PURIFICATION AND MOLECULAR CHARACTERIZATION OF LAMB PREGASTRIC LIPASE

Lamb pregastric lipase (LPGL) was purified from pharyngeal tissues. Th e purification procedure was based on an aqueous extract containing 0. 7% Tween 80 which was chromatographed on DEAE-cellulose anion-exchange r and adsorbed on HA-Ultrogel followed by gel filtration on Ultrogel A cA-54. The final enzymatic preparation, where the overall activity rec overy was 3%, showed a single protein band on SDS-PAGE with a molecula r 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 unc ontrolled proteolysis of LPGL during the purification lead to a 45 kDa form which was previously observed in human lysosomal acid lipase (HL AL) and rabbit gastric lipase (RGL). The labile bond X(54)-Leu(55) was identified. isoelectric focusing of LPGL reveals a major band corresp onding to an isoelectric point of 4.8. The pure enzyme displayed speci fic 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-imm unoreactivity of LPGL was observed with purified anti-human gastric li pase polyclonal antibodies. Determination of the amino-acid sequence o f 62 residues revealed a high degree of homology with other known pred uodenal lipases.