Regulation of fatty acid oxygen-18 exchange catalyzed by pancreatic carboxylester lipase. 2. Effects of lateral lipid distribution in mixtures with phosphatidylcholine

Abstract

The lipase-catalyzed exchange of the carboxyl oxygens of 13,16-cis,cis-docosadienoic acid (DA) was studied in the presence of a nonsubstrate matrix lipid, 1-palmitoyl-2-oleoylphosphatidylcholine. For mixed lipid films at the argon-water interface exposed to pancreatic carboxylester lipase (EC 1.1.1.13), the extent of oxygen exchange showed an abrupt increase as the abundance of DA in the interface was increased from 0.5 to 0.6 mole fraction. This compositional range was independent of the level of enzyme used and of the surface pressure, i.e., lipid packing density, of the film. Concomitant with the transition was a change in the apparent mechanism of exchange from coupled to random sequential. Like the extent of oxygen exchanged, the shift in mechanism was independent of all variables except the lipid composition of the interface. The absence of any chemical or physical change accompanying the exchange reaction precludes mechanistic explanations based on the generation of reaction products by the enzyme. Instead, the results suggest that the lateral distribution of DA in phosphatidylcholine-DA interfaces regulates the expression of carboxylester lipase activity and its apparent mechanism. Preliminary measurements give an average cluster size of 1825 molecules of DA when its mole fraction is 0.35. As the DA content of the interface reaches 0.5-0.6, there appears to be a lipid head-group based percolative transition in which DA becomes the continuum. Because this transition involves the lateral organization of the lipids themselves, other interfacially active enzymes may be regulated similarly.

Is Lateral Phase Separation Required for Fatty Acid To Stimulate Lipases in a Phosphatidylcholine Interface?

“…These are low at low mole fractions of [180]2DA and increase to values near 1.0 at [180]2DA mole fractions above 0.45. In the case of the data obtained with CEL, the increase in the parameter values is not quite as abrupt as observed earlier with [lsO]2DA-POPC monolayers (Muderhwa & Brockman, 1992a) or with PL in the present or the earlier (Muderhwa & Brockman, 1992b) study. Irrespective of this small difference, the mechanism parameter clearly shows discontinuous behavior as [180]2DA mole fraction exceeds 0.45.…”

“…This requirement is not absolute, but being able to measure substrate susceptibility to PL and CEL at monolayer surface pressures >20 mN/m minimizes potential contributions of lipase surface denaturation to the results . Using the same protocol applied earlier to DA-POPC films (Muderhwa & Brockman, 1992a;Muderhwa & Brockman, 1992b), the lipase-catalyzed exchangeability of oxygen between [lsO] 2-DA in LE monolayers of MPPC and subphase water at 24 °C and pH 6.6 was measured. Shown in Figure 8 are the extents of [1sO]2DA carboxyl oxygen atoms exchanged during 10 min following addition of CEL lipase (30 nM) or PL (10 nM) in the aqueous subphase.…”

“…For example, fatty acids and other anionic additives promote the adsorption of phospholipase A2 to zwitterionic phospholipid bilayers (reviewed in Ramirez and Jain (1991)). Effects of fatty acids on both interfacial enzyme adsorption and subsequent catalysis have been described for pancreatic carboxylester lipase (CEL)1 acting at interfaces comprised of pure films of 13,16-cis,cis-docosadienoic acid (DA) (Muderhwa et al, 1992) and mixed monolayers of DA and l-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) (Muderhwa & Brockman, 1992a). Fatty acid was the substrate in these studies, its catalyticallyproductive interaction with the enzyme having been monitored by lipase-catalyzed exchange of carboxyl oxygens with those of water (Muderhwa et al, 1992).…”

“…Fatty acid was the substrate in these studies, its catalyticallyproductive interaction with the enzyme having been monitored by lipase-catalyzed exchange of carboxyl oxygens with those of water (Muderhwa et al, 1992). As the DA mole fraction was increased from 0.4 to 0.6 in liquid-expanded (LE) monolayers of POPC, the extent of DA oxygens exchanged by CEL added to the aqueous subphase increased abruptly (Muderhwa & Brockman, 1992a). This apparently critical transition in fatty acid availability to CEL can only in part be attributed to increased adsorption of the enzyme to the interface as DA content was increased ; Muderhwa & Brockman, 1990).…”