Sodium Dodecyl Sulfate Electrophoresis Research Articles

Vitellogenin receptors during vitellogenesis in the rainbow trout Oncorhynchus mykiss.

Rainbow trout vitellogenin receptors have been characterized by ligand blotting and Scatchard analysis. Their evolution has been studied over a reproductive cycle in a broodstock of 2-year-old females. The receptors were prepared from ovarian membrane homogenates and were solubilized using n-octyl-beta-D-glucopyranoside. The visualization of the receptor by ligand blotting using 125iodine-vitellogenin after sodium dodecyl sulfate electrophoresis revealed the existence of one major binding component corresponding to a protein of 113 kDa. The Scatchard transformation of the binding data revealed a single class of binding sites with an apparent Kd of 1.8 x 10(-8) M/L. The variations of the binding characteristics (Kd and maximum binding) were investigated during vitellogenesis. This study revealed that the Kd was not affected by oocyte growth during vitellogenesis, but was highly decreased in ovulated eggs. The receptor number increased during the same period from 35 to 860 fM per oocyte, while the receptor number per mm2 of oocyte membrane surface was doubled during the same period.

Dependence of lysozyme growth kinetics on step sources and impurities

Interferometric microscopy was used to investigate the growth morphology and kinetics of {110} and {101} faces of tetragonal lysozyme crystals. Solutions were prepared from as-received Sigma and Seikagaku material, and Seikagaku lysozyme further purified by cation exchange liquid chromatography under salt-free conditions. The protein composition of the solutions was characterized by sodium dodecyl sulphate (SDS) electrophoresis with silver staining. We found that on crystals smaller than about 150 μm, 2D nucleation sites were randomly distributed over the faces. With increasing crystal size, surface nucleation became restricted to facet edges and, eventually, to facet corners. This reflects the higher interfacial supersaturation at these locations. However, on some crystals, we observed 2D nucleation at preferred non-corner sites presumably associated with defects. Upon abrupt temperature decreases, dislocation step sources formed on faces that previously had none. Within groups of dislocations, the dominating step source changed frequently. Depending on the activity of the dislocation groups, growth rates of different crystals differed by up to a factor of five during the same experiment. On facets with dislocation step sources, step generation by 2D nucleation became dominant above a critical supersaturation σ∗. In the absence of dislocations, nucleation-induced growth set in at σ < σ∗. In solutions with higher impurity concentrations, the density of the steps generated by 2D nucleation was higher and σ∗ was lower. Hence, it appears that impurity adspecies are active in surface nucleation. The presence of less than 1% of protein impurities with molecular weight (MW) ≥ 30 kD had significant effects on the crystallization kinetics. Step motion was impeded even at high σ, presumably through blocking of kink sites. In solutions without these high MW impurities, facets containing step sources did not grow below σ = ln( C / C sat) < 0.5. In the less pure solutions such a “dead zone” was not observed. Hence, it appears that in lysozyme dead zones are caused by non-protein impurities. In growth from the highly purified material no growth sector boundaries were visible, in contrast to the as-received lysozyme, and striae formation on growth temperature changes appeared drastically reduced.

Preparative electrophoresis on linear polyacrylamide‐agarose composite gels

A preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1-allyloxy-2,3-propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein-associated SDS. The gel slices are then freeze-thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polyacrylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross-linked derivatives, recoveries were virtually complete (95.4% +/- 2.2%), and were independent of molecular weights over the range tested (10(4) --10(6)).

Purification and properties of the pyruvate kinase isozyme M1 from the pig brain

There are four pyruvate kinase isozymes in vertebrate tissues, designated as L, M1, M2, and R. Although pyruvate kinases have been purified and characterized from pig liver, muscle, kidney, and heart, the brain isozyme has not. The aim of this work was to purify, characterize and make an isozymic designation for the pig brain pyruvate kinase. Purification was accomplished by chromatography on phosphocellulose, Sephadex G200, and blue-dextran agarose columns. The molecular weight of the native enzyme was determined by sucrose density centrifugation. The degree of purity, and subunit molecular weight were determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was estimated by the rapid isoelectric focusing method in sucrose gradients. The pH optimum, and kinetics in the presence and absence of fructose-1,6-diphosphate were determined spectrophotometrically. The purification scheme used resulted in a 382-fold purification of pig brain pyruvate kinase, and a final specific activity of 191 Units/mg protein. As estimated by scanning of the sodium dodecyl sulfate polyacrylamide gels, the purification scheme also resulted in a preparation that was of at least 98% purity. Pig brain pyruvate kinase has a native molecular weight of approx. 230,000, and a subunit molecular weight of approx. 60,000. The pI was determined to be approximately 8.0, while the pH optimum was estimated at pH 7.4. Fructose-1,6-diphosphate had no effect on either the Km for phospho(enol)pyruvate, or the Vmax of the reaction. Pig brain pyruvate kinase is similar in native and subunit molecular weights to the other pyruvate kinases from pig tissues. Like other vertebrate pyruvate kinases, pig brain pyruvate kinase is a tetramer. The pH optimum of 7.4 excludes pig brain pyruvate kinase as a type L isozyme, but cannot distinguish between M1 and M2 isozymes. An isoelectric point of 8.0, as well as hyperbolic kinetics and the lack of inactivation by fructose-1,6-diphosphate, however, are consistent with the pyruvate kinase from pig brain being categorized as a type M1 isozyme.

