A method for visualizing and quantifying the total complement of free and membrane-bound ribosomes in rat liver

Ramsey, James C.; Steele, William J.

doi:10.1016/0003-2697(79)90663-8

Cited by 34 publications

(12 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The protocol for separating cytosol and ER-bound polysomes in liver tissue was adapted from Ramsey and Steele [57]. Briefly, mice with or without overnight fasting were anesthetized by tribromoethanol and perfused with 20 ml of 0.25M sucrose and 1 mM MgCl 2 solution.…”

Section: Methodsmentioning

confidence: 99%

Polysome Profiling in Liver Identifies Dynamic Regulation of Endoplasmic Reticulum Translatome by Obesity and Fasting

Fan

Blanco

et al. 2012

PLoS Genet

View full text Add to dashboard Cite

Obesity-associated metabolic complications are generally considered to emerge from abnormalities in carbohydrate and lipid metabolism, whereas the status of protein metabolism is not well studied. Here, we performed comparative polysome and associated transcriptional profiling analyses to study the dynamics and functional implications of endoplasmic reticulum (ER)–associated protein synthesis in the mouse liver under conditions of obesity and nutrient deprivation. We discovered that ER from livers of obese mice exhibits a general reduction in protein synthesis, and comprehensive analysis of polysome-bound transcripts revealed extensive down-regulation of protein synthesis machinery, mitochondrial components, and bile acid metabolism in the obese translatome. Nutrient availability also plays an important but distinct role in remodeling the hepatic ER translatome in lean and obese mice. Fasting in obese mice partially reversed the overall translatomic differences between lean and obese nonfasted controls, whereas fasting of the lean mice mimicked many of the translatomic changes induced by the development of obesity. The strongest examples of such regulations were the reduction in Cyp7b1 and Slco1a1, molecules involved in bile acid metabolism. Exogenous expression of either gene significantly lowered plasma glucose levels, improved hepatic steatosis, but also caused cholestasis, indicating the fine balance bile acids play in regulating metabolism and health. Together, our work defines dynamic regulation of the liver translatome by obesity and nutrient availability, and it identifies a novel role for bile acid metabolism in the pathogenesis of metabolic abnormalities associated with obesity.

show abstract

Section: Methodsmentioning

confidence: 99%

Polysome Profiling in Liver Identifies Dynamic Regulation of Endoplasmic Reticulum Translatome by Obesity and Fasting

Fan

Blanco

et al. 2012

PLoS Genet

View full text Add to dashboard Cite

show abstract

“…Preparation ofrat liverfreepolysomes, extraction ofRNA andpreparation of poly(A)-containing RNA Free polysomes from rat liver were prepared by the method of Ramsey and Steele (1976) from a-amylase treated homogenates obtained from male, Sprague-Dawley, non-starved rats (Ramsey and Steele, 1979). In some experiments, the free polysomes were used as such for cell-free protein synthesis, but usually their RNA was extracted with a chloroform-isoamyl alcohol mixture (Oda and Joklik, 1967) after protease K treatment.…”

Section: Methodsmentioning

confidence: 99%

In vitro synthesis and post-translational insertion into microsomes of the integral membrane protein, NADH-cytochrome b5 oxidoreductase.

Borgese¹,

Gaetani²

1983

The EMBO Journal

View full text Add to dashboard Cite

RNA extracted from a free polysome fraction from rat liver was used to direct translation in nuclease‐treated rabbit reticulocyte lysates, and the [35S]methionine‐labelled, in vitro‐synthesized, cytochrome b5 reductase was isolated with specific antibodies. Analysis by SDS‐polyacrylamide gel electrophoresis, non‐equilibrium pH gradient electrophoresis and one‐dimensional peptide mapping failed to reveal any difference between the in vitro‐synthesized reductase and the enzyme endogenous to rat liver microsomes. To study the integration of the in vitro‐synthesized reductase into membranes, carboxypeptidase Y was used as a proteolytic probe. The reductase endogenous to rat liver microsomes was resistant to attack by carboxypeptidase Y, but was degraded to a smaller form when the microsomes were solubilized by detergent. Likewise, the enzyme synthesized in vitro was attacked by carboxypeptidase Y, but became largely resistant after post‐translational incubation with dog pancreatic microsomes, indicating that an integration into membranes similar to the physiological one had occurred. It is concluded that cytochrome b5 reductase is probably not synthesized as a precursor and inserts post‐translationally into the membrane. The results are discussed in relation to the particular subcellular distribution of the reductase and to the possible topology in the lipid bilayer of its C‐terminal non‐polar membrane‐binding segment.

