Employing Sodium Dodecyl Sulfate Research Articles

Alkaline urea solubilization, two-dimensional electrophoresis and lectin staining of mammalian cell plasma membrane and plant seed proteins

Plasma membranes, isolated from Chinese hamster ovary cells and seed proteins from Arachis hypogaea (L.) were analyzed by two-dimensional electrophoresis. Polypeptides were solubilized without employing sodium dodecyl sulfate (SDS), using in its place 5 m m K 2CO 3 and 9.5 m urea. After addition of dithiothreitol and the nonionic detergent Nonidet P-40, more than 95% of the total protein remained in the supernatant fraction after the preparation was centrifuged at 100,000 g. The solubilization was comparable to that achieved with boiling SDS solution. This soluble material could be used directly for either isoelectric focusing or nonequilibrium pH gradient electrophoresis in narrow bore, tubular, polyacrylamide gels crosslinked by means of N,N′-diallyltartardiamide. Up to 750 μg of protein could be analyzed in one such 3 mm gel. Electrophoresis in polyacrylamide slab gels containing SDS was used for separations in the second dimension. The method allows large amounts of both basic and acidic insoluble proteins to be solubilized and then analyzed without employing SDS as a solubilizing agent. Classes of glycoproteins on the gels were detected by incubating with small volumes of 125I-lectins in heat-sealed plastic bags. CHO cells contain several high molecular weight acidic glycoproteins that bind wheat germ agglutinin, but which do not stain with Coomassie blue. Several of the storage polypeptides in peanut seeds were also shown to bind wheat germ agglutinin and are probably, therefore, glycoproteins containing N-acetyl d-glucosamine.

POSTHARVEST STORAGE OF THE CULTIVATED MUSHROOM (Agaricus bisporus) AND ITS INFLUENCE ON QUALITATIVE PROTEIN CHANGES RELATED TO CANNED PRODUCT YIELD

ABSTRACTStorage treatments (12°C, 95% RH) were applied to freshly harvested mushrooms for 0, 24 and 72 hr. Canned product yields were determined on replicated samples processed by a standardized procedure. Canned product yields increased 19% after 72 hr storage and total free amino acid content of the tissues increased proportionately. Electrophoretic analyses employing sodium dodecyl sulfate gels demonstrated that degradation of larger molecular weight proteins occurred during storage with a corresponding increase in the lower molecular weight fractions. Water‐binding capacity and water‐holding capacity of partially purified proteins prepared from treated lots increased as storage time progressed. Data substantiated the hypothesis that qualitative changes in mushroom proteins are a factor in the increased product yields obtained by postharvest storage of mushrooms.

Activation of C55-isoprenoid alcohol phosphokinase from Staphylococcus aureus. II. Biophysical studies.

Physicochemical techniques were used to investigate the activation of C55-isoprenoid alcohol phosphokinase by synthetic lecithins. Complexes of the enzyme with phospholipids were prepared using a method employing sodium dodecyl sulfate as a protein-solubilizing agent. Circular dichorism and the intrinsic fluorescence of the kinase were used as optical probes of protein conformation with these complexes. No evidence for a major lipid-dependent conformational change in the protein was observed when these complexes were studied under conditions where the lipid mesomorphic transitions occurred. EPR studies of mixtures of synthetic lecithins and the C55-isoprenoid alcohol indicated a correlation between kinase activity and the rotational diffusion rate within the hydrophobic phase. It is concluded that the lipid physical state probably does not affect the enzyme activity by altering the protein conformation but more likely does so by affecting the motion of the molecular participants in the reaction.

Separation of inner and outer membranes of Rhodopseudomonas spheroides.

The separation of inner and outer membrane of Rhodopseudomonas spheroides has been achieved by means of sucrose density gradient (20%, 40%, 60%, w/w) centrifugation. The upper fraction of the gradient, with a specific density 1.181 (g/cm3), is high in cytochrome and succinate dehydrogenase activities, low in lipopolysaccharides and it is designated the inner membrane fraction. The bottom fraction of the gradient, with a specific density 1.240, is high in lipopolysaccharide and contains neither cytochrome nor succinate dehydrogenase activities. This fraction is the cell wall or outer membrane fraction. The intermediate band on the gradient is an unseparated fraction of inner and outer membrane fragments. This fraction has a specific denisty of 1.211 and represents less than 3% of total crude envelope. Thin sections of the vesicles of the inner membrane fraction and those of outer membrane provide morphological evidence for the identity of the individual membrane fractions. At least 22 protein bands are resolved by employing sodium dodecyl sulfate slab gel electrophoresis. Six bands are present only in the inner membrane and two bands are found exclusively in the outer membrane. Most of the remaining polypeptides are present in greater amounts in the inner membrane relative to the outer membrane fractions.