sodium-taurodeoxycholate and tributyrin

Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms, whereas very little is known about plant TAG lipases. The lipolytic activity occurring in all the laticies is known to be associated with sedimentable particles, and all attempts to solubilize the lipolytic activity of Carica papaya latex have been unsuccessful so far. However, some of the biochemical properties of the lipase from Carica papaya latex (CPL) were determined from the insoluble fraction of the latex. The activity was optimum at a temperature of 37°C and a pH of 9.0, and the specific activities of CPL were found to be 2,000 ± 185 and 256 ± 8 U/g when tributyrin and olive oil were used as substrates, respectively. CPL was found to be active in the absence of any detergent, whereas many lipases require detergent to prevent the occurrence of interfacial denaturation. CPL was inactive in the presence of micellar concentrations of Triton X-100, sodium dodecyl sulfate (SDS) and tetradecyl trimethylammonium bromide (TTAB), and still showed high levels of activity in the presence of sodium taurodeoxycholate (NaTDC) and the zwitterionic Chaps detergent. The effects of various proteases on the lipolytic activity of CPL were studied, and CPL was found to be resistant to treatment with various enzymes, except in the presence of trypsin. All these properties suggest that CPL may be a good candidate for various biotechnological applications.

Topics: Carica; Detergents; Enzymes, Immobilized; Latex; Lipase; Lipolysis; Octoxynol; Olive Oil; Plant Oils; Sodium Dodecyl Sulfate; Substrate Specificity; Taurodeoxycholic Acid; Triglycerides; Trypsin

The aim of this study was to check some biochemical and structural properties of ostrich and turkey pancreatic lipases (OPL and TPL, respectively).. Limited proteolysis of OPL and TPL was performed in conditions similar to those reported for porcine pancreatic lipase.. In the absence of bile salts and colipase, OPL failed to catalyze the hydrolysis of pure tributyrin or efficiently hydrolyze olive oil emulsion. When bile salts and colipase were preincubated with the substrate, the OPL kinetic behavior remained linear for more than 30 minutes. The enzyme presented a penetration power value into an egg phosphatidylcholine monomolecular film that was comparable to that of HPL and lower than that of TPL. Chymotrypsin, trypsin, and thermolysin were able to hydrolyze OPL and TPL in different ways. In both cases, only N-terminal fragments accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic cleavage of OPL and TPL completely degraded the enzymes. Nevertheless, chymotryptic attack generated 35-kd and 43-kd forms for TPL and OPL, respectively. Interestingly, the OPL 43-kd form was inactive, whereas the TPL 35-kd protein conserved its lipolytic activity.. OPL, TPL, and mammal pancreatic lipases share a high amino acid sequence homology. Further investigations are, however, needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of lipases.

Topics: Amino Acid Sequence; Animals; Chymotrypsin; Colipases; Deoxycholic Acid; Linoleic Acid; Lipase; Molecular Sequence Data; Olive Oil; Pancreas; Phosphatidylcholines; Plant Oils; Protein Structure, Tertiary; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Struthioniformes; Substrate Specificity; Taurodeoxycholic Acid; Thermolysin; Triglycerides; Trypsin; Turkeys

Pancreatic triglyceride lipase (PTL) requires colipase for activity. Various constituents in meals and in bile, particularly bile acids, inhibit PTL. Colipase restores activity to lipase in the presence of inhibitory substances like bile acids. Presumably, colipase functions by anchoring and orienting PTL at the oil-water interface. The x-ray structure of the colipase.PTL complex supports this model. In the x-ray structure, colipase has a hydrophobic surface positioned to bind substrate and a hydrophilic surface, lying opposite the hydrophobic surface, with two putative lipase-binding domains, Glu(45)/Asp(89) and Glu(64)/Arg(65). To determine whether the hydrophilic surface interacts with PTL in solution, we introduced mutations into the putative PTL binding domains of human colipase. Each mutant was expressed, purified, and assessed for activity against various substrates. Most of the mutants showed impaired ability to reactivate PTL, with mutations in the Glu(64)/Arg(65) binding site causing the greatest effect. Analysis indicated that the mutations decreased the affinity of the colipase mutants for PTL and prevented the formation of PTL.colipase complexes. The impaired function of the mutants was most apparent when assayed in micellar bile salt solutions. Most mutants stimulated PTL activity normally in monomeric bile salt solutions. We also tested the mutants for their ability to bind substrate and anchor lipase to tributyrin. Even though the ability of the mutants to anchor PTL to an interface decreased in proportion to their activity, each mutant colipase bound to tributyrin to the same extent as wild type colipase. These results demonstrate that the hydrophilic surface of colipase interacts with PTL in solution to form active colipase.PTL complexes, that bile salt micelles influence that binding, and that the proper interaction of colipase with PTL requires the Glu(64)/Arg(65) binding site.

Topics: Alanine; Amino Acid Substitution; Arginine; Bile Acids and Salts; Binding Sites; Colipases; Glutamic Acid; Humans; Kinetics; Lipase; Micelles; Models, Molecular; Mutagenesis, Site-Directed; Pancreas; Protein Conformation; Taurodeoxycholic Acid; Triglycerides

In the absence of colipase and bile salts, using tributyrin emulsions or monomolecular films of dicaprin at low surface pressure, we observed that no significant lipase activity can be measured with Human Pancreatic Lipase (HuPL), Horse Pancreatic Lipase (HoPL) or Dog Pancreatic Lipase (DPL). Only Porcine Pancreatic Lipase (PPL) and recombinant Guinea Pig Pancreatic Lipase Related Protein of type 2 (r-GPL) hydrolyse pure tributyrin in the absence of any additive, as well as dicaprin films at low surface pressures. The former lipases may lack enzyme activity because of irreversible interfacial denaturation due to the high energy existing at the tributyrin/water interface and at the dicaprin film surface at low surface pressures. The enzyme denaturation cannot be reflected in the number of disulfide bridges, since all the pancreatic lipases tested here contain six disulfide bridges, but behaved very differently at interfaces. We propose to use the surface pressure threshold, as determined using the monomolecular technique, as a criterion for classifying lipases in terms of their sensitivity to interfacial denaturation.

Topics: Animals; Colipases; Diglycerides; Dogs; Emulsions; Guinea Pigs; Horses; Humans; Kinetics; Lipase; Pancreas; Recombinant Proteins; Species Specificity; Stomach; Surface Properties; Surface Tension; Swine; Taurodeoxycholic Acid; Triglycerides

The kinetics of trypsin activation of pancreatic procolipase was investigated and the pH dependence of the binding of procolipase and colipase to a tributyrine-bile salt interface studied. The Km was 0.06 mM and kcat 8 s-1, and was of the same order of magnitude as for the activation of pancreatic zymogens. At basic pH values colipase had a higher affinity for the tributyrine-bile salt interface as compared to procolipase. The trypsin activation of procolipase ensures a rapid degradation of dietary lipids in the intestine.

Topics: Animals; Colipases; Enzyme Precursors; Hydrogen-Ion Concentration; Kinetics; Micelles; Protein Precursors; Proteins; Swine; Taurodeoxycholic Acid; Triglycerides; Trypsin

Topics: Animals; Bile Acids and Salts; Colipases; Fenfluramine; Lipase; Lipolysis; Pancreas; Proteins; Swine; Taurodeoxycholic Acid; Triglycerides

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.

Topics: Animals; Binding Sites; Colipases; Fat Emulsions, Intravenous; Kinetics; Models, Biological; Proteins; Swine; Taurodeoxycholic Acid; Triglycerides