Regulation of polyribosome formation and protein synthesis in the uterus. Isolation of cytoplasmic ribonucleoprotein particles and the principal properties of the cell-free protein-synthesizing system

Abstract

Three procedures for isolating ribonucleoprotein particles from the cytoplasmic fraction of rat-uterus homogenates are described. By procedure 1. ribonucleoprotein particles were isolated in the presence of 5m[image]-Mg2+ and 25 m[image]-K+, and the postmitochondrial supernatant fraction was made to 1.3% (w/v) in potassium deoxycholate. About 50% of the RNA and protein of the microsomal fraction was recovered in the monomeric ribosomes isolated. By procedure 2, ribonucleoprotein par -tides were isolated in the presence of 10m[image]-Mg2+ and 0.1 M-K+, and in the absence of detergent. The ribosomes obtained were primarily polymeric, but recovery of microsomal RNA and protein was only 32%. By procedure 3, ribonucleoprotein particles were isolated according to procedure 1 but without the use of detergent. A mixture of polymeric and monomeric ribosomes was obtained, and the recovery of microsomal RNA and protein was about 60%. Uterine polymeric and monomeric ribosomes, isolated by procedure 3 and designated polyribosomal preparation, were examined for protein-synthesizing capabilities. The principal properties of the cell-free protein synthesizing system containing the polyribosomal preparation are described. The efficiency of amino acid incorporation in the complete system incubated for 30 min. and containing the polyribosomal preparation was found to be either 2.5 molecules of [14C]leucine or 2.2 molecules of [14C]-valine incor-porated/ribosome. Assay of the preparation in the complete cell-free system containing 10m[image]-sodium fluoride indicated that 40% of the incorporation activity is a result of initiation of new polypeptide chains and 60% is due to completion of previously existing chains. Monomeric ribosomes obtained by various treatments of the polyribosomal preparation with sodium fluoride, ribonuclease and potassium deoxycholate had decreased incorporation activity in the cell-free system. However, monomeric ribosomes obtained by treatment with sodium fluoride only had an incorporation activity 50% greater than that of monomers obtained by treatment with ribonuclease only. The results indicate that uterine polymeric and monomeric ribosomes are sites of amino acid incorporation in vivo and in vitro. It is concluded that most polymeric and monomeric ribosomes occurring in the cytoplasmic fraction of the uterus are free and unattached to membranes, and that the polyribosomes are relatively unstable.