Methodes d'extraction d'enzymes, d'electrophorese en gel et de coloration specifique des enzymes utilisees pour etudier la variation genetique chez Escherichia coli et d'autres bacteries. Les procedures decrites peuvent s'appliquer, avec quelques modifications mineures, a toute espece de bacterie

Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics

The level of genetic variation revealed by two-dimensional electrophoresis of proteins from seedlings of two wheat lines strongly depends on the technical procedures. Improvements in extraction and electrophoresis procedures relative to earlier experiments on the same material led to a significant increase in the genetic variation revealed: 15.2 % instead of 6.7 % of the spots were genetically variable. The improved procedure is based on (i) precipipation of proteins from wheat seedlings with trichloroacetic acid and acetone, (ii) solubilization of the proteins with a solution containing urea, potassium carbonate and sodium dodecyl sulfate, (iii) isoelectric focusing in an optimized pH gradient, obtained with a mixture of carrier ampholytes (Pharmalyte and Servalyt), and (iv) running elecrophoresis in the second dimension on gels with increased surface.

Technical improvements in two‐dimensional electrophoresis increase the level of genetic variation detected in wheat‐seedling proteins

Aims: The mitochondrial respiratory chain is recognized today to be arranged in supramolecular assemblies (supercomplexes). Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. In the present study, we have directly addressed this issue by testing the ROS generation by Complex I in two experimental systems in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. Results: The results of our investigation provide experimental evidence that the production of ROS is strongly increased in either model, supporting the view that disruption or preve...

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Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from complex I.

DsbC is a soluble protein of the bacterial periplasm that was identified genetically as being involved in protein disulfide formation. The gene sequence was corrected to include an additional proline residue and was then consistent with the molecular weight of the purified protein. Gel filtration and subunit hybridization indicate that DsbC is a stable dimer of identical subunits. Each subunit has a -Cys-Gly-Tyr-Cys- segment that forms an unstable and reactive disulfide bond; only the first cysteine residue is accessible, similar to thioredoxin and DsbA. The other two cysteine residues of DsbC form a buried, structural disulfide bond. The reactivities and stabilities of the active site disulfide bond of DsbC have been characterized and compared to that of DsbA. Both are very unstable and can be transferred rapidly to reduced proteins and peptides, although they differ somewhat in their kinetic reactivities. The two active sites of the DsbC dimer appear to function independently. DsbC is much more active than DsbA in catalyzing protein disulfide rearrangements, and this may be its main function in vivo.

Structural and Functional Characterization of DsbC, a Protein Involved in Disulfide Bond Formation in Escherichia coli

The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.

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The effect of net charge on the solubility, activity, and stability of ribonuclease Sa

Electrophoresis buffers for polyacrylamide gels at various pH.

Fourteen myoglobins of known sequence were examined by polyacrylamide gel electrophoresis at five pH values. Gels at each pH divided the sequences into six to eight distinct classes, while the combination of the results of three gels at different pH levels distinguished 13 of 14, or 93%, of the sequences. The relative mobility of the myoglobins in the gels is significantly correlated with the charges of the proteins calculated from the pK values of the ionized groups. Major differences in mobility corresponded to expected differences in charge due to the amino acid substitutions between sequences. In addition to sequences differing in the total number of acidic and basic residues, those differing from each other in the total number of histidines were distinguished on low-pH gels. One pair of sequences differing by the exchange of lysine for arginine was separated on high-pH gels, as predicted from the differences in ionization of these two amino acids. On gels at pH 10.4, there was greater deviation of electrophoretic mobility from charge than on other gels, possibly due to the influence of amino acid substitutions in the neighborhood of lysine residues. Manipulation of the concentration and composition of the gels did not change the separation of the sequences from each other. Examination of myoglobins by gel electrophoresis at a wide range of pH values allows discrimination of nearly all amino acid substitutions and demonstrates the close relationship between titration and relative electrophoretic mobility.

Molecular Charge and Electrophoretic Mobility in Cetacean Myoglobins of Known Sequence

Two proteins with known characteristics on one-dimensional gels were studied by two-dimensional electrophoresis to compare the sensitivities of the two methods in detecting genetic variation. Two-dimensional electrophoresis was found to be less sensitive than several types of one-dimensional gels in distinguishing variants of both proteins. Denaturation of proteins in urea in the two-dimensional method makes it possible to distinguish closely related proteins that differ from each other by units of charge. Many more types of variation in protein sequences can be distinguished on one-dimensional gels in the absence of denaturants. The estimates of heterozygosity based on two-dimensional gels are lower than those based on other methods, at least in part, because of the limited types of sequence differences that can be detected on two-dimensional gels. The application of two-dimensional electrophoresis to the measurement of genetic variation and to the detection of new mutations should be made carefully, in view of the limited sensitivity of the method in finding differences in sequence.

https://www.genetics.org/content/genetics/104/2/381.full.pdf

Genetic variation in proteins: comparison of one-dimensional and two-dimensional gel electrophoresis

Fourteen myoglobins of known sequence were examined by isoelectric focusing with and without urea. The 14 sequences formed six distinct mobility classes on gels without urea and three classes on those with urea. For these proteins, isoelectric focusing provides no advantage over single, nonequilibrium, nondenaturing gels in the total number of distinguishable mobility classes. Only major charge differences, resulting from the changes in the total numbers of acidic and basic amino acids, can be detected on gels with urea, indicating that denaturation by urea alters proteins so that small differences in ionization are eliminated.

Tracy McLellan

Papers

Electrophoresis buffers for polyacrylamide gels at various pH.

Molecular Charge and Electrophoretic Mobility in Cetacean Myoglobins of Known Sequence

Genetic variation in proteins: comparison of one-dimensional and two-dimensional gel electrophoresis

The sensitivity of isoelectric focusing and electrophoresis in the detection of sequence differences in proteins.