Membrane-associated sphingomyelinase derived from bovine brain, isolation method therefor and anti-sphingomyelinase monoclonal anitibody
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United States of America Patent
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Issued Date -
Sep 26, 2002
app pub date -
Feb 5, 2001
filing date -
Feb 5, 2001
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Abandoned
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Abstract
Disclosed is a sphingomyelinase derived from the neuron membrane of the bovine brain, which is 60 kDa in molecular weight and Mg.sup.2+-dependent in activity with an optimal pH ranging from 6.0 to 9.0. It is prepared by homogenizing a bovine brain tissue and centrifuging the homogenate to remove cell debris and nuclei; separating cell membranes from the supernatant into a pellet through centrifugation; treating the cell membrane pellet with a buffer containing ammonium sulfate to disassociate the sphingomyelinase from the membrane; subjecting the extract to anion exchange chromatography; sonicating the chromatography fraction in a buffer containing Triton X-100 and centrifuging the sonicated fraction to obtain a supernatant containing the sphingomyelinase; and purifying the sphingomyelinase by subjecting the supernatant to hydrophobic interaction chromatography, anion exchange high-performance liquid chromatography, hydrophobic interaction high performance liquid chromatography, and cation exchange fast protein liquid chromatography, in due order. Also, disclosed is a monoclonal antibody against the sphingomyelinase, produced by Hybridoma SM1A5 (Deposition No. KCLRF-BT-00025).
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