Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride

Abstract

1. Treatment of washed rat liver microsomes in a medium containing 0.12m-sucrose, 12.5mm-potassium chloride, 2.5mm-magnesium chloride and 25mm-tris-hydrochloric acid buffer, pH7.6, with 2m-lithium chloride at 5 degrees for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting (14)C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH-2,6-dichlorophenol-indophenol reductase, NADH-neo-tetrazolium reductase, NADH-cytochrome c reductase and ribonuclease activities. 5. (3)H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of (3)H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap.

Studies on the Binding and Distribution of Radioactively Labelled 3′-Methylcholanthrene in Subcellular Fractions of Rat Liver

“…With regard to the binding of 3'-methylcholanthrene and its metabolites to proteins and nucleic acids of rat liver tissue, present evidence indicates that (i) cytoplasmic proteins and ribonucleic acid (RNA) interact with the hydrocarbon or its metabolites (Bresnick, Liebelt, Stevenson and Madix, 1967), the binding to RNA occurring to a very small extent, and (ii) no binding or interaction is found with deoxyribonucleic acid (DNA) (Sporn and Dingman, 1966 Scott-Burden and Hawtrey (1969). The membrane obtained, which has been shown by these workers to be devoid of ribosomes, was suspended in Medium A and washed once by centrifugation.…”

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

“…It is clear from the results reported here that the measurement of the activity of protein-disulphide isomerase can provide information on reticular [5]. membranes which cannot be obtained by any other methodology.…”

“…It can be seen that the standard procedure recommended for degranulation by LiC1 [5] destroys most of the membrane bound protein-disulphide isomerase ( fig.5C). The binding experiments reported by Scott-Burden et al [16,17] employed membranes degranulated by this procedure and the biological relevance of the conclusions drawn can be seriously questioned.…”

“…Lithium chloride degranulation was according to the method of ScottBurden [5] with modifications as described in the individual experiments. All reconstitution experiments were conducted by mixing degranulated membranes with polysomes in such proportions that the ratio of the contents of RNA in the polysomes to the quantity of protein in degranulated membranes was 0.2 to 0.4.…”

“…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.…”