Measurement of the binding of colipase to a triacylglycerol substrate.
The binding between colipase and two triacylglycerol substrates, tributyrin and Intralipid, in the presence of bile salts have been determined quantitatively by a method based on equilibrium partition in an aqueous two-phase system. In the model proposed the triacylglycerol, in the form of spherical droplets covered with bile salt, is assumed to have a certain number of independent binding sites at the surface for colipase. The binding of colipase to tributyrin at pH 7.0 in the presence of 4 mM sodium taurodeoxycholate and 150 mM NaCl had a dissociation constant Kd = 3.3 . 10(-7) M; the concentration of binding sites was 1.2 . 10(-6) M in a 102 mM tributyrin emulsion. When tributyrin was dispersed in 1 mM and 12 mM sodium taurodeoxycholate the dissociation constant was somewhat higher, 6.3 . 10(-7) M and 6.0 . 10(-7) M, respectively. Thus the binding strength was optimal at 4 mM sodium taurodeoxycholate. At the same time the concentration of binding sites decreased from 4.1 . 10(-6) M for 1 mM sodium taurodeoxycholate to 1.4 . 10(-6) M for 12 mM sodium taurodeoxycholate. This indicated that at higher bile salt concentration the bile salt acted as non-competitive inhibitors on the binding of colipase to the substrate, thus binding to other sites than colipase to the substrate. The binding of colipase to Intralipid, an emulsion of a long-chain triacylglycerol stabilized with phosphatidylcholine and glycerol, was more complex with indications of several different binding sites with different affinity. The majority of these had a dissociation constant Kd = 1.2 . 10(-6) M in the presence of 4 mM sodium taurodeoxycholate and 150 mM. With each droplet having a diameter of 10(-4) cm, the number of binding sites on each droplet was determined to 1.96 . 10(5) and the average area available for each colipase molecule to 1600 A at saturation. Colipase on denaturation has a surface of 1320 A.
D000818:Animals; D001665:Binding Sites; D003089:Colipases / Q000378:metabolism*; D005217:Fat Emulsions, Intravenous / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D011506:Proteins / Q000378:metabolism*; D013552:Swine; D013657:Taurodeoxycholic Acid / Q000494:pharmacology; D014280:Triglycerides / Q000378:metabolism*
D003089:Colipases; D005217:Fat Emulsions, Intravenous; D011506:Proteins; D014280:Triglycerides; D013657:Taurodeoxycholic Acid; C005830:tributyrin