Abstract
In the duodenum, pancreatic lipase (PL) develops its activity on triglycerides by binding to the bile-emulsified oil droplets in the presence of its protein cofactor pancreatic colipase (PC). The neutron crystal structure of a PC-PL-micelle complex (Hermoso, J., Pignol, D., Penel, S., Roth, M., Chapus, C., and Fontecilla-Camps, J. C. (1997) EMBO J. 16, 5531-5536) has suggested that the stabilization of the enzyme in its active conformation and its adsorption to the emulsified oil droplets are mediated by a preformed lipase-colipase-micelle complex. Here, we correlate the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. The method of small angle neutron scattering with D(2)O/H(2)O contrast variation was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure. In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo.
Highlights
Pancreatic lipase (PL)1 hydrolyzes triglycerides at the oilwater interface
We carried out a series of small angle neutron scattering (SANS) experiments on the pancreatic colipase (PC)-pancreatic lipase (PL)-bile salt system coupled to PL inhibition by E600 to investigate the ability of different lipids to activate the enzyme (E600 inhibition requires an exposed active site and is indicative of the opening of the flap)
Because the SANS method coupled to contrast variation is very well adapted to obtaining structural information on systems composed of two components of different scattering density (such as lipid-protein associations [21]), we characterized the formation of a PC-PL-taurodeoxycholate micelle complex in solution
Summary
The abbreviations used are: PL, pancreatic lipase; PC, pancreatic colipase; NaTDC, sodium taurodeoxycholate; NaTC, sodium taurocholate; E600, diethyl p-nitrophenylphosphate; SANS, small angle neutron scattering; Rg, radius of gyration. The structure of the homospecific porcine lipase-colipase complex [9] was solved from crystals obtained in the presence of the nonionic detergent C8E4 Under these conditions, the enzyme displays an open active site conformation. Calorimetry [10], circular dichroism [11], affinity chromatography [12], spectrofluorometry [13, 14], fluorescence [15,16,17], ultracentrifugation [18], and small-angle neutron scattering studies [19] have been reported These results suggest that PC does not bind a significant amount of bile salt monomers but interacts with a preformed micelle. Because the SANS method coupled to contrast variation is very well adapted to obtaining structural information on systems composed of two components of different scattering density (such as lipid-protein associations [21]), we characterized the formation of a PC-PL-taurodeoxycholate micelle complex in solution
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