Determining the affinity in vitro of hepatic ribosomal subunits for derivatives of the rough endoplasmic reticulum

Ekren, T.; Shires, Thomas K.; Pitot, Henry C.

doi:10.1016/0006-291x(73)90920-0

Cited by 16 publications

(12 citation statements)

References 5 publications

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“…(2) The flotation-assay procedure (Ekren et al, 1973) involves mixing polyribosomes and conditioned rough membranes in a final volume of 1.9ml of 0.44M-sucrose-T5oK25 M5 buffer. This mixture is then made 1.8M with respect to sucrose in a final volume of 7ml, transferred to a Beckman polyallomer tube and overlaid with 0.44M-sucrose-T50K25 M5 buffer (Fig.…”

Section: Conditioning Of Rough Microsomal Fractions For Polyribosome mentioning

confidence: 99%

“…Conditioned rat liver rough membranes will quantitatively bind rat kidney polyribosomes (Shires et al, 1974b) and large ribosomal subunits (Rolleston, 1972;Ekren et al, 1973) to about the same extent as they will rat liver polyribosomes. Within the liver polyribosome population, polyribosomes from the membrane-bound group bound as well as those from the free group (Shires et al, 1973;Rolleston & Mak, 1973).…”

mentioning

confidence: 95%

“…Within the liver polyribosome population, polyribosomes from the membrane-bound group bound as well as those from the free group (Shires et al, 1973;Rolleston & Mak, 1973). Only small ribosomal subunits (Rolleston, 1972;Ekren et al, 1973) and 32P-labelled polyribosomes, degraded by endogenous radiolysis (Shires et al, 1971), have been reported to bind to liver membranes less extensively than do liver polyribosomes. This performance by liver membranes is not necessarily a non-discriminIating one, unless it can be shown that all the particulate populations binding to the membranes, in fact, bind to the same sites on the membranes.…”

mentioning

confidence: 99%

“…Such an approach required a method for measurement of binding that allowed a large number of simultaneous assays. The previously described flotation-assay procedure (Ekren et al, 1973) was chosen. During its adaptation for the blockage studies, a number of factors essential for accurate stoicheiometric measurement of polyribosome-membrane interaction in vitro were investigated.…”

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confidence: 99%

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The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro

Shires¹,

McLaughlin²,

Pitot³

1975

Biochemical Journal

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Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.

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Section: Conditioning Of Rough Microsomal Fractions For Polyribosome mentioning

confidence: 99%

mentioning

confidence: 95%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro

Shires¹,

McLaughlin²,

Pitot³

1975

Biochemical Journal

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“…Centrifugation was carried out at 4 ~ for 16 hr at 183,700 • (rotor SB-283 of the IEC B 60 centrifuge). A technique not dissimilar from this one, which we have described in more detail in a previous communication [3] has also been used by Ekren, Shires and Pitot [6]. At the end of the centrifugation the gradient was fractionated with the aid of a peristaltic pump, and the samples were collected in counting vials, mixed with 1.5 ml of water and 10 ml of scintillator (Omnifluor 4 g, Toluene 700 ml and Triton X-100 600 ml) and their radioactivities counted in a Packard Tri-Carb liquid scintillation spectrometer at an efficiency of 80%.…”

Section: Incubation For the Binding Reactionmentioning

confidence: 99%

Interactions of liver endoplasmic reticulum membranes and polysomesin vitro

Schiaffonati¹,

Cajone²,

Bernelli‐Zazzera³

1975

J. Membrain Biol.

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The interactions of various preparations of endoplasmic reticulum membranes and polysomes have been studied by means of a sandwich sucrose gradient that clearly isolates free ribosomes, smooth endoplasmic reticulum (S.E.R.) and rough endoplasmic reticulum (R.E.R.) from the microsomal fraction of rat liver homogenates. Reconstructed rough membranes separate well from the native R.E.R. but occupy the same position along the gradient as the S.E.R. and the rough membranes, stripped of their ribosomes by means of LiCl. Native R.E.R. and S.E.R. do not bind any added labeled polysomes at 0 degree C; previous treatment with LiCl does not modify the behavior of S.E.R. The presence of cell sap during the binding reaction does not increase polysome fixation by stripped-rough membranes but protects in some way the polysomes and preserves all their original functional capacity of amino acid incorporation into protein.

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Functional Specialization of Membrane-Bound Ribosomes in Eukaryotic Cells

Sabatini

Kreibich

1976

The Enzymes of Bioligical Membranes

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Determining the affinity in vitro of hepatic ribosomal subunits for derivatives of the rough endoplasmic reticulum

Cited by 16 publications

References 5 publications

The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro

The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro

Interactions of liver endoplasmic reticulum membranes and polysomesin vitro

Functional Specialization of Membrane-Bound Ribosomes in Eukaryotic Cells

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