Phosphorylation of proteins of ribosomes and nucleolar preribosomal particles from Novikoff hepatmoa ascites cells

After labeling for two hours in vivo with ³²P-labeled orthophosphate, proteins from cytoplasmic ribosomes and nucleolar preribosomal particles of Novikoff hepatoma ascites cells were analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Five proteins (B2, B3, B6, B32 and B35P) were phosphorylated in the ribosomes. Approximately 19 proteins were phosphorylated in the nucleolar preribosomal particles; although four of these were ribosomal proteins, they were different from the proteins labeled in the ribosomes. The 15 additional phosphorylated nucleolar preribosomal particle proteins were non-ribosomal. These results suggest that phosphorylation of proteins of the nucleolar preribosomal particles is independent of phosphorylation of the cytoplasmic ribosomal proteins and may be a part of the maturation process of preribosomal particles.     

Two-dimensional polyacrylamide gel electrophoresis of chromatin proteins of normal rat liver and Novikoff hepatoma ascites cells

Chromatin was prepared from the citric acid nuclei of normal rat liver and Novikoff hepatoma ascites cells. After sulfuric acid extraction, the dehistonized chromatin was solubilized by digestion with deoxyribonuclease I. The proteins of normal liver and of Novikoff hepatoma chromatin fractions were analyzed by two-dimensional polyacrylamide gel electrophoresis. The liver pattern contained 69 components and the hepatoma pattern contained 84 components. Comparison of the two patterns revealed two dense protein spots migrating in the B region in the liver pattern that were absent from the tumor pattern and two dense protein spots migrating in the C region in the tumor pattern that were absent from the liver pattern.     

Comparison of plant cytoplasmic ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis

The electrophoretic mobilities of the cytoplasmic ribosomal proteins of several species of plants were compared using two-dimensional electrophoresis. The total number of proteins as well as the number of acidic and basic proteins in individual species varied markedly. Of the species examined, Triticum aestivum had the highest number of basic cytoplasmic ribosomal proteins and Hordeum vulgare had less than half as many. However, marked similarities were noted in the electrophoretic mobilities of many of the proteins, especially for wheat, rye, and barley and for peas and beans. There was a statistically significant positive correlation between the numbers of basic proteins in the species and their chromosome number.     

Two-dimensional polyacrylamide gel electrophoresis of acid extractable nuclear proteins of normal rat liver and Novikoff hepatoma ascites cells

The nuclear proteins of normal rat liver and Novikoff hepatoma nuclei were extracted with 0.4 N H₂SO₄ and subjected to two-dimensional polyacrylamide gel electrophoresis. A total of 98 protein components were found in the liver extract and 111 components in the tumor extract. A comparison of the patterns obtained revealed 11 qualitative differences and 5 quantitative differences.     

Micro-scale two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins

Summary A specially designed apparatus and conditions are described for the rapid analysis of ribosomal proteins by two-dimensional gel electrophoresis on a micro scale. The resolution of proteins in electropherograms is comparable to that obtained with other systems, but because of miniaturization, only 0.5 to 1 g of each protein is required, and the entire procedure, including electrophoresis in both dimensions, and staining and destaining can be completed in 6 to 7 hours.     

Characterisation of ribosomal proteins from HeLa and Krebs II mouse ascites tumor cells by different two-dimensional polyacrylamide gel electrophoresis techniques

