Interactions of Colipase with Bile Salt Micelles

Abstract

A detailed investigation by ultracentrifugation of the colipase-taurodeoxycholate system showed the formation of well-defined mixed associations with a sedimentation coefficient of about 2.2 S. The fact that these associations were only detectable above the critical micelle concentration of the salt indicated that micelles rather than monomers were bound to the cofactor.Two technical difficulties must be overcome before the weight of the associations could be measured with a reasonable accuracy. Firstly, the partial specific volume of the associations was determined using a digital microdensimeter and the interferometric system of the ultracentrifuge for concentration determinations. Secondly, due to the fact that micelle concentrations could not be equilibrated by dialysis, even after an extended period of time, an appropriate dilution of the ligand in the buffer compartment was necessary in order to compensate for its fixation by colipase in the solution. Then, the ionic strength dependence of the weight of the associations was found to vary in parallel with that of the micelles and to be in each case equal to the sum of the weights of one colipase molecule and one micelle. Therefore, colipase can be expected to contain a single high affinity site for bile salt micelle binding.During the last few years, several low molecular weight proteins designated colipase have been purified from porcine pancreas. The smallest and structurally best known, colipase 11, contains 84 amino acid residues [I]. It is composed of a central "core" crosslinked by four disulfide bridges and of two "tails" loosely bound by a single bridge [2]. Colipase I, which is the form used throughout this and the next work, differs from colipase I1 by the presence of ten additional residues at the end of the C-terminal tail [l]. Little is known so far about the structure of a third colipase characterized in porcine pancreas, except for the existence of seven N-terminal residues not present in colipases I and I1 [3].Flowing into the duodenum with pancreatic juice [4], colipase has been reported to prevent the inhibition of the lipase-catalyzed hydrolysis of dietary triglycerides by physiological concentrations of bile salts [5]. This effect has been assumed to imply in the first place a bile salt-induced dimerization of colipase and an association of the dimer with lipase [ 6 ] .It has been confirmed in this laboratory [7] that the apparent sedimentation coefficient and molecular weight of colipase are approximately doubled in the presence of sodium taurodeoxycholate. However, the present and following paper will show that this increase results from the binding of a bile salt micelle to the colipase molecule rather than from dimerization. MATERIALS AND METHODS ColipasePorcine pancreatic colipase I was purified to homogeneity by a modification of a recently described technique [7]. The starting material was a commercial pancreas powder (Choay, France) which was exhaustively delipidated in the laboratory prior to use. The extracts were submitted as ...

Interactions of Colipase with Bile Salt Micelles

“…In both cases, a slight increase of the absorbance below the critical micelle concentration suggested the existence of primary micelles already mentioned in a previous publication [6]. The size of sodium taurodeoxycholate micelles is known to be strongly ionic strength dependent (22 and 13 monomers, respectively, in buffer I and I1 [7,1]). …”

“…The calculation was founded upon the consideration of any couple of two points (1) and (2) arbitrarily taken on any of the curves in Fig.3 [12]. The following equations were used : tion that taurodeoxycholate micelles contain 22 monomers in buffer I and only 13 in buffer I1 [7,1]. Therefore, the single variable in Eqn (5) is the number (n) of receptors per colipase molecule.…”

Colipase and maximally activated pancreatic lipase in normal subjects and patients with steatorrhea.

“…Colipases have been isolated and purified from several species (4-7), including man (8), and appear to be quite similar, each having a molecular weight of -10,000, with 100±10 amino acid residues, five disulfide bridges, and little or no carbohydrate (9). The ability to activate pancreatic lipase depends on binding sites for both lipase (10) and lipid micelles (11), and it is thought that the cofactor functions as a bridge between the enzyme and its triglyceride substrates (12,13). In physiological solutions containing 8 mM bile salt, lipase and colipase form a 1:1 complex (10).…”

Conformational dynamics of porcine pancreatic colipase: A 360 MHz proton nuclear magnetic resonance study

“…This signal is obviously exogeneous and arises from impurities presumably firmly bound to the protein, since repeated lyophilisations and prolonged dialyses can reduce the intensity of the peak but fail to eliminate it. These methyl-(and eventually methylene-) rich contaminants could correspond to the large quantities of butanol used during colipase purification, the degradation products of diisopropylfluorophosphate used to prevent proteolytic cleavage of colipase and/or some remaining lipids which might be recognized by the high affinity binding site that colipase has been shown to possess [9,10]. A better resolution of the aromatic region is achieved at lower pH ( fig.3).…”

“…The biological role of colipase is of great importance since it acts in vivo as a cofactor preventing the inhibitory effect of physiological concentrations of bile salts on the intraduodenal lipolysis of dietary triglycerides [1,5,6]. In an attempt to explain the physiological effect of colipase, binary and ternary associations of colipase, lipase and bile salts have been recently investigated under a variety of experimental conditions [7][8][9][10][11][12][13]. Typically, colipase has been shown to bind stoichiometrical amounts of bile salt micelles and to form a binary complex that pancreatic lipase can in turn recognize.…”