Polyribosomal attachment to rat liver and hepatoma endoplasmic reticulum in vitro. A method for its study

Abstract

A system for study and measurement of the attachment in vitro of exogenous polyribosomes to membranes has been presented. Its main features are use of low temperature, post-microsomal supernatant, pyrophosphate and citric acid to remove ribosomes from the surface of rough endoplasmic reticulum, and a method for quantitative separation of unattached from membrane-associated polyribosomes. The following were found. (1) Rough endoplasmic reticulum, from which ribosomes had been removed by treatment with pyrophosphate and citrate, bound over 50% of added polyribosomes, whereas the untreated (or control) rough and smooth endoplasmic reticulum and the smooth endoplasmic reticulum treated with pyrophosphate-citrate did not bind polyribosomes. (2) The polyribosome-binding capacity of rough endoplasmic reticulum stripped of its ribosomes decayed upon storage of the membranes at 0-4 degrees C. The half-life of this decay was about 6 days whereas that of the polyribosome-binding capacity of hepatoma stripped rough endoplasmic reticulum was about 1.5 days. (3) Preparations of stripped rough endoplasmic reticulum after reassociation with polyribosomes in vitro were quite similar to preparations of native rough endoplasmic reticulum as viewed with the electron microscope. Evidence is presented to support the contention that association of polyribosomes with membranes was the result of polyribosomal reattachment to the membranes rather than trapping of the polyribosomes between vesicles of the membranes.

Interactions of liver endoplasmic reticulum membranes and polysomesin vitro

“…The RNA/protein ratios obtained with the various membrane fractions are 0.200 for native rough membranes, and 0.020 for stripped-rough membranes, in good agreement with the data of the literature [1,13,24]. Reconstructed rough endoplasmic reticulum has an RNA/protein ratio of about 0.140, the same as reported by Shires et al [24,25] and slightly above the values of Ragland et al [15]. The impossibility of attaining the original RNA/protein ratio is confirmed by the electron-micrographs, showing that the amount of ribosomes per vesicle of the reconstructed R.E.R.…”

“…A model, termed the "membron", has been proposed, which is a functioning, regulatable, translating polyribosomes complex with a specific surface area of the membrane [14]. To get a better insight into these relationships many investigations have dealt with the binding reaction between isolated polysomes and membranes obtained from rough endoplasmic reticulum by means of various treatments.Preparation techniques and properties of conditioned membranes, ion and temperature requirements of the reaction, methods of analysis of the polysome-membrane complexes, influence of various treatments in vitro have been thoroughly investigated, sometimes with conflicting results [8,10,15,17,18,20,21, 24,25], and the role played by protein, phospholipid and N-acetylneuraminic acid at the sites of polysomal attachment has also been studied [9,22]. Recently, the possibility that reconstructed rough membranes…”

A pool of naturally occurring rat liver ribosomal subunits capable of translating exogenous messenger RNA

“…Membrane bound-and free polysomes were prepared by a modification of the procedure of Ragland et al [2]. Livers from fasted rats were homog.enized in 2 vol of 0.44 M sucrose in TKM buffer (50 mM Tris-HCl, pH 7.4; 25 mM KC1; 5 mM magnesiom acetate).…”

Degranulation and sex‐specific regranulation of reticular membranes from rat liver as studied using a spectrophotometric assay of protein disulphide isomerase

“…To study ribosome-membrane binding it is first necessary to separate the interacting components in as native a form as possible. A variety of procedures have been used to attempt to achieve this purpose including treatment with puromycin plus KC1 [1 ], citrate plus pyrophosphate [2], EDTA alone [3], or plus ribonuclease [4] and LiC1 [5]. A vital consideration in this field is the validity of the methods used for measuring both degranulation and regranulation.…”