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PubMed1998

Factor structures of the Zung Self-Rating Depression Scale (SDS) for undergraduates.

In order to categorize the items of the Zung Self-Rating Depression Scale (SDS) so as to allow the construction of subscales, the Japanese version of the SDS was administered to a total of 2,258 undergraduates. Principal-component analyses of the SDS extracted three factors interpretable as cognitive, affective, and somatic symptoms. The SDS was then administered to 597 undergraduates in order to cross-validate the factor structures. The coefficient of congruence and the goodness-of-fitness index generated by a confirmatory factor analysis showed good cross-validity of the factor structures.

Adolescent↗
PubMed2005

NMR structures and orientation of the fourth transmembrane domain of the rat divalent metal transporter (DMT1) with G185D mutation in SDS micelles.

DMT1, also known as Nramp2, is an iron transporter, and belongs to the family of Nramp proteins. Disease-causing mutations both in Nramp1 and Nramp2 occurring at the conserved two adjacent glycine residues located within the fourth transmembrane domain (TM4) suggest that TM4 may serve an important biological function. In the present study, we have determined the high-resolution structures of a synthetic peptide, corresponding to the sequence of the fourth transmembrane domain of rat DMT1 with G185D mutation, in membrane-mimetic environments (e.g., SDS micelles) using NMR spectroscopy and distance-geometry/simulated annealing calculations. The spatial structures showed alpha-helices without a kink in the middle portion of the peptide, with a highly flexible and poorly defined N-terminus. Both the N-terminus and the helical core of the peptide were embedded into the SDS micelles. Interestingly, the folding and membrane location of the C-terminus was pH dependent, being well-folded and inserted into SDS micelles only at a low pH value (4.0). The peptide exhibited amphipathic characteristics, with hydrophilic residues (Asp7, Thr11, Asp14, and Thr15) lying in one side of the helix, which provide a basis for the formation of water-filled channel architectures through self-associations. The significant broadening of the resonances of the hydrophilic residues Asp7, Thr11, and Asp14, which are buried inside SDS micelles, upon addition of Mn2+ further verified the possibility of the formation of a channel through which metal ions pass. The substitution of Gly7 by an aspartate residue neither significantly altered the structure and membrane location of the peptide nor abolished its properties of channel forming and metal permeation compared with the wild-type peptide.

Amino Acid Sequence↗
PubMed2006

Simultaneous electrophoretic analysis of proteins of very high and low molecular weights using low-percentage acrylamide gel and a gradient SDS-PAGE gel.

To be able to separate and analyze giant proteins and small proteins in the same electrophoretic gel, we have used a continuous SDS-PAGE gel formed by the combination of a low-percentage acrylamide gel and a gradient SDS-PAGE gel that we have named LAG gel. To get a good resolution for proteins of more than 200 kDa, we used an acrylamide/bisacrylamide ratio of 80:1 in the low-percentage acrylamide gel. To successfully resolve proteins in the 5-200 kDa range, we used a conventional 6-15% SDS-PAGE gradient gel with the standard acrylamide/bisacrylamide ratio of 40:1. We show that the LAG system can be successfully used in general applications of SDS-PAGE electrophoresis such as proteomics and immunobloting techniques. Thus, using this continuous LAG gel, it is possible to simultaneously analyze giant proteins, such as HERC1 and dynein, big proteins like clathrin heavy chain and small proteins like ARF. The LAG system has a good resolution, low cost, and high reproducibility. Moreover, to simultaneously analyze all proteins saves time. All these characteristics, together with the use of a standard apparatus found in any biochemistry laboratory, make the LAG system an easy tool to use.

Acrylamides↗
PubMed1983

Antibody-linked polymerase assay on protein blots: a novel method for identifying polymerases following SDS-polyacrylamide gel electrophoresis.

We describe a method for correlating polymerase activity with a particular polypeptide band in an SDS-polyacrylamide gel which does not require renaturation of the SDS-denatured enzyme. The method involves the following steps: (i) transfer of proteins from an SDS-polyacrylamide gel onto nitrocellulose; (ii) incubation with excess antiserum raised against a partially purified polymerase preparation to link one Fab site of an antibody molecule to the denatured enzyme on the nitrocellulose; (iii) binding of native polymerase to the other Fab site of the antibody molecule in the immune complex to generate a specific polymerase 'sandwich'; (iv) assaying of the nitrocellulose filter for antibody-linked native polymerase activity using an appropriate template and a radioactive substrate followed by treatment with trichloroacetic acid to precipitate in situ the radioactive product. The essential feature of this method is that the use of both non-specific anti-polymerase serum and a partially purified enzyme preparation is sufficient to allow identification of a specific protein following SDS-polyacrylamide gel electrophoresis. This antibody-linked polymerase assay has been developed to identify a 130,000-dalton RNA-dependent RNA polymerase from cowpea leaves. Possible applications of this type of assay as a tool for identifying a wide variety of proteins are discussed.

