DISTRIBUTION OF NEWLY SYNTHESIZED AMYLASE IN MICROSOMAL SUBFRACTIONS OF GUINEA PIG PANCREAS

Abstract

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuglng, for 2 hr. at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC). Amylase appeared in the following positions In the gradient: (a) a light region which contained rj 35% of total enzymic activity and which coincided with a monomeric rlbosome peak; (b) a heavy region which contained [image] 10% of enzymic activity in a sharp peak but which had very little accompanying OD26O absorption; (c) a pellet at the bottom of the centrifuge tube which contained [image] 20% of the enzymic activity. After 5 to 20 min. in vivo labeling with leucine-1-C14 radioactive amylase was solubilized from these 3 fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had 5 to 10 times the specific activity (CPM/enzymlc activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted bj DOC -spermine was similar tor all three fractions. Hypotonic treatment of the fractions solubilized [image]80% of the total amylase in the fraction from the heavy region of the gradient, but only [image]20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membrances than are ribosomes devoid of nascent protein.