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Predicate | Object |
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
3
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pubmed:dateCreated |
1990-8-24
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pubmed:abstractText |
In recent years an increasing number of proteins has been shown to be membrane-anchored by a covalently attached PtdIns-glycan residue. In mammalian cells little is known about PtdIns-glycan-specific phospholipases which might play a role in the metabolism of PtdIns-glycan-anchored proteins. In order to identify PtdIns-glycan-specific phospholipases, a rapid and sensitive assay for such enzymes was developed using the PtdIns-glycan-anchored amphiphilic membrane form of acetylcholinesterase as substrate. The rate of product formation was monitored by the increase in soluble hydrophilic acetylcholinesterase in the aqueous phase after separation in Triton X-114. With this assay we established the presence of a PtdIns-glycan-specific phospholipase in bovine brain. This enzyme was soluble and could be partially purified by a heat step followed by chromatography on DEAE-cellulose and by gel filtration on Sepharose CL-6B. PtdIns-glycan-specific phospholipase had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) caused a marked decrease in the cleavage rate and methylation of the amino group of the glucosamine residue of the anchor decreased the cleavage rate to zero. Using [125I]TID-labeled substrate, diradylglycerol phosphate was identified as the second product showing that the cleavage specificity of PtdIns-glycan-specific phospholipase was that of a phospholipase D. PtdIns-glycan-specific phospholipase D was inhibited by mercurials, omicron-phenanthroline and EGTA. It was stimulated by Ca2+ in micromolar concentrations indicating that PtdIns-glycan-phospholipase D is a Ca2(+)-regulated enzyme.
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pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical | |
pubmed:status |
MEDLINE
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pubmed:month |
Jul
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pubmed:issn |
0014-2956
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
5
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pubmed:volume |
190
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
593-601
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pubmed:dateRevised |
2008-11-21
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pubmed:meshHeading |
pubmed-meshheading:2373084-Animals,
pubmed-meshheading:2373084-Brain,
pubmed-meshheading:2373084-Cattle,
pubmed-meshheading:2373084-Chromatography, DEAE-Cellulose,
pubmed-meshheading:2373084-Chromatography, Gel,
pubmed-meshheading:2373084-Hot Temperature,
pubmed-meshheading:2373084-Kinetics,
pubmed-meshheading:2373084-Phospholipase D,
pubmed-meshheading:2373084-Phospholipases,
pubmed-meshheading:2373084-Substrate Specificity
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pubmed:year |
1990
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pubmed:articleTitle |
Isolation and characterization of a phosphatidylinositol-glycan-anchor-specific phospholipase D from bovine brain.
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pubmed:affiliation |
Abteilung Neurobiochemie, Universität Bern, Switzerland.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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