Animals↗
PubMed1995

Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer.

Nitric oxide synthases (NOSs), which catalyze the formation of the ubiquitous biological messenger molecule nitric oxide, represent unique cytochrome P-450s, containing reductase and mono-oxygenase domains within one polypeptide and requiring tetrahydrobiopterin as cofactor. To investigate whether tetrahydrobiopterin functions as an allosteric effector of NOS, we have analyzed the effect of the pteridine on the conformation of neuronal NOS purified from porcine brain by means of circular dichroism, velocity sedimentation, dynamic light scattering and SDS-polyacrylamide gel electrophoresis. We report for the first time the secondary structure of NOS, showing that the neuronal isozyme contains 30% alpha-helix, 14% antiparallel beta-sheet, 7% parallel beta-sheet, 19% turns and 31% other structures. The secondary structure of neuronal NOS was neither modulated nor stabilized by tetrahydrobiopterin, and the pteridine did not affect the quaternary structure of the protein, which appears to be an elongated homodimer with an axial ratio of approximately 20/1 under native conditions. Low temperature SDS-polyacrylamide gel electrophoresis revealed that tetrahydrobiopterin and L-arginine synergistically convert neuronal NOS into an exceptionally stable, non-covalently linked homodimer surviving in 2% SDS and 5% 2-mercaptoethanol. Ligand-induced formation of an SDS-resistant dimer is unprecedented and suggests a novel role for tetrahydrobiopterin and L-arginine in the allosteric regulation of protein subunit interactions.

Allosteric Regulation↗
PubMed1981

Sexual dimorphism of SDS peptide patterns from the submaxillary glands of mice.

In mice (Mus musculus) sexual dimorphism is evident in the structure and function of the submaxillary glands, convoluted tubules being much more prominent in males than in females. Several enzymes are more abundant in the male gland. We have examined the submaxillary glands of male and female mice for the amounts of arginine peptidase and have made electrophoretic scans of SDS peptides in extracts of male and female glands during postnatal development and after androgen treatment. Larger peptides are relatively more abundant in female extracts, and smaller peptides are more abundant in the male extracts. The peptide patterns reflect the amount of arginine peptidase present in the extracts. No arginine peptidase could be detected in submaxillary salivary glands of mice from birth until 3 weeks of age. Then enzyme activity gradually increased, apparently induced by androgen hormones. The amounts of SDS peptides of high molecular weight steadily decreased with increasing age of the mouse and with increasing titers of arginine peptidase. Castration of the male led to the appearance of female SDS patterns; male patterns reappeared after treatment with testosterone. Females could be induced to form male patterns of SDS peptides by treatment with androgen hormones.

Age Factors↗
PubMed1994

A new two-step precipitation method removes free-SDS and thiol reagents from diluted solutions, and then allows recovery and quantitation of proteins.

Several polypeptides are present in a few copies per cell (i.e., membrane receptors or transcription factors), and therefore their concentration and quantitation from highly diluted solution after detergent solubilization is often an essential and difficult step in their purification by gel electrophoresis. A solution (optimized to Tris-glycine, Tris-borate, and Na-phosphate buffers) to all of these problems is here described, detailing a two-step procedure which takes advantage of the lower solubility of free potassium dodecyl sulfate (KDS) vs micellar KDS even at neutral pH. In the first step, more than 90% of free DS precipitates out from protein solution, the method being poorly sensitive to dodecyl sulfate/KCl ratio from almost no SDS to 10%. Indeed to obtain a residual 0.01% of SDS in the solution no adjustment of KCl concentration is needed from 0.1 to 2.3% SDS. When the supernatant is added with ice-cold TCA and K+, protein solubility is severely affected. Excellent protein recovery, at least 90%, is obtained with hydrophilic proteins. On the other hand, hydrophobic or low-ionic-strength-insoluble proteins are partially lost in step 1 precipitate, so that protein yield decreases, still remaining to about 80%. Furthermore our procedure simplifies determination of protein contents of diluted mercaptoethanol-SDS-solubilized proteins: since thiol reagents are discarded with the final supernatant, proteins can be quantitated in the final pellet by standard colorimetric methods.

