title: Lateral lipid distribution is a major regulator of lipase activity. Implications for lipid-mediated signal transduction.
abstract: Pancreatic carboxylester lipase catalyzes the exchange of 18O between water and 13,16-cis,cis-doco-sadienoic acid (DA) in monolayers at the argon-buffer interface (Muderhwa, J.M., Schmid, P.C., and Brockman, H.L. (1992) Biochemistry 31, 141). In mixed monolayers of 18O, 18O-DA and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), both the extent and mechanism of 18O exchange show characteristics of a critical transition in the range of 0.5-0.6 mol fraction of DA (Muderhwa, J.M., and Brockman, H. L. (1992) Biochemistry 31, 149). To determine if the regulatory behavior exhibited on this type of surface is limited to members of the carboxylester lipase gene family (cholinesterases), comparable experiments were performed with a genetically and functionally unrelated lipase, pancreatic colipase-dependent lipase (PL). PL readily catalyzed the exchange of 18O between water and the carboxyl group of DA with enzyme at either monolayer or catalytic levels in the fatty acid-buffer interface. The oxygen exchange reaction obeyed a random, sequential mechanism, indicating that the dissociation of the enzyme.DA complex is much faster than the rate-limiting step in the overall exchange process. Kinetic analysis of oxygen exchange in pure DA monolayers showed a first-order dependence on interfacial PL and DA concentrations from which kcat/Km values were calculated. The oxygen exchange reaction proceeded with a rate constant of 16 x 10(-2) cm2 pmol-1 s-1, a value comparable to that for hydrolysis of the ester substrate, 1,3-dioleoylglycerol. With a monolayer of PL adsorbed to the interfacial phase, kcat/Km for oxygen exchange was about 600-fold lower than the value obtained with catalytic levels of adsorbed enzyme, indicating a possible restriction of substrate diffusion in the protein-covered fatty acid monolayer. With constant bulk PL concentration and mixed lipid monolayers containing DA and the non-substrate lipid, POPC, the extent of oxygen exchange increased abruptly as the abundance of DA in the interface was increased from 0.5 to 0.6 mol fraction. Concomitant with this critical transition was a change in the apparent mechanism of oxygen exchange from coupled to random, sequential. For both the extent of oxygen exchange and its mechanism shift, the critical transition was independent of the lipid packing density, i.e. surface pressure, of the interface. These results show that PL responds similarly to carboxylester lipase with respect to changes in interfacial lipid mole fraction in DA-POPC surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Hormel Institute, University of Minnesota, Austin 55912.
authors.forename: J M
authors.initials: JM
authors.lastname: Muderhwa
authors.forename: H L
authors.initials: HL
authors.lastname: Brockman
mesh_terms: D000818:Animals; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D006706:Homeostasis; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D008081:Liposomes; D008433:Mathematics; D008962:Models, Theoretical; D010103:Oxygen Isotopes; D010179:Pancreas / Q000201:enzymology; D010713:Phosphatidylcholines / Q000494:pharmacology*; D015398:Signal Transduction; D013499:Surface Properties; D013552:Swine
chemical_list: D005231:Fatty Acids, Unsaturated; D008081:Liposomes; D010103:Oxygen Isotopes; D010713:Phosphatidylcholines; C078401:13,16-docosadienoic acid; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D008049:Lipase; C028694:1-palmitoyl-2-oleoylphosphatidylcholine