Sodium Dodecyl Sulfate Buffer Research Articles

Carbohydrate incorporation in plasma membranes of mouse thymocytes stimulated by concanavalin A.

Thymocytes from CBA/J mice were stimulated with concanavalin A. Incorporation of various carbohydrates, leucine and thymidine into trichloroacetic-acid-precipitable material was compared in stimulated and control cells. Incorporation of all carbohydrates, in particular of galactose, was enhanced in concanavalin-A-treated cells. Leucine and thymidine incorporation was increased 2 and 50-fold respectively in stimulated cultures. The kinetics of fucose, galactose, glucosamine, leucine and thymidine incorporation were studied using 10-h pulses at various times during the cultivation. The incorporation of fucose, galactose and thymidine showed two maxima 5 and 30 h after the beginning of the cultivation. In contrast, there were no pronounced maxima seen with glucosamine and leucine. Plasma membranes from stimulated cells were prepared by a modified procedure of McCollester (Cancer Res. 30, 2832–2840, 1970). The purity of the membrane fraction was examined by electron microscopy, by chemical analysis and by assay of marker enzymes. When prepared from cells radioactively labelled with fucose or galactose the plasma membrane fraction was enriched in carbohydrate label. Gel filtration experiments with papain treated, radioactively labelled membranes, solubilised in sodium dodecylsulphate buffer, showed that fucose was only attached to glycoprotein, while other carbohydrate label was distributed between glycoprotein and a fraction containing glycolipids. Analysis of hydrolysed, carbohydrate-labelled membranes by paper chromatography and paper electrophoresis revealed that the bulk of the fucose and galactose label had not been converted into other substances, although some (10%) galactose is converted into glucose. Mannose label had been partially (14%) converted into fucose. Glucosamine label was found to be distributed in sialic acid (25%), glucosamine (40%), galactosamine (30%) and neutral compounds (5%). The data are discussed in relation to the sequence of biochemical events occurring at the cell surface during stimulation of thymocytes with concanavalin A.

Studies on the brush border membrane of mouse duodenum. III. Iodination of membrane proteins.

Microvillus membranes were iodinated from luminally administered lactoperoxidase, H2O2 and 125I before and after neuraminidase treatment. Membranes were isolated, solubilized in sodium dodecyl sulphate buffer and electrophoresed on gels. Gels were stained for protein, and then sliced for liquid scintillation counting. When membranes were not treated with neuraminidase, the nonpermeating iodination probe attached only to a band containing protein of 150,000 daltons approximate molecular weight. This size class of protein may reside on the luminal side of the brush border membrane as opposed to the serosol side. Qualifications of this statement are discussed with reference to the location of tyrosyl residues and to the possibilities of masking molecules. Membranes treated from the luminal side with neuraminidase to remove possibly masking carbohydrate and then iodinated, appeared to contain an additional protein of estimated molecular weight 220,000 daltons which was accessible to 125I. Thus, a 220,000 dalton protein may also be on the luminal side of the membrane. An explanation is attempted for the discrepancy between the very few proteins labelled and the many proteins involved in terminal digestion and transport which would all be expected to be available to luminally administered iodination probe. Membranes were isolated, exposed to iodination and then solubilized for electrophoresis. Nearly all proteins were labelled, which indicated that there is an asymmetric distribution of proteins in the plane of the membrane. The two smallest molecular weight polypeptides which were not iodinated were proposed to be so disposed in the membrane that they were inaccessible to the probe, from either side.

Studies on the brush border membrane of mouse duodenum. II. Membrane protein metabolism.

Mouse duodenal microvillus membrane protein metabolism was measured using radioactive labelling techniques. Labelled amino acids were introduced into the lumen of ligatured duodena. Following exposure to label, brush border membranes were isolated and analyzed. Experiments measuring the specific activity of protein labelled with a single amino acid revealed that total membrane protein appeared to turnover in about 14 hr. Protein in the mucosal homogenate had a faster turnover rate. Turnover rates of individual proteins were measured with single and dual isotope experiments. Membrane protein was solubilized with sodium dodecyl sulphate (SDS) buffer. Single isotope experiments showed that all polypeptides separated on SDS-gels were maximally labelled at 6 hr after injection. Bands did not incorporate label linearly. Rates of loss (degradation) of label from membrane proteins in the seventeen bands appeared to be related to the estimated molecular size of the proteins. Rates were highest for larger polypeptides. A double isotope technique, in which proteins were allowed to incorporate the same amino acid in two isotopic forms, delivered with a set time interval intervening, revealed that the ratios of the second label to the first in the SDS-separated polypeptides were highest for larger proteins and lowest for smaller polypeptides. Certain assumptions were outlined and the ratios taken as measures of turnover of proteins. Loss of label due to cell sloughing is discussed. A mixture of labelled amino acids (excluding leucine) was used to show that differences in leucine contents of different proteins was not an explanation for the variation in level of leucine radioactivity in different bands. For specific activity measurements throughout, protein in gels was quantitated with reference to the uptake of Coomassie stain. The use of this stain was validated by the finding that, at low protein concentration, the amount of stain taken up was proportional to the amount of bovine serum albumin or membrane protein loaded.

Varietal relationships of the alpha-gliadin proteins in wheat.

AbstractThe aggregatable α‐gliadin of Bernardin et al.1 was found in only 4 of 16 wheat varieties tested by ultracentrifugation of an acetic acid extract combined with examination of the pellet by gel electrophoresis at pH 3.3. Those 4 wheat varieties were Scout 66, Cheyenne, Ponca and Itana. The name A‐gliadin was given to the aggregatable α‐gliadin to distinguish it from other gliadins with similar electrophoretic mobilities. The two‐band gel electrophoretic pattern at pH 3.2 was the same for A‐gliadin from all four varieties as was the pattern at pH 8.6. The molecular weight of A‐gliadin from all four varieties was about 36 000 as determined by gel electrophoresis in an SDS (sodium dodecyl sulphate) buffer. The genes responsible for the A‐gliadin components apparently were contributed by Crimean (Turkey) which appeared in the ancestry of all four varieties.