Purification and partial characterization of feline classical pancreatic lipase.
Classical pancreatic lipase has been purified and partially characterized in many species. The objective of this project was to purify feline classical pancreatic lipase (fPL) from pancreatic tissue and partially characterize this protein. Pancreata were collected from cats (Felis catus) euthanized for unrelated research projects. Fat was removed by trimming away grossly visible fat and by extraction in organic solvents. The delipidated pancreatic extract was further purified by extracting the enzymes in a Tris-buffer containing two different protease inhibitors, benzamidine and phenylmethylsulfonyl fluoride, followed by anion-exchange, size-exclusion, and cation-exchange chromatography. Feline pancreatic lipase was successfully purified from feline pancreatic tissue. The purified product showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of approximately 52.5 kDa. Exact molecular mass was determined by mass spectrometry as 52.4 kDa. Approximate specific absorbance at 280 nm of fPL was 1.18 for a 1 mg/ml solution. N-terminal amino acid sequence of the first 25 amino acid residues showed the sequence Lys-Glu-Ile-?-Phe-Pro-Arg-Leu-Gly-?-Phe-Ser-Asp-Asp-Ala-Pro-Trp-Ala-Gly-Ile-Ala-Gln-Arg-Pro-Leu. This sequence showed close homology with the amino acid sequence of classical pancreatic lipase in other species.
Steiner
Jörg M
JM
Gastrointestinal Laboratory, Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA. jsteiner@cvm.tamu.edu
Wilson
Benjamin G
BG
Williams
David A
DA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001550:Benzamidines / Q000494:pharmacology; D002412:Cations; D002415:Cats; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D001550:Benzamidines; D002412:Cations; D004791:Enzyme Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D008049:Lipase; C032157:benzamidine