Reductive alkylation of mammalian ribosomes

Reboud, Anne-Marie; Buisson, Monique; Arpin, Monique; Reboud, Jean‐Paul

doi:10.1016/0005-2787(77)90077-6

Cited by 17 publications

(11 citation statements)

References 13 publications

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Section: Extraction Reductivr Meihylaf Ion and Electrophoresis Of Rimentioning

confidence: 99%

“…of cold acetone [54]. Reductive methylation was performed under denaturing conditions [55]. Proteins (1 -2.5 mg) were dissolved in 400 p1 of 8 h4 guanidinium chloride before the addition of 400 p1 of 0.04 M sodium borate, pH 8.6; 80 pl of a 0.04 M formaldehyde solution was then added, followed at 30-s intervals by four successive additions of 16 p1 of NaB3H4 (5 mg/ml, with a specific radioactivity of 22Ci/mol).…”

Section: Of Ribosomal Proteinsmentioning

confidence: 99%

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Turnover Rates of Phosphoryl Groups in Ribosomal Proteins of Physarum polycephalum

Bélanger

Godin

Lemieux

1981

European Journal of Biochemistry

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The rate of phosphate exchange in individual ribosomal proteins of Physurum polyc~eplzaluni was determined in v i w . It was observed that the phosphoryl groups ofS3. lhc major plio~pliopi-oicili. had ;I turnover r a~c 01' 1 .S ' I , , per minute. The phosphoryl groups of proteins L1, L20 and L24 were stable. These results show that the phosphorylation of ribosomal proteins is regulated by at least two different mechanisms. The rapid turnover of phosphoryl groups of the major phosphoprotein is in agreement with the general observation that the phosphate content of this protein is modulated by the physiological state of the cells and possibly involved in the regulation of ribosome activity. The absence of phosphate exchange in acidic proteins suggests that these groups could play a structural role in the ribosome functions.The phosphorylation of ribosomal proteins was initially obscrved in rabbit reticulocytes and rat liver [1,2]. This phenomenon is common to all eukaryotic cells [I-l9]. In all systems, one basic protein of the small subunit, S6, or its equivalent, is the major phosphoprotein. It has also been generally observed that the phosphate content of this protein is related to the general physiology of the cells. Inhibition [3,20 -241, or stimulation of protein synthesis [25,26], addition of hormones [12,27-341, starvation [18-351, viral infection [5,24-361, growth conditions [37-401 as well as other factors [23,34,41,42] alter the phosphate content of this protein.In inany systems, the presence of additional ribosomal phosphoproteins has been reported. Although the exact number of ribosomal phosphoproteins is not yet clearly established, it is now generally accepted that the eukaryotic ribosome contains more than one phosphoprotein and that the phosphate content of these proteins is not altered by conditions known to modify the extent of phosphorylation of S6 11,3,11,14-16,31,32,43~51]. The available results suggest that the phosphorylation of ribosomal proteins is not controlled by a sole mechanism and may have different roles in ribosome activity and regulation.The precise determination of the phosphoryl groups of ribosomal proteins is one way to analyse in more detail these mechanisms. In order to study this aspect of ribosomal protein phosphorylation we have chosen to use Pliysarurn polyceplzalunz as a model system for the investigation of events occurring during the cell cycle and differentiation [52]. We have determined precisely the rate of phosphate incorporation in each ribosomal phosphoprotein. The turnover rate of phosphoryl groups in S3, the majbr phosphoprotein was 1.5 '?i per minute. The phosphoryl groups of acidic proteins L l , L20 and L24 were stable.These results confirm the existence of at least two different mechanisms involved in the phosphorylation of ribosomal f k j w w . Firefly luciferase (EC 1.13.12.7).proteins. One controls the phosphate content of S3, and could therefore selectively alter its phosphate content in rcsponse to various stimulus. The other mechanism regulates ...

