Sodium Dodecyl Sulfate-polyacrylamide Slab Gel Research Articles

Molecular structure of human seminal coagulum: the role of disulfide bonds.

Using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of 100 mM 2-mercaptoethanol, freshly ejaculated human semen showed two major protein bands of 72 and 55 kd. During liquefaction these two protein bands were degraded to low molecular weight proteins of 10-15 kd. In the absence of 2-mercaptoethanol, larger proteins such as those of 243, 221 and 195 kd were obtained which could be converted to 72 and 55 kd proteins by the action of the sulfhydryl reducing agent. Liquefaction was enhanced in the presence of 2-mercaptoethanol. These results indicated that one of the factors involved in the formation of human seminal coagulum is disulfide linkage between proteins of 72 and 55 kd.

Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540–15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540–ammonium sulfate polymer phase system by adding a retainer (10 m M acetic acid) to the upper stationary phase and an eluter (100 m M sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate–polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.

Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus.

An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.

Protein synthesis in developing ovaries of mealworm under in vivo and in vitro conditions: Effects of diflubenzuron

The effect of diflubenzuron (DFB) on protein synthesis in the ovaries of Tenebrio molitor was evaluated during oocyte maturation using in vivo and in vitro assays. When incorporated into the diet (5 and 10 mg g −1), DFB was found to affect both the weight, the protein levels and the incorporation of tritiated leucine into proteins of ovaries. In addition, electrophoretic separation of ovarian proteins by sodium dodecyl sulfate-polyacrylamide slab gels (SDS-PAGE) showed that DFB applied in vivo did not have a significant effect on the number of protein bands. When added to the culture medium (5 and 10 μg ml −1), DFB resulted in a slight but significant decrease in the rate of incorporation of tritiated leucine into proteins of ovaries cultured for 5 d compared to controls. These in vitro effects of DFB on protein synthesis were less marked than those observed in orally treated females.

Biosynthesis and characterization of type X collagen in human fetal epiphyseal growth plate cartilage

We examined in vitro collagen biosynthesis by organ cultures from human fetal epiphyseal growth plate cartilage. The biosynthetic products were characterized by NaCl fractional precipitation, limited proteolytic digestion, and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Organ cultures of human fetal epiphyseal growth plate cartilage synthesized large amounts of type X collagen in addition to type II, type IX, and type XI collagens. The individual polypeptide chains of human type X collagen migrated with an apparent M(r) of 45 kDa after proteolytic digestion with pepsin. The migration pattern of these molecules did not change when examined under reducing and nonreducing conditions, indicating that they did not contain intrahelical disfulfide bonds. Comparison of the rates at type X collagen biosynthesis at weeks 20 and 24 of human fetal development showed a marked increase of 24 weeks. Northern hybridization analysis of total RNA from freshly isolated epiphyseal growth plate chondrocytes with a cDNA corresponding to the carboxyl terminus of human type X collagen indicated that the developmental increase of type X collagen production is determined by pre-translational mechanisms.

Activity and solubility of bone morphogenetic protein derived from porcine bone matrix.

Bone morphogenetic protein (BMP) was extracted from porcine bone matrix and purified by liquid chromatography. The final purified fraction was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE). The molecular weight of porcine bone matrix-derived BMP was estimated to be about 20 kDa by SDS-PAGE. The final purified BMP was highly soluble in vivo, so that it dispersed immediately after implantation and exerted no effect on bone induction. The other crude active fractions obtained in the process of purification induced new bone in three weeks when implanted into muscle pouches of Wistar rats. These findings suggested that pure BMP requires an appropriate carrier (delivery system) for clinical use; hence, experiments using atelopeptide type-I collagen as carrier were conducted.

Identification of a bovine serum mannan-binding protein reactive with a Ra chemotype strain of Salmonella serovar typhimurium.

To study the relationship between a bovine serum mannan-binding protein (MBP) and a serum protein reactive with a Ra chemotype strain of Salmonella serovar Typhimurium (Ra-reactive factor, RaRF), both proteins were isolated by use of their affinity for yeast mannan and the Salmonella cells followed by affinity chromatography on mannobiose-Sepharose 4B. Both purified proteins showed a major protein band with a molecular weight of 33,000 and a few faint bands in sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis under reducing conditions. In Western blotting with rabbit anti-bovine MBP antibody, the major subunit of both proteins were found to be immunologically identical. Similar findings were also obtained with purified human MBP and RaRF. From these findings, bovine and human serum MBP are suggested to be electrophoretically and immunologically the same as their corresponding RaRF.