Animals↗
PubMed1999

Micellar Binding of alpha-Diimine-Chromium(III) Complexes (Cr(NN)3+3) to Sodium Dodecyl Sulfate (SDS).

Binding interactions of alpha-diimine-chromium(III) complexes with sodium dodecyl sulfate (SDS) were studied in air-saturated solution and in N2-purged solution using lifetime measurements. The lifetime titration curves consisted of two regions with different slopes: one of them negative and more pronounced at low concentrations of SDS until reaching a minimum and the other, at a later stage, with a less pronounced positive slope till a plateau was reached. This biphasic behavior of lifetime-SDS concentration data revealed the presence of premicellar aggregates at low SDS concentration and the formation of normal micelles at high surfactant concentration. The results were analyzed with a model that includes binding of the sensitizer to micelles and to small premicellar aggregates. Binding constants obtained by fitting of lifetime titration curves are in agreement with the electrostatic interactions between the complexes and the surfactant and with the hydrophobic interactions between the ligands of the complexes and the formed micelle. Copyright 1999 Academic Press.

Journal Article↗
PubMed2002

Burst-phase expansion of native protein prior to global unfolding in SDS.

Although numerous studies have been directed at understanding early folding events through the characterization of folding intermediates, there are few reports on the very late folding events, i.e. on the events taking place on the native side of the folding barrier and on alternative conformations of the folded state. To shed further light on these issues, we have characterized by protein engineering the structure of an expanded but native-like intermediate that accumulates transiently in the unfolding reaction of the small protein S6 in the presence of SDS. The results show that the SDS micelles attack the native protein in the dead-time of the denaturation experiment, causing an expansion of the hydrophobic core prior to the major unfolding transition. We distinguish two forms of the unfolding intermediate that are correlated with the micellar structure. With spherical micelles, the expansion is seen mainly as a weakening of the interactions which anchor the two alpha-helices to the core of the S6 structure. With cylindrical micelles, prevalent at higher SDS concentrations, the expansion is more global and produces a species which closely resembles the transition-state structure for unfolding in GdmCl. Despite the highly weakened core, the micelle-associated intermediate displays cooperative unfolding, indicating a significant structural plasticity of the species on the native side of the folding barrier in the presence of SDS.

Circular Dichroism↗
PubMed1984

The isolation of immunogenic molecular entities from immunogenic and nonimmunogenic tumor homogenates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

YAC, a Moloney-virus-induced tumor of A-strain mice, is a nonimmunogenic tumor. Mice injected with the inactivated neoplastic cells and challenged with viable tumor cells did not survive longer than mice that received the challenge dose alone. The homogenate of this nonimmunogenic tumor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gel slices containing isolated molecular entities were injected into various groups of mice. The mice were challenged with low doses of viable tumor cells (10-30 cells) and their survival time was recorded. Small but significant numbers of mice injected with apparent 80-90 K SDS-PAGE-isolated molecular entity rejected the tumor or survived longer than the control groups of mice. Spleen cells from mice injected with 80-90 K molecular entity inhibited the YAC tumor cotransferred with them to naive recipients (Winn assay). Spleen cells from mice injected with monoclonal antibody against nonspecific T-cell helper factor and immunized with 80-90 K SDS-PAGE-isolated molecular entity failed to inhibit the tumor growth in naive recipients, indicating that helper T cells are involved in induction of the antitumor resistance. Nylon-wool-passed splenocytes from mice injected with 80-90 K inhibited tumor growth in some of the recipient mice. Spleen cells from these mice treated with anti-Thy-1 and complement also inhibited the tumor growth in some of the recipients, suggesting that the effector cells were both T and non-T cells. C57BL/6 mice immunized with apparent 20 K SDS-PAGE-isolated molecular entity of RBL5 tumor also induced in vivo resistance to the syngeneic viable RBL5 cells, but not to the syngeneic B16 melanoma cells, indicating the specificity of the protective effect. The practical and theoretical implications of these findings are discussed.

Animals↗
PubMed1991

Anomalous mobilities of Na,K-ATPase alpha subunit isoforms in SDS-PAGE: identification by N-terminal sequencing.

