Two-dimensional Gel System Research Articles

Effect of inhibitors of elongation factor Tu on the metabolic regulation of protein synthesis in Escherichia coli

By using inhibitors of elongation factor Tu (L-1-tosylamido-2-phenylethyl chloromethyl ketone [TPCK] and kirromycin), we determined the effect of elongation factor Tu inhibition on the synthesis of individual components of the translation machinery. The rates of synthesis of individual proteins were measured in double-label experiments using a two-dimensional gel system. TPCK inhibition produce a coordinate decrease in the differential synthesis rates of all components of the translation machinery examined in these experiments. On the other hand, kirromycin inhibition increased the differential synthesis rates of some translation components and decreased the differential synthesis rates of others. These results suggest that the metabolic regulation of synthesis of various translation proteins is not mediated through a common signal.

Cell fractionation studies on the guinea pig pancreas. Redistribution of exocrine proteins during tissue homogenization.

A double-label protocol was used to estimate the extent of leakage and relocation artifacts that affect exocrine pancreatic proteins in cell fractionation experiments. Guinea pig pancreatic lobules were pulsed in vitro with a mixture of 14C-amino acids to enable the lobules to produce and process endogenously labeled exocrine proteins. At the end of the pulse (10 min) or after an appropriate chase interval, the lobules were homogenized in 0.3 M sucrose to which a complete mixture of 3H-labeled exocrine pancreatic proteins was added as an exogenous tracer. The distribution of both labels was studied in each cell fraction of interest at the level of TCA-insoluble proteins and individual exocrine proteins resolved by using a two-dimensional gel system. Based on the premises that the exogenous and endogenous label behave identically during homogenization-fractionation and that all endogenously labeled exocrine proteins found in the postmicrosomal supernate come from intracellular compartments ruptured during tissue homogenization, a series of equations was derived to quantitate leakage and adsorption and to define the ratio of endogenous label still in its primary location to total label (primary location index or PLI) for each cell fraction. Leakage was found to be uniform for all exocrine proteins, but unequal in extent from different cell compartments (condensing vacuoles is greater than zymogen granules is greater than rough endoplasmic reticulum) ; it increased with exposure to shearing forces especially in the case of zymogen granules and condensing vacuoles, and was substantially reduced from rough microsomes by adding 10 mM KCl to the homogenization media. Relocation of exogenous label by adsorption to other subcellular components was extensive (approximately 55%), uneven (free polysomes is greater than rough microsomes is greater than smooth microsomes and zymogen granules), preferential (cationic proteins are massively adsorbed to ribosomes and membranes, resulting in a complementary enrichment of the post-microsomal supernate with anionic exocrine proteins), and reversible (with successive 50-100 mM KCl washes). After correction for adsorption and leakage, the kinetics of intracellular transport derived from cell fractionation data were found to be nearly identical to those obtained from quantitative autoradiographic studies.

Synthesis of tubulin and actin during the preimplantation development of the mouse

The relative rates of synthesis of actin and tubulin during mouse preimplantation development have been investigated utilizing O'Farrell's two-dimensional polyacrylamide gel system and internal protein markers. During mouse preimplantation development, rates of protein synthesis remain low and are little changed until the 8-cell stage when a rapid increase is evident. From the 8-cell stage on, a much higher rate of synthesis is maintained. The rate of synthesis of actin remains also at a steady low level in the unfertilized and fertilized ovum. However, by the 8-cell stage actin synthesis has increased 10-fold. Our measurements include the blastocyst, at that point in development actin synthetic rates are almost 90-fold higher than in the unfertilized ovum. While this precipitous increase is proceeding, incorporation of [ 3H]leucine into total protein increases only 7-fold. Synthesis of actin in the blastocyst represents 5.7% of total protein synthesis. The rate of tubulin synthesis, unlike actin, more closely parallels the increments in total protein synthetic rates. At the blastocyst stage it has increased 14-fold and its synthesis represents almost 2% of total protein synthesis. These results are discussed with reference to some of the physiological changes taking place during preimplantation development.

Altered E2 glycoprotein of Sindbis virus and its use in complementation studies.

We have detected a Sindbis virus variant that contains a smaller-molecular-weight form of the viral glycoprotein E2. The molecular weight of the PE2 precursor and the glycosylation pattern of the smaller E2 are normal, thus indicating that this E2 is formed by an aberrant proteolytic cleavage. The altered E2 was detected in an RNA+ temperature-sensitive mutant that was defective in proteolytic cleavage, but the abnormal PE2-to-E2 reaction could be separated from the ts mutation and is not itself a temperature-sensitive defect. We used the variant E2 as a marker to monitor the complementation reaction between an RNA+ and an RNA- mutant and discovered that complementation was not reciprocal; the RNA defect was corrected by the RNA+ mutant gene products but the RNA+ defect was not complemented by any RNA- gene products. Other studies have shown that the smaller E2 is not preferentially selected during viral maturation and budding. No significant changes have been detected in the biological activity of virions with this altered E2 protein. Comparison of the electrophoretic migration of the E1 and E2 Sindbis viral glycoproteins in a two-dimensional polyacrylamide slab gel system that was first run in the absence of sulfhydryl-reducing reagent and then with beta-mercaptoethanol indicated that the mobility of E1, but not that of E2, was significantly altered by reduction.