Changes in the Polypeptide Patterns of Barley Seedlings Exposed to Jasmonic Acid and Salinity

Soluble and thylakoid membrane proteins of jasmonic acid (JA)-treated and salt-stressed barley (Hordeum vulgare L.) seedlings were investigated using 15% sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. High JA concentrations induced marked quantitative and qualitative changes in polypeptide profiles concerning mainly the proteins with approximately equal mobility, as in NaCl-stressed plants. The most obvious increase in thylakoid polypeptide band intensity was at 55 to 57 kilodaltons (kD). The relative share of some polypeptides with apparent molecular masses above 66 kD and of polypeptides with lower molecular masses in the region of 20.5 to 15 kD was enhanced. At the same time, one new band at 31 to 31.5 kD was well expressed at 25 and 250 micromolar JA concentrations and became discernible in the 100 micromolar NaCl-treated plants. The intensity of some polypeptides of soluble proteins (molecular masses of 60, 47, 37, 30, and 23.4 kD) increased with increasing JA concentration, whereas the intensities of other polypeptide bands (55, 21.4, and 15 kD) decreased. Enhanced levels of 60-, 47-, 34-, and 30-kD polypeptides and reduced levels of 55- and 15-kD polypeptides were present in NaCl-treated plants. The appearance of one new polypeptide, of 25.1 kD, was observed only in NaCl-treated plants. At 100 millimolar NaCl, an eightfold increase in proline content was observed while at 250 micromolar JA, the proline content was threefold over the control. It is hypothesized that exogenously applied jasmonates act as stress agents. As such, they provoke alterations in the proline content and they can modulate typical stress responses by induction of stress proteins.

Modification of a discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide gel system for minigel electrophoresis

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system was recently described by J. P. Doucet and J. M. Trifaró ((1988) Anal. Biochem. 168, 265–271). The system was developed to separate with high and broad resolution the components from large volume samples after an overnight electrophoresis. This system was found to have many advantages. However, when used directly as a minigel system, this method cannot sustain the high voltage inherent to minigel electrophoresis and produces artefacts, namely a double front and a loss of resolution in the low molecular weight range. These problems were eliminated using the buffer system of M. A. Porzio and A. M. Pearson ((1977) Biochem. Biophys. Acta 490, 27–34) in the separating gel and in the electrode chambers. The resulting modified discontinuous minigel system has the same advantages as the technique described for large slab gel electrophoresis, including the effective and rapid transfer of high molecular weight proteins to nitrocellulose membranes, as well as the advantages of the minigel format.

Collagenase and Collagenolytic Cathepsin in Normal and Fibrotic Rat Liver

Collagenase and collagenolytic cathepsin activities in normal and carbon tetrachloride-induced fibrotic livers of rats were simultaneously determined at 35 and 25 degrees C for 18 h, using the same 14C-labeled neutral soluble collagen as a substrate. Collagenolytic cathepsin had higher activity under the assay conditions at both 35 and 25 degrees C than collagenase in normal and fibrotic livers. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the collagen was visibly degraded by collagenolytic cathepsin, but not by collagenase. These results indicate that, unlike collagenase, collagenolytic cathepsins exist as active forms in the rat liver, and can participate in the degradation of collagens, especially of soluble collagens including procollagens.

Preparative electrophoresis of human apolipoprotein E: an improved method.

Apolipoprotein E was isolated from human very low density lipoproteins by a two-step electrophoretic procedure derived from that of Méndez (1982. Anal. Biochem. 126: 403-408). It included separation in a sodium dodecylsulfate polyacrylamide slab gel, transfer into an agarose gel, and extraction by ultracentrifugation for 30 min. No protein labeling, dialysis, or concentration procedures were needed. The method was fast, showed an excellent protein recovery, and could be suitable as a general method of protein isolation by polyacrylamide gel electrophoresis.

Purification of rabbit bone morphogenetic protein derived from bone, dentin, and wound tissue after tooth extraction

Bone morphogenetic protein (BMP) was extracted from bone matrix, dentin matrix, and wound tissue after tooth extraction in rabbits, and purified. These purified fractions were shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE), and induced new bone in situ in 3 weeks when implanted into the calf muscles of Wistar rats. The dentin matrix-derived BMP was different from the other two types in molecular weight and the properties revealed in the process of purification. However, each tissue-derived BMP was shown to induce new bone growth in a bioassay of xenogenic implantation. For this reason, BMP is thought to have subunits with certain commonalities in different tissue.