Three isoforms of the alpha subunit of Na,K-ATPase, alpha 1, alpha 2, and alpha 3 have been characterized at the DNA, mRNA and protein levels. In admixtures, isoforms migrate as doublets (i.e. alpha 1 and another band originally designated alpha +, comprising alpha 2 + alpha 3) when analyzed by SDS-PAGE. As deduced from cDNA sequences their masses range from 111.7 to 112.6 kDa. With conventional protein standards, however, SDS-PAGE yields nominal masses of 85-105 kDa. In this system, the presence of a doublet that reacted with a polyclonal anti-Na,K-ATPase antibody in the kidney was interpreted as indicating two molecular or conformational species of the kidney alpha sub-unit (Siegel, G.J. and Desmond, T.J. (1989) J. Biol. Chem. 264, 4751-4754). We report that Na,K-ATPase purified from dog, guinea pig and rat kidney medulla or from rat brain, can yield two distinct bands when analyzed by SDS-PAGE or STS-PAGE, migrating between 85 and 105 kDa. An additional band migrating at 117 and 120 kDa appears often in enzyme purified from rat and guinea pig kidney medulla. The apparent molecular weights and relative intensities of these bands vary with temperature and duration of incubation during sample preparation. N-terminal sequencing and monospecific antibody probes revealed that the two distinct bands obtained from the kidney enzyme consist only of the alpha 1 isoform. The band appearing at 117-120 kDa also contains only the alpha 1 N-terminal sequence. In contrast, as reported earlier (Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6067), the doublet seen in brain preparations consists of alpha 1 and alpha 2 or (alpha 2 + alpha 3). We conclude that monospecific antibody probes or N-terminal sequencing must be used to identify Na,K-ATPase isoforms by SDS- or STS-PAGE. In addition, gel conditions that may affect the mobilities of the isoforms are discussed.

Amino Acid Sequence↗
PubMed1992

Three-dimensional structure of (1-36)bacterioopsin in methanol-chloroform mixture and SDS micelles determined by 2D 1H-NMR spectroscopy.

Spatial structures of proteolytic segment A (sA) of bacterioopsin of H. halobium (residues 1-36) solubilized in a mixture of methanol-chloroform (1:1), 0.1 M LiClO4 organic mixture, or in perdeuterated sodium dodecyl sulfate (SDS) micelles, were determined by 2D 1H-NMR techniques. 324 and 400 NOESY cross-peak volumes were measured in NOESY spectra of sA in organic mixture and SDS micelles, respectively. The sA spatial structures were determined by local structure analysis, distance geometry calculation with program DIANA and systematic search for energetically allowed side chain rotamers consistent with NOESY cross-peak volumes. The structures of sA are similar in both milieus and have the right-handed alpha-helical region from Pro8 to Met32 with root mean square deviation (RMSD) of 0.25 A between backbone heavy atoms and fit well with Pro8 to Met32 alpha-helical region in electron cryo-microscopy model of bacteriorhodopsin. The N-terminal region Ala2-Gly6 of sA in organic mixture has a fixed structure of two consecutive gamma-turns as 2 * 2(7)-helix (RMSD of 0.25 A) stabilized by the Thr5 NH...O = C Gln3 and Ile4 NH...O = C Ala2 hydrogen bonds while this region in SDS micelles has disordered structure with RMSD of 1.44 A for backbone heavy atoms. The C-terminal region Gly33-Asp36 of sA is disordered in both milieus. Torsion angles chi 1 of sA were unequivocally determined for 13 (SDS) and 11 (organic mixture) of alpha-helical residues and are identical in both milieus.

Amino Acid Sequence↗
PubMed1977

Detection of antigens and antibodies by an immuno-peroxidase method applied on thin longitudinal sections of SDS-polyacrylamide gels.

This paper describes a method for detection of antigens in thin sections of SDS-polyacrylamide gels. This method, called SGIP, involves longitudinal sectioning of SDS gels, fixation of the proteins in the gels, removal of the SDS, incubation of the sections with an antiserum and detection of antigens by the indirect immuno-peroxidase technique. The method is useful for assessing the affinity spectrum of a given antiserum against a heterogeneous mixture of proteins, and for the detection of proteins in tissue homogenates or other protein mixtures by means of well defined antisera. By applying the method to serial sections from a single SDS-polyacrylamide gel, a dilution dependent reactivity of antisera is demonstrated.

