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.     

Study on proteins from yeast cytoplasmic ribosomes by two-dimensional gel electrophoresis

Summary Proteins of yeast cytoplasmic ribosomes were analyzed by two different methods of two-dimensional gel electrophoresis: run at pH 8.6 in 1-D¹ and at pH 4.6 in 2-D (Method A); run at pH 5.0 in 1-D and in the presence of sodium dodecyl sulfate in 2-D (Method B). The numbers of proteins estimated were 28 (Method A) and 29 or 30 (Method B) in the 40S small subunit, and 40 (Method A) and 41 (Method B) in the 60S large subunit, respectively. Molecular weights of proteins in the small and the large subunits were found to be less than 40,000 and 60,000 respectively.     

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.     

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.     

Characterization of N-ethylmaleimide-reactive proteins from human tonsillar ribosomes by two-dimensional polyacrylamide gel electrophoresis.

Human tonsillar 80-S ribosomes were 17% and 43% inactivated by 1 mM N-ethylmaleimide after 12 min at 30 or 37 degrees C, respectively. The ribosomes were unaffected by the reagent during the same period of time at 0 or 20 degrees C. 4, 12, 27 and 59 sulfhydryl groups per 80-S ribosomes were found labeled by 1 mM N-ethyl[14C] maleimide after 12 min at 0, 20, 30 or 37 degrees C, respectively. The analysis of radioactively labeled proteins by two-dimensional gel electrophoresis revealed the following: after 3 min at 37 degrees C only two 40-S proteins, S3 and S7, displayed a significant amount of label. After 12 min at 37 degrees C, there was a several-fold increase in the extent of radioactivity found in each of these proteins and, additionally, S1, S2, S4, S5, S15, S22 and S31 were also found among labeled 40-S proteins. S3 appeared to be the most N-ethylmaleimide-reactive 40S protein. After 3 min at 37 degrees C, L10, L17, L20 (and/or S20), L26, L32 and L33, and after 12 min at 37 degrees C, additionally L1, L2, L7, L9, L11, L15, L16, L18, and L25 were labeled among 60-S proteins. l17 and 32 were the most N-ethylmaleimide-reactive proteins under these conditions. After 12 min at 37 degrees C, approx. 26% and 39% of the radioactivity incorporated into the 80 S or 60 S ribosomal protein, respectively, was found in these two proteins. After 12 min at 0 degrees C, S3, L17, L32 and L33 were the only labeled proteins.     

Triple-spot proteins in two-dimensional gel electrophoresis.

A triple-spot pattern of polypeptides occurring in two-dimensional gel electrophoresis of proteins is described. The presence of a mutant protein, Pc 1 Duarte, which results in a splitting of all three polypeptides, is evidence that they are produced by the same gene. This pattern is seen in about 1% of the proteins from a variety of sources. Typically, about 50% of the protein occurs as a single major spot, the remainder occurring as two polypeptides with an additional negative charge and slightly different molecular weight. The reproducibility of this pattern implies a functional significance which is presently unknown. The implication of this configuration for patterns seen by one-dimensional gel electrophoresis is discussed.     

Studies on mitochondrial proteins I. Separation and characterization by polyacrylamide gel electrophoresis

Rat liver mitochondria were fractionated into inner and outer membranes and soluble intermembrane space and matrix. The protein components of these fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mitochondria contained at least 20 components ranging in molecular weights from 10 000 to 140 000. Inner membranes differed markedly from outer membranes both in number of components and size distribution. The intermembrane space contained a few polypeptide species. These were of low molecular weight. The matrix was characterized by a high molecular weight component (130 000) which comprised 30% of this fraction. A major carbohydrate-containing polypeptide with an approximate molecular weight of 93 000 was detected in outer membrane preparations.