Isolation and characterization of messenger ribonucleic acid species for guinea-pig milk proteins from free and membrane-bound polyribosomes.

Abstract

Morphological and cytochemical evidence has suggested the existence of two distinct classes of polyribosomes in secretory cells, those attached to the endoplasmic reticulum (bound polyribosomes) and those apparently free in the cytoplasmic matrix (free polyribosomes). Siekevitz & Palade (1960) proposed that in secretory cells, membranebound polyribosomes were engaged primarily in the synthesis of proteins for export, whereas free polyribosomes synthesized primarily proteins for intracellular requirements. To study the functional specificity of the two polyribosomal classes it is necessary to isolate undegraded polyribosomes and to demonstrate in vitro the ability or otherwise of their mRNA species to direct the synthesis of specific secretory proteins. Free and membrane-bound polyribosomes were isolated from lactating guinea-pig mammary gland by differential centrifugation (see Fig. l ) , essentially as described by Blobel & Potter (1967). The criteria for the successful preparation of intact polyribosomes were based on our ability to isolate undegraded mRNA from the preparations, as judged both by polyacrylamide-gel electrophoresis under denaturing conditions and the ability of these mRNA species to direct the synthesis of protein in cell-free translational systems. The separation of the polyribosomal classes involved a prolonged centrifugation, increasing the susceptibility, particularly of the membrane-bound polyribosomes, to degradation by intracellular ribonuclease activities. In order to inhibit such activities all isolation solutions contained heparin (250,ug/ml) and 0.2~01. of guinea-pig liver high-speed supernatant. Examination of polyribosomes isolated in this manner on 1040% sucrose gradients revealed high-molecular-weight polyribosomal species with little evidence of monoribosomes, diand tri-mers. Similar profiles were obtained from both free and membranebound polyribosomal preparations mRNA isolated from both polyribosomal fractions by chromatography on oligo(dT)-cellulose directed the synthesis of protein in a cell-free system derived from Krebs I1 ascites cells. Immunoprecipitation with antibodies specific for the guinea-pig-milk casein fraction (Table 1 ) clearly demonstrated the presence of mRNA species for secretory proteins in both free and bound polyribosomal fractions. However, on the basis of units recovered after phenol/chloroform extraction, 15 %