Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy.
Phospholipase A2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.
Grainger
D W
DW
Institut für Organische Chemie, Universität Mainz, F.R.G.
Reichert
A
A
Ringsdorf
H
H
Salesse
C
C
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000032:analysis; D001665:Binding Sites; D004134:Dimyristoylphosphatidylcholine / Q000032:analysis; D004546:Elapid Venoms / Q000032:analysis; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007089:Image Enhancement; D008856:Microscopy, Fluorescence; D010316:Particle Size; D010714:Phosphatidylethanolamines / Q000032:analysis; D010740:Phospholipases / Q000032:analysis*; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D010743:Phospholipids / Q000032:analysis*
D004546:Elapid Venoms; D005456:Fluorescent Dyes; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D015060:1,2-Dipalmitoylphosphatidylcholine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D004134:Dimyristoylphosphatidylcholine; C018525:1,2-dimyristoylphosphatidylethanolamine