Low-level endoglucanase contamination in a Trichoderma reesei cellobiohydrolase II preparation affects its enzymatic activity on β-glucan

A routinely employed and specific affinity chromatography purification method was used to isolate cellobiohydrolase II (CBHII) from a Trichoderma reesei culture supernatant. Two different identically purified batches of CBHII were found to have distinctly different properties in barley β-glucan hydrolysis. The abilities of the two preparations to produce small oligosaccharides and reduce the viscosity of β-glucan were substantially different, but no contamination or heterogeneity was detected in sodium dodecyl sulfate-electrophoresis used to assess the protein purity. Furthermore, the specific activities of the preparations on cellotetraose substrate were similar. Careful analysis with specific chromogenic oligosaccharide substrates showed that the considerable variation in β-glucan hydrolysis was caused by a minor endoglucanase contamination consisting of only 0.4% of the total protein. The contamination is possibly caused by nonspecific interactions between the proteins and chromatographic column material or by unfavorable protein-protein interactions during the chromatographic separation. The data demonstrate clearly the caution needed in the interpretation of hydrolysis studies on polymeric substrates with cellulolytic enzyme preparations.

Purification and characterization of guanine nucleotide-exchange factor, eIF-2B, and p37 calmodulin-binding protein from calf brain.

Eukaryotic initiation factor 2B, or guanine nucleotide-exchange factor, has been purified for the first time from the brain by a novel procedure that allows the purification of initiation factor 2 as well and uses a salt wash postmicrosomal supernatant as starting material. The procedure includes a three-part chromatographic step in heparin-Sepharose and in SP-5PW and diethylaminoethyl-5PW ion-exchange high-performance chromatographies. The purification of the factors was followed by measuring activity in the guanine nucleotide-exchange assay and the capacity of initiation factor 2 to form a ternary complex with the initiation form of methionyl-tRNA and GTP. The method yields guanine nucleotide-exchange factor (75%) and highly purified initiation factor 2 (> 95%), which are separated in the last step. The exchange factor from the brain is a multimeric protein with five subunits of molecular masses of 82, 65, 52, 42, and 30 kDa; it stimulates ternary complex formation in the presence of GDP, and this activity is inhibited by N-ethylmaleimide. A 37-kDa protein that copurifies with initiation factors is characterized in this study as a new calmodulin-binding protein (p37); it is highly phosphorylated by casein kinase activities and can comigrate with the alpha subunit of initiation factor 2 under standard sodium dodecyl sulfate electrophoresis conditions.

The corpuscles of Stannius in arawana ( Osteoglossum bicirrhosum), an ancient teleost

The corpuscles of Stannius of arawana ( Osteoglossum bicirrhosum), an ancient teleost, were examined by routine light and electron microscopy and following their immunoreactivity to salmon and trout stanniocalcin antisera. Periodic acid-Schiff positive cells of the corpuscles of Stannius had a follicular arrangement and demonstrated a strong immunohistochemical reaction with both stanniocalcin antisera. Fine structural analysis of the paired, posteriorly located, and perirenal ovoid glands revealed two morphologically distinct cell types the basal laminae of which were ramified by nerve terminals. Immunocytochemistry demonstrated that osmiophilic secretory granules in both cell types were immunoreactive to the stanniocalcin antisera. When extracts of arawana corpuscles of Stannius were subjected to sodium dodecyl sulphate electrophoresis and Western blot analysis a diffuse molecular weight band was evident (∼68 kDa) in the non-reduced condition. In all cases, immunoreactivity was abolished by preabsorption of the antisera with salmon stanniocalcin or with a crude extract of arawana corpuscles of Stannius. The corpuscles of Stannius of arawana are similar to those in more recent teleosts with respect to cell structure and their anatomical distribution but their stanniocalcin is more similar in molecular weight to that present in at least one other non-teleost actinopterygian (the gar) which has an ancient lineage.

