Paul Canioni

Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improved method for the determination of pancreatic lipase and colipase

The rate of hydrolysis of long chain triglycerides by pure bovine pancreatic lipase has been determined in the presence of variable amounts of bile salts and colipase. Cofactor-free lipase is strongly inhibited by sodium taurodesoxycholate and by mixed bovine bile salts at concentrations higher than the critical micellar concentration. Bile salt inhibited lipase is reactivated by the addition of bovine colipase. Gel filtration of pancreatic juice from several species (Cow, dog, pig) on Sephadex G 100 allows the separation of lipase from colipase. It is found that the enzyme catalyzed hydrolysis of long chain triglycerides by pancreatic lipase from one species is activated by the addition of colipase from other species. Studies on the activation of pancreatic lipase by colipase in the presence of bile salts allowed the re-evaluation of optimal conditions for the determination of lipase and the development of a procedure to assay colipase.

Horse pancreatic lipase. Interaction with colipase from various species.

Horse pancreatic lipase has been purified from tissue homogenates. Molecular and catalytic properties of horse lipase are comparable to those of the pancreatic lipases previously isolated. Kinetic studies of the inhibition of horse lipase activity by bile salts and of reactivation by pure colipase from three species (horse, ox and pig) allowed to calculate the apparent dissociation constant (Kd) of the lipase-colipase complex in the presence of the substrate (triolein). Identical values of Kd were found in all three cases (Kd = 1.1 10(-9) M). These values are lower by several orders of magnitude than that published for the binding between lipase and colipase in the absence of substrate. Qualitative experiments show that the activation of horse lipase can be accomplished by rat, dog and chicken colipase as well. The interaction between lipase and colipase is enhanced when the complex is adsorbed at the lipid-water interface. This specific protein-protein interaction is preserved in heterologous mixtures using colipases from other animal species.

Pancreatic and microbial lipases: a comparison of the interaction of pancreatic colipase with lipases of various origins.

Conjugated bile salts inhibit the hydrolysis of triglycerides (TG) by the lipases fromRhizopus arrhizus andGeotrichum candidum. This occurs for detergent concentrations similar to those which suppress the action of mammalian pancreatic lipases upon the same substrates. However, in opposition with what is observed with the latter enzymes, the activity is not restored by the addition of pancreatic colipase. Both pancreatic andR. arrhizus lipases are inactivated at tributyrin/water interface, but only the first enzyme is protected against this surface denaturation by the pancreatic cofactor. These observations suggest that colipases synthesized in mammalian pancreas display specific interaction towards the lipases made by the same organ.

Characterization of Triton X 100 extracted colipase from porcine pancreas.

Colipase was isolated from porcine pancreas homogenate prepared in the presence of detergent (Triton X 100). After precipitation by ammonium sulfate and ethanol, the cofactor was purified by chromatography on SP-Sephadex in the presence of Triton X 100 and on DEAE-cellulose in the absence of detergent. Two molecular forms of porcine colipase were obtained. They represent 80 per cent (colipase A) and 20 per cent (colipase B), respectively, of the total colipase. Valine is the N-terminal residue of both proteins. Their aminoacid composition is similar to that found by Borgstrom for the two forms of porcine colipase. Determination of the sequence of the first sixteen residues at the N-terminal end of colipase A indicates that the cofactor undergoes no proteolytic degradation in this region of the molecule when extraction is carried out in the presence of detergent. The recovery of colipase is about 30 per cent.

Studies on bovine pancreatic lipase and colipase

1. n1. Purification of bovine pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3) was achieved from pancreas and pancreatic juice. Purification includes (NH4)2SO4 fractionation, chromatography on DEAE-cellulose at pH 7.0 and CM-cellulose at pH 5.0 and gel filtration on Sephadex G-100. Gel filtration allowed an estimated molecular weight of the enzyme as 48500. n n2. n2. Purified bovine lipase displays partial activity toward emulsified short and long chain triglycerides. Maximal activity is reached upon addition of a fraction separated from the enzyme during the DEAE-cellulose chromatography. Properties of this fraction are analogous to those of the already well characterized porcine cofactor of lipase (colipase). Porcine and bovine colipase are equally effective for activation of bovine lipase. From the observed effect of colipase on maximum rate of hydrolysis of emulsified triglycerides, it is suggested that the cofactor might be involved in the rate-limiting step of lipolysis.

