Purification of classical pancreatic lipase from dog pancreas.
The purification of canine classical pancreatic lipase from canine pancreatic juice, but not from pancreatic tissue, has been reported previously. Given the logistic difficulties associated with collection of pancreatic juice in dogs and efforts to minimize experiments in live animals the objective of this project was to purify canine classical pancreatic lipase from dog pancreas. Dog pancreata were collected from research dogs that had been sacrificed for unrelated research projects. Pancreatic tissue was delipidated using organic solvents. The delipidated pancreatic extract was further purified by extracting the enzymes in a Tris-buffer containing two different protease inhibitors, benzamindine and phenylmethylsulfonyl fluoride (PMSF), followed by anion exchange chromatography, gel-filtration, and cation exchange chromatography. The purified protein showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of approximately 50.7. Isoelectric focusing showed isoelectric points ranging from 6.0 to 6.2. N-terminal amino acid sequencing of the first 25 amino acid residues showed the sequence Lys-Glu-Val-X-Phe-Pro-Arg-Leu-Gly-X-Phe-Ser-Asp-Asp-Ser-Pro-Trp-Ala-Gly-Ile-Val-Glu-Arg-Pro-Leu. This sequence showed close homology with classical pancreatic lipase in pigs, horses, and human beings. We conclude that canine classical pancreatic lipase can be successfully purified from canine pancreatic tissue.
Steiner
Jörg M
JM
Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, TAMU 4474, College Station, TX 77843-4474, USA. jsteiner@cvm.tamu.edu
Williams
David A
DA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000838:Anions; D001550:Benzamidines / Q000494:pharmacology; D002412:Cations; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D003089:Colipases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000187:drug effects / Q000201:enzymology*; D010189:Pancreatic Juice / Q000187:drug effects / Q000201:enzymology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D000596:Amino Acids; D000838:Anions; D001550:Benzamidines; D002412:Cations; D003089:Colipases; D004791:Enzyme Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D008049:Lipase