Determining the authenticity of raw reformed breaded scampi ( Nephrops norvegicus) by electrophoretic techniques

A comparison was made between isoelectric focusing of aqueous extracts and sodium dodecyl sulphate (SDS) electrophoresis of SDS extracts of the ‘meat’ of raw reformed breaded scampi ( Nephrops norvegicus) as a possible means of detecting adulteration with Pacific scampi ( Metanephrops andamanicus) or tropical shrimp ( Penaeus indicus). Only SDS electrophoresis gave profiles from mixed species products in which the characteristic protein zones of the component species could be identified.

Sodium dodecyl sulfate capillary electrophoresis of proteins in entangled solutions of poly(vinyl alcohol)

A novel polymer network is described for efficient sieving of sodium dodecyl sulfate (SDS)-protein complexes: poly(vinyl alcohol) (PVA; average M r 133 000). The entanglement threshold of these solutions was found to be at 3% (w/v) PVA. Solutions from 4 to 6% PVA offer excellent resolution in the 14 400-94 000 protein molecular mass interval. Ferguson plot analysis showed that the separation is indeed based on mass discrimination, as it should in SDS electrophoresis, with extrapolated (at 10% polymer) limit values of mobility for all particles in the range (2.34-2.87) · 10 −8 m 2 V −1s −1. The advantages of PVA are full transparency in the UV region down to 200 nm and extremely low viscosities (e.g., a 5% PVA solution has a viscosity 25 times greater than that of buffer at 30°C). A unique wall effect was found, by which, on decreasing the inner diameter of the capillary from 75 to 25 μm, the apparent entanglement threshold was shifted to extremely dilute PVA solutions, since in 25-μm capillaries efficient sieving was obtained below 1% PVA, i.e., at concentrations well below the entangled regime. It is hypothesized that residual, free silanols present (even in a coated capillary) act as nucleation sites for H-bond formation and aggregation of free PVA molecules.

24] Purification and assay of kinases that interact with Rac/Cdc42

This chapter describes a method to detect a new family of Rho p21 interacting proteins. It also describes the use of the assay for expression screening and in the purification of an abundant Cdc42 and Rac1 target, which is the brain serine/threonine kinase p65-PAK. The GTPase-activating protein (GAP) overlay technique not only detects tissue levels of GAPs after sodium dodecyl sulfate (SDS) electrophoresis but also detects GTPase-inhibitory proteins. For these proteins to exert such an effect on the intrinsic GTPase activity of p21, they must remain bound (that is, they cannot act in a catalytic manner as do GAPs). After autoradiography of the nitrocellulose overlay filter, which holds the image of the nucleotide state of the test p21 within the original filter, some of these proteins can be observed as signals darker than the background. The process of detection of SDS–polyacrylamide-fractionated GTP-p21-binding proteins is described in the chapter.

Sensitive quantification of proteins by electrophoresis in gels by use of chemiluminescence.

For protein quantitation in gels or blotting membranes, chemiluminescence (CL) offers the advantages of a substantial improvement of detection limits. Easy-to-use CL-chemicals and specific probes such as antibodies conjugated to enzymes, e.g. alkaline phosphatase (AP) may be used in combination with a newly developed luminometer. CL was found to have low detection limits and a linear relation between relative light units (RLU) and the concentration of the antibody enzyme complex present over a wide concentration range. Measurements of the immunoglobulin IgE in dot blots and in blots after sodium dodecyl sulfate (SDS) electrophoresis under nonreducing conditions in agarose gels are described.

Micropreparative one- and two-dimensional electrophoresis: improvement with new photopolymerization systems.

To improve the efficiency of one- and two-dimensional electrophoresis for micropreparative purposes, the use of gels polymerized with other initiators than the standard N,N,N',N'-tetramethylethylenediamine (TEMED)/persulfate systems for sodium dodecyl sulfate electrophoresis has been investigated. We show here that the recently described photoinitiator system, composed of methylene blue, toluene sulfinate and diphenyliodonium chloride, leads to a decreased resolution. Resolution can be restored if methylene blue is replaced by riboflavin. Two-dimensional electrophoresis with mg loadings of proteins has also been evaluated with these systems. Independently of the polymerization system, resolution for the first dimension is low with rod gels, increases with gel strips and is further improved when immobilized pH gradients are used. Here too, only the riboflavin/sulfinate/iodonium system results in a resolution that matches the one obtained with the standard TEMED/persulfate system. Gels polymerized with the riboflavin/sulfinate/iodonium system yield better results upon N-terminal microsequencing after blotting than gels polymerized with the standard TEMED/persulfate system.

Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt): the state of the art and the controversy of vertical versus horizontal systems.

After having established the basic protocol of two-dimensional electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt) in 1988 (A. Görg et al., Electrophoresis 1988, 9, 531-546), some critical parameters of the actual IPG-Dalt protocols as well as the results obtained with horizontal and vertical second-dimensional sodium dodecyl sulfate-electrophoresis are demonstrated and discussed.

On the separation mechanism of capillary sodium dodecyl sulfate-gel electrophoresis of proteins.

Polyethylene oxide-mediated capillary sodium dodecyl sulfate-electrophoresis is a recently established, high-resolution separation method for fast purity check and molecular mass assessment of protein molecules. The effects of the sieving polymer chain length and concentration on the separation mechanism of sodium dodecyl sulfate-protein complexes were examined. The studies aimed to clarify whether the separation can be described by either the Ogston sieving theory, or the reptation or reptation-with-stretching theory. Polyethylene oxides with molecular masses of 100,000, 300,000 and 900,000 Da were used as separation matrices at various concentrations ranging from 1-4%, 0.5-2% and 0.25-1%, respectively. The separation phenomena was examined using a standard protein test mixture containing six proteins in the molecular mass range of 14,200-97,400 Da. A possible separation mechanism of reptation with stretching is suggested, where separation performance was improved with increasing sieving polymer chain lengths and/or concentration.

Accumulation of HLA-DM, a regulator of antigen presentation, in MHC class II compartments

The HLA-DM genes encode an unconventional HLA (human leukocyte antigen) class II molecule that is required for appropriate binding of peptide to classical HLA class II products. In the absence of DM, other class II molecules are unstable upon electrophoresis in sodium dodecyl sulfate and are largely associated with a nested set of peptides derived from the invariant chain called CLIP, for class II-associated invariant chain peptides. DMA and DMB associated and accumulated in multilaminar, intracellular compartments with classical class II molecules, but were found infrequently, if at all, at the cell surface. Thus, DM may facilitate peptide binding to class II molecules within these intracellular compartments.

Characterization of growth hormone binding sites in rat brain

The binding of 125I-labelled rat growth hormone (GH) to different areas in the brain was studied in male Sprague-Dawley rats. A high density of GH binding was found in the choroid plexus, hypothalamus, hippocampus, pituitary and spinal cord, whereas a lower binding density was observed in the cortex. Binding of the hormone to the various brain regions was age dependent. Binding was also dependent on time, pH and protein concentration. The binding affinity of the labelled hormone to choroid plexus was 4.3 per nmol/l and the binding capacity was 33.4 nmol/mg protein. The corresponding figures for binding of 125I-labelled GH to hypothalamus were 5.6 per nmol/l and 21.6 nmol/mg protein. By sodium dodecyl sulphate electrophoresis of the cross-linked hormone-receptor complexes, molecular weights of 60,000 and 61,000 were determined for the binding units in the choroid plexus and hypothalamus, respectively. It was further indicated that the binding unit for rat GH was distinct from that for prolactin.

Protein carboxyl methylation in Saccharomyces cerevisiae: evidence for STE14-dependent and STE14-independent pathways.

We incubated yeast cells (Saccharomyces cerevisiae) with the methyl donor S-adenosyl-L-[methyl-3H]methionine and then fractionated their cellular components by gel electrophoresis in sodium dodecyl sulfate. By analyzing gel slices for [3H]methyl esters by a vapor-phase diffusion assay, we detect major methyl-esterified species that migrate at apparent polypeptide sizes of 24 and 22 kDa and minor species of 49, 38, 35, 33, 31, and 26 kDa. Incubation of extracts from labeled cells with ribonuclease A or proteinase K revealed that the 24- and 22-kDa species represent methyl-esterified RNAs, whereas the other species are methyl-esterified polypeptides. The 38-, 33-, 31-, and 26-kDa polypeptides were not methyl-esterified in an isogenic yeast strain lacking the STE14 gene encoding a C-terminal isoprenylcysteine methyltransferase, suggesting that they are substrates for the STE14 methyltransferase. On the other hand, the amount of the methylated 49-kDa polypeptide is reduced in the ste14 mutant, indicating that at least two methylated polypeptides are present--one a substrate of the STE14 methyltransferase and one a substrate of a STE14-independent methyltransferase. The 35-kDa polypeptide also appears to be methylated by a STE14-independent methyltransferase. When cells were incubated in the presence of the protein synthesis inhibitor cycloheximide, little or no methylation of the STE14-dependent species was detected while the methylation of the STE14-independent substrates was unaffected. Pulse-chase studies revealed significant turnover of all of the methylated species in a 4-h period, with the exception of the 38-kDa polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal cations and abnormal membrane lipids in the red blood cells of dogs with familial stomatocytosis-hypertrophic gastritis

