Association of the intestinal brush-border membrane phospholipase A2 and lysophospholipase activities (phospholipase B) with a stalked membrane protein.
We have attempted to determine the size and membrane orientation of a recently described rat jejunal brush-border protein possessing phospholipase A2 and lysophospholipase activities (phospholipase B) (Pind, S. and Kuksis, A. [1988] Biochim, Biophys. Acta 938, 211-221). The phospholipase A2 and lysophospholipase activities were renatured following nonreducing sodium dodecyl sulphate polyacrylamide gel electrophoresis of the total membrane proteins and were shown to migrate as a component of a protein band having a relative molecular mass of 170 kDa. This band accounted for approximately 1% of the total Coomassie Blue staining proteins. Phospholipase B was also shown to be solubilized from the membranes, in an active form, by a proteolytic digestion with papain. Papain solubilization resulted in a loss of the hydrophobic properties observed for the intact phospholipase. These results suggest that the active site of the phospholipase projects from the luminal surface of the membrane vesicles. In support of this, phospholipase activity towards exogenous, detergent-solubilized phosphatidylcholine was demonstrated under conditions in which the membranes remained intact. We conclude that the phospholipase B has the characteristics of a stalked, brush-border membrane protein and may be considered as another digestive enzyme anchored in this membrane.
Department of Biochemistry, University of Toronto, C.H. Best Institute, Ont., Canada.
S
S
Pind
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Kuksis
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007583:Jejunum / Q000201:enzymology*; D008245:Lysophospholipase / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism; D010740:Phospholipases / Q000032:analysis*; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D051381:Rats; D012995:Solubility
D008565:Membrane Proteins; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D008245:Lysophospholipase; D010206:Papain