Transient rates of synthesis of individual polypeptides in E. coli following temperature shifts

The effect of temperature shifts on the rates of synthesis of selected individual polypeptides in wild-type E. coli was studied through pulse-labeling and resolution of protein by the O'Farrell two-dimensional gel system. The overall rate of protein synthesis quickly accelerated to its new steady state rate after temperature shifts, but the differential rates of synthesis of most individual polypeptides studied either increased transiently (as much as 50 fold) or decreased transiently (as much as 10 fold). Their new steady state values, usually the pre-shift values, were reched 20 min after the shift. The magnitude of the responses depended upon the size of the temperature shift, and a shift downward generated responses opposite to those of a shift upward. The ten aminoacyl-tRNA synthetases and five transcriptional or other translational factors studied responded with greater similarity than did a group of nine unidentified polypeptides chosen from different regulatory classes. Guanosine tetraphosphate accumulated transiently upon a temperature shift upward, but reducing this accumulation did not in general alter the quantitative responses of the polypeptides. For some proteins, these transients accomplish rapid attainment of a new steady state level. Many of the transients seem to be active regulatory responses.

Specialized transducing phage for the initiation factor 3 gene in Escherichia coli.

A previously isolated thermosensitive mutant [Springer, M., Graffe, M. & Grunberg-Manago, M. (6977) Mol. Gen. Genet. 151, 17-26] exhibits two defects in vitro, one in the initiation factor IF3 and the other in the L-phenylalanine: tRNA-Phe ligase (EC 6.1.1.20). Specialized lambda transducing phages that transduced this mutant to thermoresistance were selected. In vitro studies showed that the transductants had a normal IF3 activity. One of these transducing phages was shown to code for a protein synthesized under the control of Escherichia coli promoters, which has the same molecular weight as IF3. This protein crossreacts specifically with IF3 antisera and comigrates with pure IF3 in a two-dimensional gel system.

Transient rates of synthesis of five amionacyl-transfer ribonucleic acid synthetases during a shift-up of Escherichia coli

The steady-state levels of a number of aminoacyl-transfer ribonucleic acid synthetases are known to be positively correlated with growth rate in Escherichia coli. To describe the regulation of these enzymes during a nutritional shift-up, use was made of the recent identification of polypeptide chains of several synthetases in whole cell lysates resolved by the O'Farrell two-dimensional gel system. A culture growing in acetate minimal medium was shifted to glucose-rich medium and pulse labeled with [3H]leucine and [3H]isoleucine for 30- or 6-s intervals during the 20 min after the shift. After mixing with a uniformly [35S]sulfate-labeled reference culture, the samples were subjected to two-dimensional gel electrophoresis. The 3H/35S ratio in the resolved synthetase polypeptides provided an accurate estimation of their transient rates of synthesis. Five aminoacyl-transfer ribonucleic acid synthetases (those for argnine, glycine, isoleucine, phenylalanine, and valine) exhibited an increase in formation within 30 to 90 s after the shift-up. The magnitude of the increases corresponded to the final steady-state values and were reached within 2 to 3 min. The addition to rifampin revealed that the increase in the differential rate of valyl-transfer ribonucleic acid synthetase formation was the result of increased messenger ribonucleic acid transcription and not of a relaxation of some translation restriction.

Chemical measurement of steady-state levels of ten aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli

Polypeptide chains of 10 aminoacyl-transfer ribonucleic acid synthetases (those for arginine, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, phenylalanine, threonine, and valine) have been identified in lysates of Escherichia coli resolved by the O'Farrell two-dimensional gel system. By labeling cells uniformly with [14C]glucose and by measuring the total amounts of these polypeptides by their radioactivity, estimations of the steady-state, molecular amounts of these enzymes were made and compared to the number of ribosomes and elongation factors in these cells. Portions of a reference culture grown on glucose and labeled with [14C]leucine or [35S]sulfate were mixed with four cultures grown in widely different media containing [3H]leucine or [3H]leucine plus [3H]isoleucine. From the isotope ratios of the total protein and of the spots containing the synthetase chains, the chemical amount of each synthetase relative to that of the reference culture was determined. The results, where comparable, show reasonable agreement with enzyme activity measurements. In general, these synthetases each exhibit a positive correlation with growth rate in unrestricted media, indicating a strong tendency for the levels of transfer ribonucleic acid, synthetases, elongation factors, and ribosomes to remain approximately, though not exactly, in balance at different growth rates.

Ribosomal proteins. VII: Two-dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins

A two-dimensional polyacrylamide gel system has been developed, improving the analytical separation of complex protein mixtures as obtained from ribosomes. An equipment is described in detail (Figs. 1–4) which permits a simultaneous electrophoretic separation of five protein mixtures under identical conditions. By this method the protein mixtures of E. coli ribosomes can be resolved into about 50 components (Fig. 5). The use of this fingerprinting technique facilitates and accelerates considerably a number of investigations on the structure of ribosomes.