Free and membrane-bound polysomes of rat liver: Separation in nearly quantitative yield and analysis of structure and function

Venkatesan, Natarajan; Steele, William J.

doi:10.1016/0005-2787(72)90298-5

Cited by 43 publications

(18 citation statements)

References 39 publications

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“…In agreement with previous reports (Loeb et al, 1967;Talal & Kaltreider, 1968;Ragnotti et al, 1969;Tanaka et al, 1970;Lee et al, 1971;Venkatesan & Steele, 1972;Mishra et al, 1972), our results suggest first that the two classes of polyribosomes present within the liver cell, though morphologically distinct, are not functionally separate and secondly that the membrane-ribosome interaction is a dynamic relationship that shifts as the functional requirements of the cell demand.…”

Section: Vol 146supporting

confidence: 93%

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats

Ragnotti

Aletti

1975

Biochemical Journal

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1. The effect of phenobarbitone on the rate of protein synthesis and on the sedimentation patterns of various liver subcellular fractions containing ribosomes was studied in rats. 2. Phenobarbitone treatment increased the incorporation of [14C]leucine into protein by all preparations, provided they had not been subjected to preliminary treatment with Sephadex G-25. The phenobarbitone-induced effect on incorporation was associated with a gain in liver weight and a higher degree of polyribosomal aggregation. 3. Preparations that were treated with Sephadex G-25 incorporated more radioactivity into protein, but did not show the response to phenobarbitone treatment. 4. When the influence of starvation and phenobarbitone was studied separately on membrane-bound and membrane-free polyribosomes, it was shown that whereas both classes of polyribosomes were affected by starvation, apparently only the former class was susceptible to phenobarbitone stimulation of protein synthesis. 5. The decreased capacity for protein synthesis of polyribosomes from starved rats was independent of their association with the membranes of the endoplasmic reticulum, but resulted from polyribosomal disaggregation, from an intrinsic defect of the polyribosomes themselves and from changes in composition of the cell sap. 6. The results are discussed in relation to the problem ofthe control ofprotein biosynthesis and of the functional separation of membrane-bound and membrane-free polyribosomes.

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Section: Vol 146supporting

confidence: 93%

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats

Ragnotti

Aletti

1975

Biochemical Journal

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“…and 12min at 135000gmax. (SW27.1 rotor; Beckman) to separate free polyribosomes from the rough microsomal fraction (Venkatesan & Steele, 1972b). The supernatant containing free polyribosomes was decanted and stored at 0°C.…”

Section: Methodsmentioning

confidence: 99%

Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes

Ramsey¹,

Steele²

1977

Biochemical Journal

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Free loosely bound and tightly bound polyribosomes were separated from rat liver homogenate by salt extraction followed by differential centrifugation, and several of their structural and functional properties were compared to resolve the existence of loosely bound polyribosomes and verify the specificity of the separation. The free and loosely bound polyribosomes have similar sedimentation profiles and polyribosome contents, their subunit proteins have similar electrophoretic patterns and their products of protein synthesis in vitro show a close correspondence in size and amounts synthesized. In contrast, the tightly bound polyribosomes have different properties from those of the free and loosely bound polyribosomes; their average size is significantly smaller; their polyribosome content is higher; their 60S-subunit proteins lack two components and contain four or more components not found elsewhere; their products of protein synthesis in vitro differ in size and amounts synthesized. These observations show that rat liver membranes entrap a large fraction of the free polyribosomes at low salt concentrations and that these polyribosomes are similar to those ofthe free-polyribosome fraction and are different from those of the tightly bound polyribosome fraction in size, structure and function.

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“…Methods for the preparation and analysis of hepatic polysomes from Xenopus are similar to those described by others (19,20). The details of our methods will be published elsewhere.…”

Section: Methodsmentioning

confidence: 99%

Regulation by estrogen of the vitellogenin gene.

Skipper¹,

Hamilton²

1977

Proc. Natl. Acad. Sci. U.S.A.

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The vitellogenin gene is inactive in the liver of male Xenopus laevis, unless exogenous estrogen is administered. We have previously shown that conventional doses of estradiol-17ft result in the appearance of new hepatic messenger RNAs, some of which are encoded for vitellogenin. We now report that much higher doses of the hormone (2 mg/frog per day for 4 days) are required to elicit maximal responses. The relative levels of membrane-bound polysomes and vitellogenin mRNA were determined as a function of time and dose of hormone. Translation of total polysomal RNA in a cell-free system derived from wheat germ was used to estimate the relative levels of vitellogenin messenger RNA. Faithful translation of this messenger RNA was indicated by two lines of evidence: labeled cell-free products were immunoprecipitated with antivitellogenin antibody, and the migration of the major labeled product in sodiui dodecyl sulfate/acrylamide gels was identical to that of native vitellogenin. Our results establish conditions for maximal estrogen-induced responses in this system, and are compatible with the hypothesis that a major regulatory mechanism of steroid hormones in the control of protein synthesis is that of gene activation and regulation of messenger RNA levels.Investigations of a variety of experimental systems used to study the molecular mode of action of sex steroid hormones have led some investigators to conclude that a major mechanism for controlling protein synthbesis is the hormone-dependent regulation of specific mRNAs (1-4). Definitive results relevant to this problem have been obtained from work with target tissues in which the steroid hormone evokes the synthesis of a specific protein in large quantities. The male African clawed frog (Xenopus laevis), lacking endogenous estrogen, does not synthesize vitellogenin. However, treatment of male frogs with exogenous hormone evokes a prodigious and prolonged synthesis of vitellogenin, thfe precursor of the yolk proteins. This response of the male liver offers a valuable experimental system for the study of hormonal activation of a specific gene.These considerations have prompted us (4) and others (5-7) to exploit this system (for recent review, see ref. 8). We recently demonstrated that estrogen administered to the male Xenopus caused the appearance of new mRNA in the liver (4). This mRNA, microinjected into living Xenopus oocytes, directed the synthesis de novo of the yolk proteins. Our observation has been confirmed and extended by Berridge and Lane (9), who also demonstrated that the primary translational product of this estrogen-induced mRNA is vitellogenin. This precursor is processed within the oocyte into the individual yolk proteins, lipovitellin and phosvitin (10).In general, the isolation and fractionation of polysomes have been useful intermediate steps in the purification of various species of mRNAs (11,12) effects of large doses of estrogen on the relative levels of polysomes and vitellogenin mRNA. Finally, we present evidence that estrogen-induce...

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Free and membrane-bound polysomes of rat liver: Separation in nearly quantitative yield and analysis of structure and function

Cited by 43 publications

References 39 publications

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats

Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes

Regulation by estrogen of the vitellogenin gene.

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