Summary Electrophoresis of ribosomal proteins according to Kaltschmidt and Wittmann, 1970a, b (pH 8.6/pH 4.5 urea system) yielded 29 proteins for the small subunits and 35 and 37 proteins for the large subunits of Krebs II ascites and HeLa ribosomes, respectively. Analysis of the proteins according to a modified technique by Mets and Bogorad (1974) (pH 4.5/pH 8.6 SDS system) revealed 28 and 29 proteins in the small subunits and 37 and 38 proteins in the large subunits of Krebs II ascites and HeLa ribosomes.The molecular weights of the individual proteins were determined by: 1. three-dimensional gel electrophoresis; 2. two-dimensional gel electrophoresis at pH 4.5/pH 8.6 in SDS. The molecular weights for 40S proteins ranged from 10,000 to 39,000 dalton (number average molecular weight: 21,000). The molecular weights for the 60S proteins ranged from 14,000 to 44,000 dalton (number average molecular weight: 23,000) using the three-dimensional technique. A molecular weight range from 10,000 to 38,000 dalton (number average molecular weight: 21,000) was obtained for the 40S subunits, whereas the molecular weights for the 60S ribosomal proteins (average molecular weight: 26,000) ranged from 12,000 to 69,000 dalton using the pH 4.5/pH 8.6 SDS system.The molecular weights of Krebs II ascites and HeLa ribosomal proteins are compared with those obtained by other authors for different mammlian species.     

Negatively charged phosphopeptides of nucleolar nonhistone proteins from Novikoff hepatoma ascites cells.

To characterize the sites phosphorylated by endogenous kinases, phosphopeptides of isolated nucleolar nonhistone proteins were analyzed. Major phosphoprotein bands C23 and B23 were ³²P labeled $\text{in vitro}$ and electrophoretically isolated. Tryptic phosphopeptides were resolved by DEAE-Sephadex chromatography into fractions A, B and C for band C23 and α and β for band B23. Each of these fractions contained phosphoserine, had a distinct amino acid composition of 49–65% glx + asx and 4–11% lys, and had molecular weights of 7–11,000 determined on Sephadex G50. These data indicate that two nucleolar nonhistone proteins have similar phosphorylated regions of high negative charge density.     

Effects of 5-azacytidine on nucleolar RNA and the preribosomal particles in Novikoff hepatoma cells.

Examination of nucleolar RNA from cultured Novikoff hepatoma cells treated for 3 hr with 5 x 10-4 M 5-azacytidine shows that significant amounts of analog-substituted 45S RNA are processed to the 32S RNA species, but 28S RNA formation is completely inhibited. Under these conditions of analog treatment 37% of the cytidine residues in the 45S RNA is replaced by 5-azacytidine. During coelectrophoresis of nucleolar RNA from 5-azacytidine-treated and control cells, the analog-substituted 45S RNA and 32S RNA display reduced mobilities compared to the control 45S RNA and 32S RNA. Coelectrophoresis of analog-substituted and control RNA after formaldehyde denaturation shows no differences in electrophoretic mobility between the two RNA samples, suggesting that 5-azacytidine incorporation may alter the secondary structure of the 45S RNA and the 32S RNA. 5-Azacytidine at 5 x 10-4 M severely inhibits protein synthesis in Novikoff cells by 3 hr. After this length of treatment, however, CsCl buoyant density analysis reveals no difference in density of either the 80S or 55S preribosomal ribonucleoprotein particles when compared to normal particles. Also 5-azacytidine treatment does not appear to cause major changes in the polyacrylamide gel electrophoresis patterns of the proteins in the 80S and 55S preribosomal particles. These results together with previous findings suggest that 5-azacytidine's inhibition of rRNA processing is possibly related to its alteration of the structure of the ribosomal precursor RNAs and is not a consequence of a general inhibition of ribosomal protein formation.     

Analysis of the protein composition of yeast ribosomal subunits by two-dimensional polyacrylamide gel electrophoresis

The number of proteins in yeast ribosomal subunits was determined by two-dimensional polyacrylamide gel electrophoresis. The 40S subunit obtained after dissociation of ribosomes at high ionic strength contains 30 different protein species (including six acidic proteins). The 60S subunit, obtained in the same way contains 39 different species (including 1 acidic protein). While the total number of protein species found in yeast ribosomes, thus, is in close agreement with those reported for other eukaryotic organisms, the distribution between acidic and basic proteins is quite different. When the ribosomes were dissociated at low ionic strength, four extra protein spots appeared in the electropherograms of both 40S and 60S subunits. We consider these proteins to be nonribosomal.