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Section: Extraction Reductivr Meihylaf Ion and Electrophoresis Of Rimentioning

confidence: 99%

Section: Of Ribosomal Proteinsmentioning

confidence: 99%

Turnover Rates of Phosphoryl Groups in Ribosomal Proteins of Physarum polycephalum

Bélanger

Godin

Lemieux

1981

European Journal of Biochemistry

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“…Preparation of ribosomes and separation of active 40-S and 60-S subunits was performed as previously described [3]. The subunits were stored in buffer A (1 mM potassium phosphate pH 7.3, 30 mM KCl, 1.5 mM MgC12, 20 mM 2-mercaptoethanol).…”

Section: Ribosomes Und Subunitsmentioning

confidence: 99%

“…The number of methyl groups incorporated into the samples was calculated on the assumption that one hydrogen atom derived from sodium borohydride is incorporated per methyl group per lysine residue [I]. The total number of lysine residues per 40-S and 60-S subunit, respectively, were 462 and 975; the number of methylated lysine residues per protein moiety were determined as previously described [5,2].…”

Section: Reductive Methylationmentioning

confidence: 99%

“…Among the numerous chemical reactions already carried out on bacterial ribosomes, reductive alkylation is of particular interest, because it is a very mild method ; in particular it makes possible the preparation of labelled ribosomes that remain functionally active [l]. In a previous study [2], we analysed the properties of eucaryotic ribosomes labelled by this method. It was observed that ribosomes kept all their activity in polyphenylalanine synthesis after this treatment, and that all the ribosomal proteins were alkylated to a varying but very low extent.…”

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Change of Protein Reactivity in Mammalian Ribosomal Subunits as a Function of Temperature

Reboud

Buisson

Marion

et al. 1977

European Journal of Biochemistry

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Reductive methylation of rat liver ribosomal subunits was carried out at different temperatures and the reaction was followed as a function of time. The percentage of protein lysine residues which were methylated reached a definite plateau for each temperature, and was considerably increased by heating (four times at 40 "C). This increase was not observed when free ribosomal proteins were heated under the same conditions. Ribosomal subunits kept their biological activity (elongation steps of protein synthesis) even when methylated to a high extent. Half of the subunit activity was still found when 67 % of the lysine residues were methylated.Proteins were divided into different classes on the basis of their alkylation in response to temperature. Those which reacted poorly within subunits even at high temperature should interact directly with rRNA. The results suggest that the protein reactivity modifications induced by heating (up to 37 "C), which were often totally or partially reversible, reflect a conformational change of the proteins themselves within the subunits. Besides protein unfolding, higher temperatures produced structural modification and inactivation of the subunits, which were restrained by the presence of aminoacyltRNA.Chemical modifications of ribosomes with protein reagents, which have often been used to study the accessibility of ribosomal proteins on the organelle, are also a very useful tool for studying conformational changes of ribosomal subunits. Among the numerous chemical reactions already carried out on bacterial ribosomes, reductive alkylation is of particular interest, because it is a very mild method ; in particular it makes possible the preparation of labelled ribosomes that remain functionally active [l]. In a previous study [2], we analysed the properties of eucaryotic ribosomes labelled by this method. It was observed that ribosomes kept all their activity in polyphenylalanine synthesis after this treatment, and that all the ribosomal proteins were alkylated to a varying but very low extent.In this communication, it is shown that the number of reactive lysine residues considerably increases if ribosomal subunits are heated during methylation (instead of being treated by formaldehyde and borohydride at 4 "C as in all the experiments mentioned above). This temperature effect was not observed when free proteins were heated. Therefore the method was used to study the conformational change of subunits induced by heating, the reversibility of this change and the protective effect of tRNA binding. MATERIALS AND METHODS Ribosomes und SubunitsPreparation of ribosomes and separation of active 40-S and 60-S subunits was performed as previously described [3]. The subunits were stored in buffer A (1 mM potassium phosphate pH 7.3, 30 mM KCl, 1.5 mM MgC12, 20 mM 2-mercaptoethanol). Reductive MethylationThe reaction was carried out in a fume cupboard as follows. Ribosomes or ribosomal subunits (1 mg of protein/ml) were suspended in buffer A containing 20 mM sodium borate, the pH of this buffer b...

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Interaction, topography and function of ribosomal components

Bielka¹

1982

The Eukaryotic Ribosome

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Reductive alkylation of mammalian ribosomes

Cited by 17 publications

References 13 publications

Turnover Rates of Phosphoryl Groups in Ribosomal Proteins of Physarum polycephalum

Turnover Rates of Phosphoryl Groups in Ribosomal Proteins of Physarum polycephalum

Change of Protein Reactivity in Mammalian Ribosomal Subunits as a Function of Temperature

Interaction, topography and function of ribosomal components

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