Examination of the red blood cells (RBCs) of eight dogs with familial stomatocytosis-hypertrophic gastritis (FS-HG), a multiorgan disease associated with hemolytic anemia, hereditary stomatocytosis (HSt), and hypertrophic gastritis resembling Ménétrier's disease in man, showed abnormal osmotic fragility, normal mean corpuscular volume, slightly increased cell water, and normal cation content and cation fluxes. Cholesterol was decreased in RBC and increased in plasma. In both RBCs and plasma, total phospholipid (PL) was normal, phosphatidylcholine (PC) decreased, and sphingomyelin increased. The palmitic acid content of PC was increased, and the stearic acid content of PC was decreased. Sodium dodecyl sulfate electrophoresis of RBC membrane proteins was normal. These findings have not been described previously in HSt. They suggest that in FS-HG, abnormal composition of the PL in RBCs secondary to abnormal PL in plasma causes defective membrane function and stomatocytic shape-change. This conclusion was supported by a shortened half-life of 51Cr-labeled RBCs from normal dogs after transfusion in dogs with FS-HG. It was concluded (1) that not all hereditary forms of stomatocytosis are necessarily associated with an intrinsic structural defect of the RBC membrane, but that the change in shape of RBC may also be induced by abnormal composition of the plasma; (2) that stomatocytosis may be caused by loss of membrane surface area rather than by the increased cation uptake such as has been shown in some human kindreds with HSt, (3) that FS-HG is a disorder of lipid metabolism, and by consequence, (4) that abnormal lipid metabolism might be involved in the pathogenesis of Ménétrier's disease.

Normal Cations and Abnormal Membrane Lipids in the Red Blood Cells of Dogs With Familial Stomatocytosis-Hypertrophic Gastritis

Abstract Examination of the red blood cells (RBCs) of eight dogs with familial stomatocytosis-hypertrophic gastritis (FS-HG), a multiorgan disease associated with hemolytic anemia, hereditary stomatocytosis (HSt), and hypertrophic gastritis resembling Menetrier's disease in man, showed abnormal osmotic fragility, normal mean corpuscular volume, slightly increased cell water, and normal cation content and cation fluxes. Cholesterol was decreased in RBC and increased in plasma. In both RBCs and plasma, total phospholipid (PL) was normal, phosphatidylcholine (PC) decreased, and sphingomyelin increased. The palmitic acid content of PC was increased, and the stearic acid content of PC was decreased. Sodium dodecyl sulfate electrophoresis of RBC membrane proteins was normal. These findings have not been described previously in HSt. They suggest that in FS-HG, abnormal composition of the PL in RBCs secondary to abnormal PL in plasma causes defective membrane function and stomatocytic shape-change. This conclusion was supported by a shortened half-life of 51Cr-labeled RBCs from normal dogs after transfusion in dogs with FS-HG. It was concluded (1) that not all hereditary forms of stomatocytosis are necessarily associated with an intrinsic structural defect of the RBC membrane, but that the change in shape of RBC may also be induced by abnormal composition of the plasma; (2) that stomatocytosis may be caused by loss of membrane surface area rather than by the increased cation uptake such as has been shown in some human kindreds with HSt, (3) that FS-HG is a disorder of lipid metabolism, and by consequence, (4) that abnormal lipid metabolism might be involved in the pathogenesis of Menetrier's disease.

Deterioration of bioprosthetic heart valves.

The aim of this study was to detect biologic factors in the structural deterioration of bioprosthetic heart valves. Prostheses were removed from patients after 4-8 years of implantation and submitted to biochemical and morphologic assays. Successive staining of biologic sections revealed colocalization of lipids and glycosaminoglycans underneath calcifications in the disintegrated extracellular matrix. On biochemical assays, the amidolysis of synthetic peptide substrates indicated thrombin, plasmin, and tissue plasminogen activator activities in the nonhemocompatible leaflets; 0.15 mol NaCl, 0.05 mol Tris, and 5 mmol CaCl2 extracts from the prostheses cleaved the peptide substrate for collagenase and lysed gelatin gels. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate disclosed the presence of low molecular mass polypeptides in extracts of the deteriorated prostheses. The detection of plasmin and collagenolytic enzyme(s), and the known broad proteolytic activity of plasmin, may point to the role of activation of the fibrinolytic system in the proteolytic degradation of bioprosthetic valves.