α-Amylase et lipase du pancreas de rat. Purification chromatographique, recherche du poids moleculaire et composition en acides amines

Abstract

1. 1.|A rapid and efficient method of preparation of the cationic enzymes of the rat exocrine pancreas is described. The hydrolases are solubilized quantitatively and afterwards chromatographed on 2 coupled columns of Sephadex G-25 and DEAE-cellulose in 13 mM Tris-HCl buffer (pH 8.2). Filtration on Sephadex G-100 separates 7 or 8 constituents and among them are amylase and lipase. The latter enzymes are purified on Bio-Gel P-60 and on CM-cellulose respectively, after which they show homogeneity by disc electrophoresis. 2. 2.|Amylase and lipase constitute approx. 10.9% and 0.4% of the pancreatic proteins in rats fed a balanced diet. Starting from 30 animals (15 g of tissue) a yield of about 100 mg of amylase and 4 mg of lipase is usually obtained within 2 days after a 10-fold purification of the first enzyme and a 140-fold purification of the second. 3. 3.|Amylase has an excess of 3.5 basic residues % over acidic residues, contains 3.1% of tryptophan and has a E1 cm1 % value of 20 at 280 mμ. The specific activity is 600 units/mg at 25°. Amylase is increasingly adsorbed on Sephadex and Bio-Gels, when the porosity of these resins increases and when the ionic strength of the buffer decreases. Lipase has an excess of 3.2 basic residues % over acidic residues, a E1 cm1 % value of 12 at 280 mμ and a specific activity of 2500 units/mg at 25°. The molecular weight, determined by filtration on Sephadex G-100 and Bio-Gel P-150, is approx. 43 000. This value includes 14% of lipids made of free fatty acids (37%), phospholipids (35%), cholesterol esters (20%) and glycerides (8%). Lauric, myristic and palmitic acids are prominent in these lipids. 4. 4.|The rapid and efficient recovery of rat pancreatic amylase indicates the usefulness of a method that would be valid for other amylases: after filtration of a crude extract on a Sephadex-retaining amylase, this enzyme is filtered on a Bio-Gel from which it is more rapidly excluded than in the preceding filtration. Pig pancreatic amylase has been easily purified in this manner with an efficiency of 80%. © 1968.