:Chlorides; D005290:Ferric Compounds; D005679:G(M3) Ganglioside; D010743:Phospholipids; D013107:Sphingolipids; D017967:Zinc Compounds; D000077410:Aluminum Chloride; D003300:Copper; C016837:zinc chloride; D000535:Aluminum; D013108:Sphingomyelin Phosphodiesterase; D015032:Zinc; C029892:cupric chloride; C024555:ferric chloride