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.     

Characterisation of the ribosomal proteins from Schizosaccharomyces pombe by two-dimensional polyacrylamide gel electrophoresis

Summary The 80S ribosome of Schizosaccharomyces pombe contains 93 proteins as determined by twodimensional electrophoresis on polyacrylamide gels. Of these, 76 are basic and 17 are acidic at pH 8.7. 38 proteins could be assigned unambiguosly to the large sub-unit and 19 to the small.67 proteins were extracted from the two-dimensional gels and their molecular weights determined by electrophoresis on calibrated SDS-gels. Values varied from 11,000 to 52,000 daltons, the number average being 25,000 daltons. Hence the total protein content of the 80S ribosome must be at least 1.67x10⁶ daltons.Three drug resistant strains are known, cyh1, anil and tri5 (resistant to cycloheximide, anisomycin and trichodermin respectively), in which resistace is conferred by an altered ribosome, in each case by an altered 60S sub-unit. When 80S ribosomal protein patterns from these strains were compared with that of wild type, in only one case was a clear difference seen. This involved a large sub-unit, basic protein (designated number 66 on our classification) which, in the cyh1 strain, had a reduced mobility in the second dimension when compared to the wild type. The mutant form of protein 66 had a molecular weight of 25,000 daltons compared to the 22,000 of the wild type protein. Production of a larger protein by the mutant strain could either be due to a readthrough event or to an alteration in the specificity of a modifying or processing enzyme.     

Subpicogram analysis of sweat proteins using two-dimensional polyacrylamide gel electrophoresis

Sweat collected from six normal volunteers was analyzed to determine if reproducible protein patterns could be obtained using two-dimensional polyacrylamide gel electrophoresis of ¹²⁵I-labeled sweat proteins. This method has the capability of easily detecting picogram quantities of protein. Once the methods of collection of the sweat had been standardized, reproducible patterns were obtained from these volunteers. Over 100 discrete spots were revealed by a combination of fluorography and rare earth screen radioautography of dried two-dimensional gels. This method will allow analysis of sweat for qualitative and quantitative variations in protein content in pathologic conditions such as cystic fibrosis, renal failure, and diabetes.     

Preparative two-dimensional polyacrylamide gel electrophoresis of rat liver ribosomal proteins and determination of their amino acid compositions

A maize root fraction which inactivates nitrate reductase has been shown to have protease activity which can be measured by the hydrolysis of azocasein. This inactivating enzyme was also found to inactivate yeast tryptophan synthase. Yeast proteases A and B, which inactivate this latter enzyme, also gave a specific inactivation of the maize nitrate reductase. The maize root inactivating enzyme, like yeast protease B, degraded casein, and was inhibited by phenylmethylsulphonyl fluoride. A partially-purified yeast inhibitor prevented catalysis by the yeast proteases and maize root inactivating enzyme, but purified yeast inhibitors were without effect on the latter protein. The level of nitrate reductase-inactivating activity, and associated azocasein-degrading activity, increased with age of the maize root. Evidence was obtained for a heat stable inhibitor which maintained them in an inactive state, especially in the young root tip cells.     

Two-dimensional polyacrylamide gel electrophoresis: an improved method for ribosomal proteins

A two-dimensional electrophoresis system for analysis of ribosomal proteins with several advantages over previous systems is described. The general features of this system are: (1) first-dimension separation on the basis of mobility at pH 5.0 in 8 m urea and 4% polyacrylamide; (2) second-dimension separation on the basis of molecular weight using dodecyl sulfate detergent; (3) rapid electrophoretic shift between first- and second-dimension separation conditions; (4) high resolution separation can be obtained on 10-cm² slabs with proteins from approximately 100 μg of ribosomal subunits; (5) capacity for handling up to 10 samples at a time, with electrophoresis complete within about 10 hr; and (6) the apparatus is relatively simple and inexpensive to construct and use.     

Resolution of rat mitochondrial matrix proteins by two-dimensional polyacrylamide gel electrophoresis.

The mitochondrial matrix subfractions from rat liver, kidney cortex, brain, heart, and skeletal muscle were isolated and their protein components were resolved by two-dimensional polyacrylamide gel electrophoresis, revealing between 120 and 150 components for each matrix subfraction. Excellent resolution was obtained utilizing a pH 5 to 8 gradient in the first dimension and in 8 to 13% exponential acrylamide gradient in the second dimension, increasing the number of mitochondrial matrix proteins observed 3-fold over one-dimensional systems. Protein components tentatively identified by co-migration with pure enzymes and by known tissue distributions are carbamoyl-phosphate synthetase (EC 2.7.2.5), ornithine transcarbamylase (EC 2.1.3.3), glutamate dehydrogenase (EC 1.4.1.3), pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7), fumarase (EC 4.2.1.2), aconitase (EC 4.2.1.3), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoyl transsuccinylase (EC 2.3.1.12), lipoamide dehydrogenase (EC 1.6.4.3), glutamate-aspartate aminotransferase (EC 2.6.1.1), and the two subunits of pyruvate dehydrogenase (EC 1.2.4.1). Protein components unambiguously identified by peptide mapping are citrate synthase, aconitase, and pyruvate carboxylase. The inner membrane subfraction from rat liver mitochondria was also resolved two dimensionally; the alpha and beta subunits of ATPase (F1) (EC 3.6.1.3) were identified by peptide mapping.     

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.     

Separation of t-RNAs by two-dimensional polyacrylamide gel electrophoresis

A pH 5.8 polyacrylamide gel electrophoresis buffer is described. Electrophoresis in this MES-citrate system at pH 5.8 separates E. coli transfer RNAs into 15 bands using 15% acrylamide gels. Polyacrylamide gel electrophoresis in a second dimension at pH 8.3 further resolves E. coli t-RNAs into 20 spots.     

Analysis of ribosomal proteins and ribosomal subunits of pea seeds by electrophoresis in polyacrylamide gel]

The amino acid composition of overall protein of ribosomes and ribosomal subunits of pea seeds has been found typical of ribosomal protein. Electrophoresis in polyacrylamide gel demonstrates that proteins extracted by the solution of 3 M LiCl-4 M urea from purified ribosomes of pea seeds move towards the cathode at pH 2.2 and separate into 41 components. Electrophoresis in a tris-glycine buffer at pH 9.2 does not reveal any substance corresponding to acid proteins. Similar distribution patterns are observed when ribosomal particles are isolated with or without triton (0,5%). The treatment of ribosomes by deoxycholate results in some changes, depending on the detergent concentration. All the protein components detected in ribosomes, except one, are present in the subunits. Proteins of large and small ribosome subunits produced 26 and 21 components respectively in polyacrylamide gel electrophoresis. The distribution patterns of proteins of the two subunits appear to be different. The majority of the components of the large and small subunits differ in mobility. The data obtained suggest considerable specificity of the protein composition of 60S and 40S subunits of 80S ribosomes in higher plants.     

Dodecyl maltoside-sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis of chloroplast thylakoid membrane proteins

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-beta-D-maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-beta-D-maltoside and separated in 4-7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-beta-D-maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5-20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3',5,5'-tetramethylbenzidine-H2O2, Coomassie blue, and silver staining. A number of protein components were identified on "Western blots" of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a/b protein complex, photosystem I, photosystem II, the cytochrome b6/f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.