Purification of zymogen granules from monkey parotid glands

Abstract

A method giving highly purified zymogen granules from Macaca irus and Cercopithecus aethiops parotid glands in reported. A 0.3 M sucrose medium for homogenization was supplemented with 10 mM tris/HCl, pH 7.3, and 0.1 mM lauric acid to stabilize the fragile monkey zymogen granules. Nuclei and cell debris were sedimented at 150 times g. A "crude" zymogen granule fraction was trapped in the 1.0 M sucrose layer of a discontinuous sucrose gradient at 1000 times g. Equilibrium centrifugation in a continuous sucrose gradient gave a fraction of zymogen granules at 1.85 M sucrose. Compared to the homogenate, this fraction exhibited about two-fold increase in zymogen granule marker, whereas mitochondrial marker was reduced to 1/4 and lysosomal marker to 1/2. A low level of contamination from other cell organelles was confirmed by electron microscopic investigation.