J. Decaro et al., PURIFICATION AND MOLECULAR CHARACTERIZATION OF LAMB PREGASTRIC LIPASE, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1252(2), 1995, pp. 321-329
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.