show abstract

“…Free or membrane-bound polysomes were prepared from unstarved rats according to the methods of Ramsey and Steele [37,38]. Polysomal RNA was extracted using a chloroform/ isoamyl alcohol mixture [39] and poly(A)containing mRNA was purified by affinity chromatography on oligo(dT)-cellulose according to Aviv and Leder [40].…”

Section: Preparation Of Polysomes and M R N A From Rat Livermentioning

confidence: 99%

“…A guanidine thiocyanate procedure [42] was used to obtain total liver RNA. The same method was employed to extract RNA of free and membrane-bound polysomes from the membrane or supernatant fractions obtained by the Ramsey and Steele [38] fractionation procedure, which was carried out using 1.5 mM hydroxystilbamidine 1431 in the perfusion and homogenization buffers as an RNase inhibitor. After homogenization in 4 M guanidine thiocyanate containing 10 mM 2-mercaptoethanol, RNA was recovered by centrifugation through a caesium chloride cushion, resuspended in guanidine hydrochloride and reprecipitated [44].…”

Section: Preparation Of Polysomes and M R N A From Rat Livermentioning

confidence: 99%

Studies on the Biosynthesis of Microsomal Membrane Proteins

Okada

Frey

Guenthner

et al. 1982

European Journal of Biochemistry

113

View full text Add to dashboard Cite

The site of synthesis and mechanism of insertion into membranes of several microsomal polypeptides was studied using translation system programmed in vitro with polysomes or with mRNA extracted from free and membrane-bound rat liver polysomes. Primary translation products of cytochrome bs, NADH : cytochrome b5 oxidoreductase, NADPH : cytochrome P-450 oxidoreductase and epoxide hydrolase were isolated by specific immunoprecipitation and compared with the mature proteins. The following observations were made :1. While cytochrome b5 and NADH : cytochrome bs oxidoreductase are synthesized in free polysomes, NADPH : cytochrome P-450 oxidoreductase and epoxide hydrolase are made in membrane-bound polysomes.2. In all cases the radioactively labelled products synthesized in vitro comigrated with the purified native polypeptides. Since these proteins are not glycoproteins, it can be inferred that in vivo they do not undergo proteolytic cleavage during or after translation.3. Levels of translatable mRNA coding for NADPH : cytochrome P-450 oxidoreductase or epoxide hydrolase were increased by phenobarbital or by various carcinogens respectively. 4. A comparison of amino-terminal segments of the native epoxide hydrolase and that synthesized in vitro showed that this protein retains an amino-terminal polypeptide sequence which resembles the transient insertion signal found in presecretory proteins.5. The amino-terminal region of NADPH : cytochrome P-450 oxidoreductase contains 7 leucine residues in its first 15 amino acids and can, therefore, be presumed to be the hydrophobic portion which anchors this polypeptide to the membrane.Mechanisms which may account for the spatial disposition of the membrane proteins and for their selective accumulation in endoplasmic reticulum membranes are discussed.There is now considerable interest in the mechanisms which serve to ensure the specific incorporation of newly synthesized polypeptides into the different organellar membranes which characterize the eukaryotic cell. The endoplasmic reticulum plays an important role in the biogenesis of membranes by providing sites for cotranslational insertion of a variety of polypeptides, which are synthesized by membrane-bound ribosomes and are subsequently transferred to other subcellar compartments (cf. [l]).In the hepatocyte and in many other cell types the endoplasmic reticulum constitutes the most extensive membrane system of the cell and contains membrane-associated enzymes which play an important role in metabolic and biosynthetic processes affecting a variety of substrates. The biosynthesis of integral membrane proteins which are characteristic of the endoplasmic reticulum raises interesting questions concerning the incorporation of the polypeptides into the membranes and the means which allow for their retention in the endoplasmic reticulum by distinguishing between them and those to be transferred to other membrane systems [2].To study these questions we have selected several proteins of the hepatocyte endoplasmic reticulum membranes which...

show abstract

A method for visualizing and quantifying the total complement of free and membrane-bound ribosomes in rat liver

Cited by 34 publications

References 12 publications

Polysome Profiling in Liver Identifies Dynamic Regulation of Endoplasmic Reticulum Translatome by Obesity and Fasting

Polysome Profiling in Liver Identifies Dynamic Regulation of Endoplasmic Reticulum Translatome by Obesity and Fasting

In vitro synthesis and post-translational insertion into microsomes of the integral membrane protein, NADH-cytochrome b5 oxidoreductase.

Studies on the Biosynthesis of Microsomal Membrane Proteins

Contact Info

Product

Resources

About