Quantitation of eukaryotic ribosomal proteins separated by two‐dimensional polyacrylamide gel electrophoresis

Martini, Oskar H. W.; Temkin, R.J.; Jones, A. E.; Riley, Kate; Gould, Hannah J.

doi:10.1016/0014-5793(75)81092-1

Cited by 9 publications

(4 citation statements)

References 15 publications

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“…For some of the ribosomal proteins listed in Fig. 8 the percentage contribution of central absorbances to the sum of all central absorbances was calculated previously by Martini et al (1975), whose preparation was comparable with our LiCl-extracted dialysed sample. For the sake of comparison we computed our results for the corresponding proteins in a similar manner.…”

Section: Discussionmentioning

confidence: 99%

“…protein L, in one pattern with protein L, in a second pattern, protein SI with protein SI and so on. This is based on the observations of Martini et al (1975) that, below a certain total protein loading, the central absorbance of a spot is proportional to the amount of protein (and this was demonstrated for the spots of several small subunit proteins). -This method has the advantage that the difficulties encountered in determining the area of the spot are avoided.…”

Section: Quantitative Protein Determinationmentioning

confidence: 99%

“…Photography of gel, scanning on a densitometer, and treatment of data on a Nova 800 computer, were performed as described by Martini et al (1975).…”

Section: Quantitative Protein Determinationmentioning

confidence: 99%

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Effect of various methods of preparation on the apparent protein composition of eukaryotic ribosomes. An essential preliminary to stoicheiometric measurements

Huvos¹,

Fey²,

Hardwicke³

1978

Biochemical Journal

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1. We investigated whether there is any change in the relative amounts of ribosomal proteins during the isolation or extraction of the ribosomes by different methods, or during electrophoresis of the proteins. 2. To see whether proteins are lost (or gained) during the preparation of the ribosome we compared the two-dimensional protein pattern of three preparations: (a) ribosomes conventionally prepared by ultracentrifugation; (b) crude ribosomes obtained by pH5 precipitation; (c) crude ribosomes prepared by gel filtration. 3. To see whether proteins were lost during protein extraction we compared the two-dimensional pattern of ribosomes by using three different extraction methods (LiCl/urea, acetic acid and guanidine hydrochloride). 4. In all experiments listed above the relative amounts of the great majority of the proteins remained unchanged. We interpret this as showing that the relative amounts of ribosomal proteins (as we observed them on a two-dimensional gel) correspond to the proportions existing in the particle in vivo.

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Section: Discussionmentioning

confidence: 99%

Section: Quantitative Protein Determinationmentioning

confidence: 99%

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Effect of various methods of preparation on the apparent protein composition of eukaryotic ribosomes. An essential preliminary to stoicheiometric measurements

Huvos¹,

Fey²,

Hardwicke³

1978

Biochemical Journal

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“…It follows as closely as possible the nomenclature introduced for rabbit reticulocyte ribosomal proteins [32] analysed by the same electrophoresis technique. The main difference between the patterns shown here and those cited above [32,33] are: (a) the particularly basic highmolecular-weight protein L3b of rabbits is missing in the MPC 11 pattern; (b) the particularly basic MPC 11 protein L8b was not observed in rabbit patterns, but is also found in rats (unpublished observations); (c) mouse and rabbit patterns differ also with respect to protein position L7a; (d) the detection of protein L12e (not enumerated in earlier studies [33,34]) may be due to an improved resolution in an 'overpopulated' region of the gel; (e) the small-subunit protein S18b, previously omitted from enumeration [32 -341, has now been included, because we observed it regularly in several species when protein loads were sufficiently high. Since we named this protein S18b, conforming to the principles of our nomenclature [32], we had to rename S18, which we now call S18a.…”

Section: The Ribosomal Protein Patternsmentioning

confidence: 99%

Ribosomal Phosphoproteins of Mouse Myeloma Cells

Martini

Kruppa

1979

European Journal of Biochemistry

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Mouse myeloma cells (MPC 11) in culture, labelled with I3'P]i for several hours, were subjected to hypertonic initiation block by raising the salt concentration of the culture medium from 110 to 210 mM NaCl.The relative amounts of messenger-free and bound ribosomes of the cells were estimated from ribosome profiles following sucrose density centrifugation. The initiation block caused the proportion of messenger-bound ribosomes to decrease substantially. The extent of the decrease was larger under suboptimal (50 %) than under satisfactory (25 %) nutritional conditions. Analysis of the ribosomal proteins by two-dimensional gel electrophoresis, autoradiography and radiospectrometry showed, that in the small subunit only protein S3 had incorporated 32P and that S3 was the most strongly phosphorylated ribosomal protein in control cells. Upon salt treatment of the cells the radioactivity of S3 from both messenger-free and bound ribosomes decreased to low levels. The lowest levels (15% of the controls) were reached when the nutritional conditions were suboptimal. The results show that there is a positive correlation between the number of ribosomes engaged in protein synthesis and the degree of phosphorylation of S3. However, phosphorylation of S3 does not seem to be a prerequisite for ribosomes to be attached to mRNA. Under all experimental conditions S3 from messenger-bound ribosomes was about twice as strongly labelled as S3 from messenger-free ribosomes.The large-subunit phosphoproteins were identified as L28, the protein thought to be homologous to the Escherichia coli proteins L7/L12, and as L5, which was only weakly labelled. In contrast to S3 the radioactivity of L28 increased slightly upon the tonicity shift.Of the approximately 70 different species of ribosomal proteins from eukaryotic cells some 4 or 5 are phosphorylated in vivo [1,2] of which at least two, namely the small-subunit protein S3 (denoted S6 in the nomenclature of Sherton and Wool [3]) and the acidic, large-subunit protein(s) (likely to be homologous to proteins L7/L12 of Escherichia coli [4]) can become phosphorylated at more than one site ([5,6] and [7,8] respectively).As to the biological significance of ribosomal protein phosphorylation, neither experiments in vitro [9 -11 ] nor the numerous studies in vivo [5,6, have yielded conclusive results. In particular, it has remained controversial to which, if any, parameter of protein metabolism phosphorylation of ribosomal proteins may be related [5,13,15,17,18].Abbreviations. Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Tris/K/Mg, buffer containing 50 mM Tris-HCI, pH 7.6, 25 mM KCI, 5 mM magnesium acetate, 1 mM dithiothreitol.Of the three principal stages of peptide chain synthesis, initiation has generally been regarded the most likely to be under regulatory control. Experimentally, the frequency of peptide chain initiation can be drastically reduced in cultured cells by increasing the tonicity of their growth medium, socalled hypertonic initiation block 119 -221. Because of the...

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Chemical components

Bielka¹

1982

The Eukaryotic Ribosome

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Quantitation of eukaryotic ribosomal proteins separated by two‐dimensional polyacrylamide gel electrophoresis

Cited by 9 publications

References 15 publications

Effect of various methods of preparation on the apparent protein composition of eukaryotic ribosomes. An essential preliminary to stoicheiometric measurements

Effect of various methods of preparation on the apparent protein composition of eukaryotic ribosomes. An essential preliminary to stoicheiometric measurements

Ribosomal Phosphoproteins of Mouse Myeloma Cells

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