scholarly journals Enzymic iodination of eukaryotic ribosomal subunits. Characterization and analysis by two-dimensional gel electrophoresis

1975 ◽  
Vol 152 (2) ◽  
pp. 373-378 ◽  
Author(s):  
David P. Leader
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Small Subunit ◽  
Suitable Method ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Ribosomal Subunits

1. Conditions are described for the enzymic iodination of ribosomal subunits from rat liver. The reaction is relatively insensitive to broad changes in the concentration of KCl, allowing subunits to be studied under conditions which minimize their dimerization. 2. Mixtures of extracted ribosomal proteins were iodinated with 125I, the proteins separated by two-dimensional gel electrophoresis and the radioactivity in each protein was determined. Thus 19 out of 23 of the proteins of the small subunit and 25 out of 33 of the proteins of the large subunit were labelled. Iodination should therefore be a suitable method for studying the topography of the ribosomal proteins of rat liver. 3. When the intact 40S subunit (rather than the extracted mixture of proteins) was iodinated, 18 of the 19 proteins were still labelled. However five of these were labelled less strongly than before. When the intact 60S subunit was iodinated, 17 of the 25 proteins were still labelled, although six of these were labelled less strongly. 4. These results show that in rat liver most of the ribosomal proteins of both subunits are at least partially at the surface of the particles. They are also consistent with the idea that the proportion of the ribosomal proteins in the interior of the particle may be greater for the 60S subunit than for the 40S subunit.

scholarly journals Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins.

1979 ◽  
Vol 82 (1) ◽  
pp. 17-31 ◽  
Author(s):  
A M Lambowitz ◽  
R J LaPolla ◽  
R A Collins
Keyword(s):  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Protein S ◽  
Electrophoretic Analysis ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Mitochondrial Ribosome

Recent results with Neurospora crassa show that one protein (S-5, mol wt 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria (Lambowitz et al. 1976. J. Mol. Biol. 107:223-253). In the present work, Neurospora mit ribosomal proteins were analyzed by two-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 MKCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI greater than 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol was found to rapidly inhibit the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits (LaPolla and Lambowitz. 1977. J. Mol. 116: 189-205). Two-dimensional gel analysis shows that the more slowly sedimentaing CAP-30S particles are deficient in S-5 and in several other proteins, whereas these proteins are present in normal concentrations in mature small subunits from the same cells. Because S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol, the results tentatively suggest that S-5 plays a role in the assembly of mit small subunits. In addition, the results are consistent with the idea that S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and six additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. However, [poky] mit small subunits are deficient in S-5 and also contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].

Separation of cytoplasmic ribosomal proteins of Microsporum canis

1987 ◽  
Vol 33 (4) ◽  
pp. 339-343 ◽  
Author(s):  
Valsan Mandiyan ◽  
G. Ramananda Rao
Keyword(s):  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Polyacrylamide Gel Electrophoresis ◽  
Small Subunit ◽  
Polyacrylamide Gel ◽  
Microsporum Canis ◽  
Two Dimensional ◽  
Molecular Weights

The cytoplasmic ribosomal proteins of Microsporum canis were characterised in basic–acidic and basic–SDS two-dimensional polyacrylamide gel electrophoresis systems. The small subunit contained 28 proteins and the large subunit 38 proteins. The molecular weights of these proteins were in the range of 32 500 to 7600 and 48 000 to 11 000 in the small and large subunits, respectively. The 80S ribosomes showed 65 and 66 protein spots in basic–acidic and basic–SDS gel systems, respectively.

Analysis of Ribosomal Proteins from a Yellow Mutant of Chlamydomonas reinhardii by Two-dimensional Gel Electrophoresis

1979 ◽  
Vol 174 (9) ◽  
pp. 822-830 ◽  
Author(s):  
István Gyurján ◽  
Géza Erdös ◽  
Nadiezda P. Yurina ◽  
Marina S. Turischeva ◽  
Margarita S. Odintsova
Keyword(s):  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Chlamydomonas Reinhardii

scholarly journals The proteins of Xenopus ovary ribosomes

1971 ◽  
Vol 125 (4) ◽  
pp. 1091-1107 ◽  
Author(s):  
P J Ford
Keyword(s):  
Potassium Chloride ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Buoyant Density ◽  
Small Subunit ◽  
Chemical Methods ◽  
Ribosomal Subunits ◽  
Molecular Weights ◽  
Caesium Chloride ◽  
Chloride Treatment

1. The preparation of ribosomes and ribosomal subunits from Xenopus ovary is described. 2. The yield of once-washed ribosomes (buoyant density in caesium chloride 1.601g·cm-3; 44% RNA, 56% protein by chemical methods) was 10.1mg/g wet wt. of tissue. 3. Buoyant density in caesium chloride and RNA/protein ratios by chemical methods have been determined for ribosome subunits produced by 1.0mm-EDTA or 0.5m-potassium chloride treatment and also for EDTA subunits extracted with 0.5m-, 1.0m- or 1.5m-potassium chloride, 4. Analysis of ribosomal protein on acrylamide gels at pH4.5 in 6m-urea reveals 24 and 26 bands from small and large EDTA subunits respectively. The actual numbers of proteins are greater than this, as many bands are obviously doublets. 5. Analysis of the proteins in the potassium chloride extract and particle fractions showed that some bands are completely and some partially extracted. Taking partial extraction as an indication of possible doublet bands it was found that there were 12 and 20 such bands in the small and large subunits respectively, making totals of 36 and 46 proteins. 6. From the measured protein contents and assuming weight-average molecular weights for the proteins of large and small subunits close to those observed for eukaryote ribosomal proteins it is possible to compute the total numbers of protein molecules per particle. It appears that too few protein bands have been identified on acrylamide gels to account for all the protein in the large subunit, but probably enough for the small subunit.

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