Purification and characterization of pregastric esterase from calf.
Calf pregastric esterase (PGE) was purified from calf gullet tissues. The tissue was excised and lyophilized, and lipid materials were extracted with acetone and n-butanol at -20 degrees C. Proteins were extracted from the delipidated tissue with a buffer containing a chaotropic salt (NaSCN) to solubilize hydrophobically bound protein aggregates. Calf PGE precipitated from the crude extract at pH 5.0. The precipitated, solubilized proteins were subjected to anion-exchange chromatography on DEAE-Sephacel, and the enzymatic activity was eluted using a linear gradient from 0.10 to 0.50 M NaCl at pH 8.0. Fractions with high specific activities were then chromatographed twice using gel filtration on Sephadex G-100. The resultant enzyme was shown to be pure upon discontinuous electrophoresis in 12% polyacrylamide containing 0.1% sodium dodecyl sulfate (SDS-PAGE). From SDS-PAGE gel patterns, a molecular weight of 49,000 was determined. The amino acid composition of the enzyme allowed calculation of an "average hydrophobicity" (Bigelow index) of 1150 cal/residue. This indicates that calf PGE is relatively hydrophobic, being similar to proteins such as alpha-lactalbumin and bovine serum albumin in average hydrophobicity.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004947:Esophagus / Q000201:enzymology*; D008049:Lipase / Q000302:isolation & purification*; D008970:Molecular Weight; D012997:Solvents
D000596:Amino Acids; D012997:Solvents; D008049:Lipase; C043101:gastric triacylglycerol lipase