Acrylamides↗
PubMed1990

Immobilization of the surfactant-degrading bacterium Pseudomonas C12B in polyacrylamide gel beads: I. Effect of immobilization on the primary and ultimate biodegradation of SDS, and redistribution of bacteria within beads during use.

The surfactant-degrading bacterium Pseudomonas C12B was immobilized in polyacrylamide gel beads. Conditions were established for minimizing the apparent loss of sodium dodecyl sulfate (SDS)-degrading activity accompanying polymerization, while still retaining durable gel beads. Apparent losses in SDS-degrading activity compared with free untreated bacteria were attributed largely to substrate diffusion limitations imposed by the gel matrix. Changes in the rate and extent of conversion of radiolabel from [1-14C]SDS to 14CO2 were attributed to diffusional restrictions on O2 availability within the gel beads. Scanning electron microscopy was used to show that beads (3 mm3) repeatedly exposed to SDS for 35 days contained a high cell density in a sub-surface layer 0.4-0.7 mm deep, with relatively few bacteria either at greater depth or at the bead surface.

Biodegradation, Environmental↗
PubMed1989

Selective separation of virus proteins and double-stranded RNAs by SDS-KCl precipitation.

The total viral structural polypeptides and the double-stranded genomic RNAs of bluetongue virus can be selectively separated by a single SDS-KCl precipitation step. This simple, rapid and highly reproducible method enables greater than 95% recovery and purity of both viral proteins and dsRNAs within 30 min. The serotypic identity of the separated dsRNAs can be analyzed by SDS-PAGE electrophorogram immediately. After a single phenol/chloroform extraction, the dsRNA can also be used as hybridization probes, templates for molecular cloning and direct RNA sequencing. The SDS-KCl-precipitated viral proteins could be used readily for peptide mapping and as immunogens. Polyclonal and monoclonal antibodies raised against SDS-KCl-precipitated viral structural polypeptides were useful in Western immunoblots.

Blotting, Northern↗
PubMed1995

Unfolding, conformational change of active sites and inactivation of creatine kinase in SDS solutions.

It has been reported that inactivation occurs before noticeable conformational change can be detected during denaturation of creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) and other enzymes by guanidinium chloride or urea. Therefore, Tsou suggested that enzyme active sites may display more conformational flexibility than the enzyme molecules as a whole [Tsou (1986) Trends Biochem. Sci. 11, 427-429; Tsou (1993) Science 262, 380-381]. In this study, the conformational change of the active site, the unfolding of the whole molecule and the inactivation of creatine kinase in solutions of different concentrations of SDS are compared. The results show that, at low SDS concentrations, the conformational change of the active site and inactivation of the enzyme occur to nearly the same extent. However, both of these changes occur at much lower concentrations of SDS than required to significantly unfold the enzyme molecule. The rates of conformational changes of enzyme active sites are markedly faster than those of inactivation. However, at the same SDS concentration, both the inactivation rate and the rate of the active site conformational change are much faster than that of the unfolding of the enzyme molecule as a whole. The above results provide direct evidence of the flexibility of the active site of creatine kinase.

Animals↗
PubMed2006

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation.

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein-surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein-surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein-surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein-surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.

Animals↗
PubMed2004

Laser flash photolysis and time-resolved fluorescence measurements of the aggregation number of SDS-biopolymer complexes.

Aqueous solutions of 0.5% sodium carboxymethyl cellulose, NaCMC, and 2-hydroxyethyl cellulose, HEC, and variable concentration of sodium dodecyl sulfate, SDS, were studied by the intensities ratio of pyrene fluorescence bands (I/III and monomer/excimer) and conductance measurements to determine the critical aggregation concentration, cac, and the degree of micellar dissociation, alpha, respectively. The cac of these systems is close to 2-4 x 10(-3)M and values of alpha are consistent with the formation of SDS micelles adsorbed cooperatively to the polymer backbone. Laser flash photolysis (LFP) and time-resolved fluorescence (TRF) techniques were employed to determine the micellar aggregation number, N, using the probes flavone and pyrene, respectively. The obtained N for HEC/SDS and NaCMC/SDS were 48 and 68, respectively. The presence of the counterions at the NaCMC backbone is the main factor responsible for this number. Besides, the transient spectra of flavone and present in 0.5% HEC or NaCMC with and in absence of SDS are discussed. Flavone triplet state exit rate constant from the biopolymer/SDS complexes showed that these systems are completely different from a pure SDS micelle.

Biopolymers↗