Identification of Different Forms of Human C4b-Binding Protein Lacking β-Chain and Protein S Binding Ability

Human C4b-binding protein (C4BP) is a multimeric regulatory component of the complement system that circulates in plasma either as a free protein or in a noncovalent complex with the vitamin K-dependent protein S. The major form of C4BP is composed of seven identical alpha-chains (70 kDa) and one beta-chain (45 kDa). C4BP was purified from human plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity chromatography. C4BP-high and low Mr forms were both obtained from the barium citrate precipitate and the supernatant. C4BP-high and low forms from the barium citrate precipitate were separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis and extracted with Triton X-100. Both forms contained the beta-chain as was demonstrated on sodium dodecylsulfate polyacrylamide slab gel electrophoresis under reduced conditions after silver-staining and with Western-blotting using monoclonal antibodies specific for the beta-chain. The C4BP-high and low forms demonstrated similar protein S binding affinity (KA: 3.18 x 10(8) and 3.21 x 10(8) M-1, respectively) in a C4BP-protein S binding assay and a protein S ligand blot using a peroxidase-conjugated monoclonal anti-protein S antibody. The barium citrate supernatant contained two forms of C4BP-high and one form of C4BP-low. One form of C4BP-high did contain the beta-chain and was capable of protein S binding (KA: 4.35 x 10(8) M-1). The two other forms of C4BP lacked the beta-chain and were unable to bind protein S.

Antigenicity of Basement Membrane Fractions Obtained From Rat Seminiferous Tubules

A noncollagenous fraction of basement membrane (D-STBM) obtained from rat testes was submitted to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE), and eight well-defined bands were detected. A cross-reaction with an antiserum against laminin was revealed by immunoblotting in five bands, with molecular weights ranging from 54 to 64 kDa. No further resolution of these components could be obtained by size exclusion and ionic exchange chromatography. Fifty-two percent of the rats immunized with D-STBM and adjuvants developed a mild multifocal damage of the testis. The lesions were characterized by foci of seminiferous tubules with different degrees of sloughing and/or atrophy of the germinal epithelium. Giant multinucleated cells were frequently seen, and mild interstitial mononuclear cell infiltrates were also detected. By immunofluorescence, deposits of rat IgG with a faint discontinuous linear pattern were observed along the walls of the seminiferous tubules. Circulating antibodies to D-STBM were detected by ELISA in 100% of the rats, whereas in a cross-reaction with laminin antibodies were detected in only 63%. All rats studied revealed a positive delayed type of hypersensitivity (DTH) response to D-STBM. None of the control rats injected with saline and adjuvants presented circulating antibodies to D-STBM or laminin or a positive DTH reaction to D-STBM. Some control group rats (10%) revealed few isolated seminiferous tubules with some degree of sloughing of the germinal epithelium.

Purification and characterization of an ethanol-induced UDP-glucuronosyltransferase

A UDP-glucuronosyltransferase (GT) enzyme was isolated from ethanol-induced male New Zealand white rabbit hepatic protein. The animals were pretreated for 2 weeks with 10% ethanol in their drinking water. The GT enzyme was purified by anion-exchange and affinity chromatography and was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The molecular mass of the ethanol-induced UDP-glucuronosyltransferase was determined to be 57,000 Da. Tryptic digests of the ethanol-induced GT and a similarly purified GT from control rabbit liver appeared to be different by HPLC analysis, even though the molecular masses of the enzymes were indistinguishable. Amino acid compositions of the two proteins were different for six amino acids. The apparent K m values for the ethanol-induced GT enzyme for 1-naphthol and morphine as substrates were 43 and 109 μ m, respectively. The apparent V max values for the ethanol-induced GT enzyme for these substrates were 83 and 4.6 nmol/min/mg protein. The increases in catalytic efficiencies, apparent V max K m for 1-naphthol and for morphine, for the ethanol-induced isozyme compared to the control isozyme activities were 2.0- and 2.4-fold. A polyclonal antibody raised in sheep to the rabbit ethanol-induced GT demonstrated a 520-fold selectivity for precipitation of the ethanol-induced protein rather than the control protein. These results demonstrate the production of an unique isozyme of UDP-glucuronosyltransferase that is produced in rabbits as a result of chronic ethanol exposure.

Purification of the Ah receptor from C57BL/6J mouse liver.

The photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, previously has been shown to selectively label two peptides in the cytosol fraction of C57BL/6J mouse liver: a 95-kDa peptide, the ligand binding moiety of the Ah receptor, and a 70-kDa proteolytic fragment formed from the larger peptide (Poland, A., Glover, E., Ebetino, F. H., and Kende, A.S. (1986) J. Biol. Chem. 261, 6352-6365). These two peptides were partially purified to an approximately 20,000-fold enrichment with a 15-20% yield by the following scheme: 1) photoaffinity labeling of the 35-55% ammonium sulfate fraction of liver cytosol; 2) chromatography on polyethyleneimine-Sepharose coupled at low charge density and heparin/Mn2+ precipitation of the dilute column eluate; 3) DEAE-Sepharose chromatography to remove heparin; 4) chromatography on heparin-Sepharose; 5) preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by electroelution of the protein and ion pair extraction to remove sodium dodecyl sulfate; and 6) high performance liquid chromatography on a reverse-phase C-4 column. Following initial chromatography on polyethyleneimine Sepharose, it was found that substantial subsequent purification could only be achieved under denaturing conditions.

Inactivation of human plasma kallikrein and factor XIa by protein C inhibitor.

The inhibition of kallikrein and factor XIa by protein C inhibitor (PCI) was studied. The method of Suzuki et al. [Suzuki, K., Nishioka, J., & Hashimoto, S. (1983) J. Biol. Chem. 258, 163-168] for the purification of PCI was modified in order to avoid the generation of proteolytic activity and subsequent inactivation of PCI. With the use of soybean trypsin inhibitor, an efficient inhibitor of kallikrein and factor XIa, the generation of proteolytic activity was avoided. The kinetics for the inactivation of activated protein C (APC), kallikrein, and factor XIa by PCI were determined. In the absence of heparin, no inactivation of APC was observed, in contrast to kallikrein and factor XIa, which are inhibited with second-order rate constants of (11 +/- 4) X 10(4) and (0.94 +/- 0.07) X 10(4) M-1 s-1, respectively. Addition of heparin potentiated the inhibition of APC [(1.2 +/- 0.2) X 10(4) M-1 s-1] and factor XIa [(9.1 +/- 0.7) X 10(4) M-1 s-1] by PCI, whereas the inhibition of kallikrein by PCI was unchanged [(10 +/- 1) X 10(4) M-1 s-1]. The second-order rate constants for the inhibition of kallikrein or factor XIa by PCI were similar to the second-order rate constants for the inhibition of their isolated light chains by PCI, indicating a minor role for the heavy chains of both molecules in the inactivation reactions. With sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and immunoblotting, complex formation of APC, kallikrein, and factor XIa with PCI could be demonstrated. APC and kallikrein formed 1:1 molar complexes with PCI.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and some properties of cytotoxin produced by Clostridium difficile.

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.

A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described. The slab gel consists of two porous layers of acrylamide of the following composition: 4% acrylamide, 0.04% bisacrylamide for the stacking gel, and 10% acrylamide, 0.1% bisacrylamide for the separating gel, both layers having different buffers. The separating gel mixture (final pH 9.0) and the buffers of the electrode chamber (pH 8.45) consist of Tris and glycine in such a ratio that no acid or base is necessary to adjust the pH. The resulting gel system has the following advantages: (a) it is able to resolve the components from large-volume samples (up to 200 μl) after an overnight electrophoresis run while still maintaining the capacity to produce very sharp bands; (b) it has a high and broad resolution, allowing the separation on the same gel of proteins with apparent molecular masses between 10,000 and 450,000 Da; (c) it is very easy to prepare and shows excellent reproducibility in the electrophoretic patterns; (d) when used as a second dimension in tandem with isoelectric focusing, it improves the resolving power of two-dimensional gel electrophoresis; and finally, (e) its low crosslinker-to-acrylamide ratio allows the effective and rapid transfer of proteins to nitrocellulose membrane, thus improving the usefulness of protein blotting. In all cases, adrenal medullary chromaffin cell proteins were used as test samples.

Detection and localization of actin in Toxoplasma gondii.

The immunological cross-reactivity of the protein from whole-cell extracts of Toxoplasma gondii with antisera against actins from chicken-gizzard smooth muscle and from Ascaris body-wall muscle was demonstrated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE) and immunofluorescence studies. A protein of about 42 kDa, with the same mobility as that of rabbit smooth-muscle actin in SDS-PAGE showed cross-reactivity with anti-actin antisera on the electroblotted nitrocellulose sheet. Indirect fluorescent antibody staining of the parasites clearly showed that actin localized strongly in the anterior third of the organism in both extra- and intrahost cellular stages. From both a biochemical as well as structural point of view, the results obtained in the present experiments might explain the locomotive features of the parasite such as twirling and sliding, with most of the activity at the anterior end.