Hydrolysis of monodisperse phosphatidylcholines by phospholipase A2 occurs on vessel walls and air bubbles.
Hydrolysis of monodisperse short chain phosphatidylcholines, far below their critical micelle concentration, by phospholipase A2 (PLA2) and other interfacial enzymes is characterized. Results show that virtually all the observed hydrolysis by pancreatic and human inflammatory PLA2 occurs on surfaces of the reaction vessel or air bubbles. Conditions to eliminate such extraneous contributions at low substrate concentrations are established. Premicellar aggregates are apparently formed near the critical micelle concentration. The observation window at low substrate concentrations is used to obtain an upper limit estimate of the rate of hydrolysis through the monodisperse Michaelis complex. A limit estimate of <0.1 s-1 is obtained for the hydrolysis of monodisperse substrates by pig pancreatic phospholipase A2. These results show that the observed rate of hydrolysis of dihexanoyl- and diheptanoylphosphatidylcholines with pig pancreatic phospholipase A(2) through the monomer path is insignificant compared to the rate of >1000 s-1 seen at the saturating levels of the micellar substrate. These protocols should be useful for evaluating reactions catalyzed at vessel walls. Implications of these results for assays and models of interfacial activation of pancreatic PLA2 are discussed.
Yu
B Z
BZ
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Berg
O G
OG
Jain
M K
MK
D000327:Adsorption; D000388:Air; D000818:Animals; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008823:Micelles; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D010969:Plastics; D011485:Protein Binding; D011791:Quartz; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods; D013314:Stress, Mechanical; D013499:Surface Properties; D013552:Swine
D008823:Micelles; D010713:Phosphatidylcholines; D010969:Plastics; D012996:Solutions; D011791:Quartz; D010741:Phospholipases A; D054467:Phospholipases A2