alpha-chymotrypsin and tributyrin

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

In the intestine, the hydrolysis of triglycerides by pancreatic lipase is performed only in the presence of colipase, whose function is to anchor lipase to the bile-salt-coated lipid interface. Biochemical and crystallographic data on porcine and human lipases have shown that the molecule is made of two well-delimited domains. In order to get more information on the role of the domains in catalysis and colipase binding, we performed limited proteolysis on lipase from various species and obtained different patterns of cleavage. In the case of porcine and human lipases, only the C-terminal domain (12 kDa) could be obtained after chymotryptic attack, whereas in the horse enzyme the cleavage of the Leu410-Thr411 bond gave rise to a large N-terminal (45 kDa) and a small C-terminal (4 kDa) fragment. The isolated porcine and human C-terminal domains were completely inactive towards emulsified tributyrin, though were able to bind colipase. Conversely, the horse 45 kDa fragment retained the lipase activity but failed to correctly bind colipase. This work definitely proves that catalysis and colipase binding are separate events involving topographically distinct regions of the molecule and focuses attention on the role of the C-terminal domain in colipase binding.

Topics: Amino Acid Sequence; Animals; Cattle; Chymotrypsin; Colipases; Horses; Humans; Lipase; Molecular Sequence Data; Pancreas; Peptide Fragments; Peptide Mapping; Protein Conformation; Swine; Triglycerides