Binding of porcine pancreatic lipase and colipase in the absence of substrate studies by two-phase partition and affinity chromatography.

Abstract

M), and was inhibited by bile salts at concentrations above their critical micelle concentration. Below their critical micelle concentration bile salts strengthened binding slightly. Bile salt inhibition of binding was independent of the presence of lecithin in the micelles but could be reversed by fatty acid. At physiological pH (measured at 6.8 + 0.6), 150 InM NaCl and 8 XnM taurodeoxycholate, lipase, and colipase bind together to form a 1:l complex with a dissociation constant of 3.6 X 10m6 M. With this constant and in the absence of substrate, significant lipase-colipase binding in vivo would not be expected to occur at the duodenal protein concentrations measured (lipase = 1.6 + 1.1 X 10e7 M, colipase = 2.0 & 1.7 X 10m7 M). These results support the sequential idea of lipolysis whereby colipase binds substrate first, then lipase binds to colipase. A possible consequence of this system is the uniform distribution of enzymatic attachment sites (colipase) over the surface and among fat droplets which might not occur if lipase and colipase bound substrate simultaneously. Colipase, isolated from the pancreas of the dogfish (Squab acanthius), was found to activate and to bind in solution to porcine pancreatic lipase.