Phosphorus-31 nuclear magnetic resonance study of post mortem catabolism and intracellular pH in intact excised rabbit muscle

Phosphorus-31 nuclear magnetic resonance has been used to study the post mortem catabolism of high-energy phosphate compounds and the associated intracellular pH variation in pure fast- and slow-twitch rabbit muscles and in rabbit muscle with mixed fiber types. Comparative results from pure fiber types are reported for the first time. Large amounts of glycerophosphorylcholine (14.1 mumol/g fresh tissue) are found in the internal conoidal bundle (ICB), a pure oxidative slow twitch muscle, whereas the m. psoas major (PM), a pure glycolytic fast twitch muscle and the m. gastrocnemius caput medialis (GCM), with mixed fiber types, are devoid of the same metabolite. The total content of phosphorylated metabolites is constant among the three muscle types. The time-dependent post mortem changes in phosphorylated metabolites display the expected rapid drop in phosphocreatine and a simultaneous increase in intracellular inorganic phosphate. However, the ATP level remains constant during more than 2 h. Rate constants for metabolite breakdown and apparent ATPase activity have been determined. The comparative kinetics of intracellular acidosis at 25 degrees C yield rates of 3.3 X 10(-3) pH unit/min for PM, 2.7 X 10(-3) pH unit/min for GCM and 3.0 X 10(-3) pH unit/min for ICB. Initial intracellular pH values are 7.07, 7.20 and 7.02, respectively. Upon aging, the heterogeneity of the Pi signal reflects the existence of cellular compartments with different internal pH. The results suggest that the more intense low-pH Pi signal arises from the sarcoplasmic reticulum while the less intense resonance would reflect the sarcoplasmic higher pH. The temperature effect on post mortem catabolism in the 15-25 degrees C range has been documented. As expected, phosphocreatine and ATP breakdown increase with temperature but at a higher rate for slow-twitch ICB than for fast-twitch PM.

LIMITED TRYPSINOLYSIS OF PORCINE AND EQUINE COLIPASES - SPECTROSCOPIC AND KINETIC-STUDIES

Porcine and equine colipases have been submitted to mild tryptic digestion. Proteolysis occurs at the Arg5-Gly6 bond with the loss of the N-terminal pentapeptide. Studies of native and trypsin-treated colipases by circular dichroism and laser chemically induced dynamic nuclear polarization indicate that proteolysis induces conformational changes in the region of the tyrosine cluster. Experiments in the presence of phospholipid provide further evidence showing that these residues are in or close to the region of the protein interacting with aggregated lipids. Kinetic studies of the reaction of bile salt-inhibited lipase with emulsified triolein in the absence and in the presence of lecithin show that tryptic hydrolysis of the protein cofactor increases its affinity for the enzyme in the presence of lipid substrate. In both cases, it was found that the apparent dissociation constant of the lipase-colipase complex is decreased by one order of magnitude. Our results confirm that the biological activity of the lipase cofactor is enhanced by specific tryptic cleavage in the amino terminal region of the polypeptide and support the suggestion by Borgström et al. (Borgström, B., Wieloch, T., Erlanson-Albertsson (1981) FEBS. Lett. 108, 407-410) that the secreted form of colipase is a precursor.

Isolation and partial structural characterization of chicken pancreatic colipase

Colipase has been isolated from acidic extracts of chicken pancreatic tissue homogenized with Triton X-100. The cofactor fully activates bile salt inhibited mammalian lipases. The amino terminal sequence of the avian protein has been determined up to position 39 and compared to the homologous region of the mammalian colipases (pig, horse, man) previously studied. From this comparison, it appears that a high degree of homology exists between the proteins.

Ovine pancreatic lipase : purification and some properties.

Lipase has been isolated from sheep pancreas. The lipoprotein complex formed in pancreas homogenates by the enzyme and endogenous lipids is split by treatment with acetone. Lipase is further purified by ion-exchange chromatography and gel filtration. The molecular weight and the amino-acid composition of ovine lipase are very similar to that of the porcine and bovine enzymes. As previously found in bovine lipase, no carbohydrate is covalently bound to the polypeptide chain which has a N-terminal residue of lysine. The study of the catalytic properties of ovine pancreatic lipase indicates that the enzyme is fully activated by colipase from various species in the presence of conjugated bile salt micellar solutions.

Conformation of colipase. Prediction of the secondary structure, circular dichroism and 360 MHz proton NMR studies of porcine colipase A.

The secondary structure of porcine colipase (93 residues) was established according to the predictive method of Chou and Fasman (Chou, P.Y. and Fasman, G.D. (1974) Biochemistry 13, 211--222 and 222--245). The relative composition of the conformational regions was as follows: 5% alpha-helix (region 39--44), 25% beta-sheet (three regions, 7--11, 49--57 and 77--85) and eight beta-turns corresponding to 32% of the polypeptide. Colipase contains a large proportion (about 35%) of unordered structure. Estimated values for the alpha-helix and beta-sheet contents from the circular dichroism spectrum were in good accordance with the predicted model. A less satisfactory value was found for the beta-turns. A characteristic feature of the far ultraviolet dichroic spectrum is the presence of an unusual positive band at 225 nm that might be indicative of a particular spatial arrangement of the chromophores in the molecule. Two tyrosines (Tyr56 and Tyr57) and one histidine (His86) are at close vicinity in the three dimensional structure of the protein as shown by proton NMR studies. These residues are located at the end of two beta-sheet hydrophobic regions(49--57 and 77--85) which might play a role in the association of colipase with the lipid-water interface as indicated by results of the NMR studies of the taurodeoxycholate-colipase complex.