Purification and molecular characterization of lamb pregastric lipase.
Lamb pregastric lipase (LPGL) was purified from pharyngeal tissues. The purification procedure was based on an aqueous extract containing 0.7% Tween 80 which was chromatographed on DEAE-cellulose anion-exchanger and adsorbed on HA-Ultrogel followed by gel filtration on Ultrogel AcA-54. The final enzymatic preparation, where the overall activity recovery was 3%, showed a single protein band on SDS-PAGE with a molecular mass of 50 kDa. LPGL is a glycoprotein containing approx. 14% (w/w) of carbohydrate. Extensive deglycosylation using peptide N-glycosidase F yielded a protein with an apparent molecular mass of 43 kDa. An uncontrolled proteolysis of LPGL during the purification lead to a 45 kDa form which was previously observed in human lysosomal acid lipase (HLAL) and rabbit gastric lipase (RGL). The labile bond X54-Leu55 was identified. Isoelectric focusing of LPGL reveals a major band corresponding to an isoelectric point of 4.8. The pure enzyme displayed specific activities of 950 U mg-1, 300 U mg-1 and 30 U mg-1 at pH 6.0, using tributyroylglycerol, trioctanoylglycerol and trioleoylglycerol as substrates, respectively. Using Western blot analysis, a cross-immunoreactivity of LPGL was observed with purified anti-human gastric lipase polyclonal antibodies. Determination of the amino-acid sequence of 62 residues revealed a high degree of homology with other known preduodenal lipases.
De Caro
J
J
Laboratoire de Lipolyse Enzymatique, UPR 9025, CNRS, Marseille, France.
Ferrato
F
F
Verger
R
R
De Caro
A
A
D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010614:Pharynx / Q000201:enzymology*; D016415:Sequence Alignment; D012756:Sheep; D013379:Substrate Specificity
D000596:Amino Acids; D008049:Lipase; C043101:gastric triacylglycerol lipase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase