Effect of Rhamnolipids on Growth of Pseudomonas aeruginosa Mutant Deficient in n-Paraffin-utilizing Ability

Egg white solids freeze-dried with or without the addition of glucose were stored at 50°C under 65 % relative humidity to study the effect of the Maillard reaction on the solubility and heat stability and the formation of aggregates. A stimulative effect on the former properties was observed on the sample with glucose in the initial stage of the Maillard reaction. By comparing the SDS-polyacrylamide gel electrophoretic patterns, solubilities in SDS and SDS/2-mercaptoethanol and the s_20,w values, it was found that glucose, in addition to the effect due to the changes of charged groups in glucose-protein complex occurring in the course of the Maillard reaction, might have a protective effect against aggregate formation through a stable cross-linking, which was not dependent on the SS bond, and through a stable non-covalent bond.     

Inhibition of bFGF activity by complement C1s: covalent binding of C1s with bFGF

The first complement component C1s formed large aggregates with bFGF when bFGF and C1s were incubated at 37°C overnight. Under non-reducing conditions, a part of the aggregates did not penetrate into 5% polyacrylamide gel in the presence of SDS, and the rest penetrated into 5% gel but not into 12% gel. The aggregates were dissociated into monomers by reducing with 2-mercaptoethanol. Both active and inactive C1s formed aggregates with bFGF. In addition, a portion of bFGF was degraded by active C1s but not by inactive C1s. Aggregates were not formed when 2-mercaptoethanol (2 mM &base;) was added to the incubation mixture. After the incubation with C1s the growth-stimulating activity of bFGF was measured by using human umbilical vein endothelial cells (HUVEC) as indicator cells. The aggregate formation between C1s and bFGF significantly reduced the activity of bFGF. © 1998 John Wiley & Sons, Ltd.     

An isoelectric focusing procedure for erythrocyte membrane proteins and its use for two-dimensional electrophoresis

Procedures are described and evaluated for one-dimensional isoelectric focusing of erythrocyte membrane dissolved in lysine, urea, and Triton X-100 without using sodium dodecyl sulfate (SDS) and for two-dimensional electrophoresis with SDS in the second dimension. The membrane was completely dissolved, most of the proteins including the anion porter(s) entered the focusing gel, and complex, well-resolved patterns were seen. Ampholines, 2-mercaptoethanol, or SDS in the applied sample each seriously reduced focusing resolution and phenylmethylsulfonyl fluoride blurred the patterns. The two-dimensional patterns showed more and sharper spots than did patterns obtained from membrane initially dissolved with SDS. Anion porter spots were seen with both procedures. However, major cytoskeletal proteins were much less well recovered with the former procedure than with the latter.     

Removal of artifactual bands associated with the presence of 2-mercaptoethanol in two-dimensional polyacrylamide gel electrophoresis

A method for eliminating artifactual bands due to the presence of 2-mercaptoethanol in two-dimensional gels is described. The method is based on a modification of the procedure of application of the first dimension gel to the SDS slab gel. 2-Mercaptoethanol is removed during equilibration and replaced by iodoacetamide. The use of iodoacetamide improves the recovery of proteins and results in a better detection of them.     

Electrophoretical Analysis of Zein and Isolation of Its Components

Zein was resolved into several components by electrophoresis on polyacrylamide gel in the presence of sodium dodecylsulfate (SDS). Treated with 2-mercaptoethanol (2-ME) in advance, zein was resolved into only two components by the electrophoresis. These two components were tentatively named α- and β-zein. Both were isolated by gel filtration on Sephacryl S-200 in the presence of SDS and 2-ME. Amino acid analysis showed that α- and β-zein were similar to each other, except that the number of methionine residue was three in the former and one in the latter. When protein bodies isolated from corn endosperms were subjected to electrophoretical analysis, the same characteristics as those found in zein were observed.     

Characterization of a macromolecular aggregate of ovine pituitary corticotropin-beta-lipotropin common precursor

A macromolecular aggregate of corticotropin-beta-lipotropin common precursor had been observed in ovine pituitary preparations as an excluded fraction of Sephadex G-200 gel filtration. This fraction could not penetrate a 10% gel during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, when 2-mercaptoethanol or other disulfide-cleaving agents were not present in the buffer used to solubilize the protein preparation prior to the electrophoresis. On a 4.6% gel (acrylamide:bisacrylamide, 20:1), the material migrated as a diffuse band to a position between those of beta-galactosidase (Mr 130 000) and myosin (Mr 200 000). Both observations were consistent with an apparent Mr greatly in excess of that of the corticotropin-beta-lipotropin common precursor reported by many investigators. Neither 5% SDS nor 1% Triton X-100 could dissociate the macromolecular aggregate, but 2-mercaptoethanol and urea, either alone or in combination, were able to dissociate it to two main protein components, one of which was identified as corticotropin-beta-lipotropin with an apparent Mr of 34 000. The fact that urea alone could dissociate this macromolecular aggregate led us to believe that it might be a non-covalent aggregate and that 2-mercaptoethanol probably did not achieve the dissociation through the cleavage of an interchain disulfide bond but by bringing about conformational changes as a result of reduction of intrachain disulfide bonds so that aggregation became unfavorable. Moreover, the dissociation by urea or by 2-mercaptoethanol was found to be irreversible. The origin of the macromolecular aggregate of corticotropin-beta-lipotropin common precursor remains obscure.     

Human liver manganese superoxide dismutase. Purification and crystallization, subunit association and sulfhydryl reactivity

Manganese superoxide dismutase (Mn-SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low-angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form. Four of the eight cysteine residues in the tetramer reacted with 5,5'-dithiobis(2-nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2-mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs2-reactive peptides isolated by activated thiol-Sepharose covalent chromatography. Incubation of Mn-SOD in 1% SDS for 2 or 3 days at 25 degrees C or 5 min at 100 degrees C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2-mercaptoethanol heated in SDS produced only the 25-kDa monomer species. Essentially, no thiol groups were detected in the 23-kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.     

Cuticular Collagenous Proteins of Second-stage Juveniles and Adult Females of Meloidogyne incognita: Isolation and Partial Characterization

Cuticles isolated from second-stage juveniles and adult females of Meloidogyne incognita were purified by treatment with 1% sodium dodecyl sulfate (SDS). The juvenile cuticle was composed of three zones differing in their solubility in β-mercaptoethanol (BME). Proteins in the cortical and median zones were partially soluble in BME, whereas the basal zone was the least soluble. The BME-soluble proteins from the juvenile cuticle were separated into 12 bands by SDS-polyacrylamide gel electrophoresis and characterized as collagenous proteins based on their sensitivity to collagenase and amino acid composition. The adult cuticle consisted of two zones which were dissolved extensively by BME. The basal zone was completely solubilized, leaving behind a network of fibers corresponding to the cortical zone. The BME-soluble proteins from the adult cuticle were separated by electrophoresis into nine bands one of which constituted > 55% of the total BME-soluble proteins. All bands were characterized as collagenous proteins. Collagenous proteins from juvenile cuticles also contained glycoproteins which were absent from the adult cuticles.     

Various physico-chemical properties of inorganic pyrophosphatase from the mouse spleen

Manganese, calcium and mercury ions, as well as p-chloromercury benzoate and dithiothreitol are studied for their effect on the activity of inorganic pyrophosphatase (EC 3.6.1.1) of mice spleen. It is shown that Ca2+ and Mn2+ are inhibitors of this enzyme, but Mn2+ in low concentrations may replace Mg2+ in the pyrophosphatase reaction. Hg2+ and p-chloromercury benzoate inhibit the pyrophosphatase activity essentially but not completely. Mice spleen pyrophosphatase is very labile: its preincubation without the substrate for 30 min at 37 degrees C leads to a complete loss of the activity. Neither glycerol, nor glutathione and cysteine but magnesium ions, dithiothreitol and 2-mercaptoethanol protect the enzyme from inactivation. The enzyme is purified by the sulphate ammonium salting-out, gel filtration on Sephadex G-100 as well as by isoelectrofocusing in 5% PAAG. Then pyrophosphatase is eluted from gel and subjected to electrophoresis in the plane layer of the linear gradient of 5-15% PAAG with SDS or 5-25% PAAG without denaturing conditions. One zone corresponding to the molecular mass of 70 kDalton is obtained. It is splitted into two zones in electrophoresis with SDS and 2-mercaptoethanol.     

Two-dimensional gel electrophoresis of proteins for genetic studies in douglas fir (Pseudotsuga menziesii)

A method of two-dimensional gel electrophoresis of proteins from Douglas fir needles is described. Extraction in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol followed by heating at 100°C produces reliable two-dimensional gels which are convenient for genetic studies. Three genotypes from different geographical origins have been compared: among 225 loci expressed, 22 display regulatory variations and 7 show allelic variations. Thus it is now possible to undertake the genetic study of Douglas fir using this powerful technique.This work was supported in part by Grant ATP PIRDES 508 444 from the INRA-CNRS.     

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins myofibrillar proteins

Electrophoresis of the high-molecular-mass proteins (>500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoresic dye front. Inclusion of 10 mm 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mm 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.     

DNA cleavage in chromatin spacer segments by a non-enzymic probe, 1,10-phenanthroline-copper complex

The action of 1,10-phenanthroline-copper complex under aerobic conditions in the presence of 2-mercaptoethanol on chromatin DNA in murine thymocyte nuclei was studied. At limited oxygen supply primarily multiple single-strand breaks of DNA in the spacer segments are observed while the core DNA segments in chromatin remain intact. After the single-strand breaks accumulate in both DNA strands (under conditions of improved oxygen supply), double-strand cleavage of DNA to fragments of nucleosomal size becomes apparent. This regular character of DNA degradation in chromatin is apparently due to the preferential binding of the 1,10-phenanthroline-copper complex to DNA of the spacer segments and to the localized generation of damaging radicals.Abbreviations OP 1,10-phenanthroline - OP-Cu 1,10-phenanthroline-copper complex - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulphate - 2-ME 2-mercaptoethanol     

Isolation,Stabilization, and Characterization of a Toxin from Timber Rattlesnake Venom

A rapid and convenient method for the purification of a toxin from timber rattlesnake, Crotalus horridus horridus, venom using carboxymethyl cellulose ion-exchange chromatography has been devised. The toxicity of this venom component is labile, but it is stabilized by the addition of 20% V/V glycerol to the buffer solution. This toxin has a molecular weight of 15,000 + 700 as determined by SDS gel electrophoresis. It is both heat and protease resistant. Treatment of this venom component with 2-mercaptoethanol followed by G-50 Sephadex chromatography causes no loss of toxicity although incubation of the toxin with 1% SDS and 1% 2-mercaptoethanol prior to electrophoresis does result in a faster migrating species. The toxin does not affect neuromuscular junctions but does appear to act on the nervous system. It causes no local responses in mice.     

Isolation, stabilization, and characterization of a toxin from timber rattlesnake venom.

A rapid and convenient method for the purification of a toxin from timber rattlesnake, Crotalus horridus horridus, venom using carboxymethyl cellulose ion-exchange chromatography has been devised. The toxicity of this venom component is labile, but it is stabilized by the addition of 20+ V/V glycerol to the buffer solution. This toxin has a molecular weight of 15,000 +/- 700 as determined by SDS gel electrophoresis. It is both heat and protease resistant. Treatment of this venom component with 2-mercaptoethanol followed by G-50 Sephadex chromatography causes no loss of toxicity although incubation of the toxin with 1% SDS and 1% 2-mercaptoethanol prior to electrophoresis does result in a faster migrating species. The toxin does not affect neuromuscular junctions but does appear to act on the nervous system. It causes no local responses in mice.     

PURIFICATION AND PROPERTIES OF THE MUCUS OF EUGLENA GRACILIS (EUGLENOPHYCEAE)1

Anionic groups were demonstrated in the mucus of Euglena gracilis Klebs var. bacillaris Cori by histochemical staining with alcian blue or diaminobenzidine tetrahydrochloride and were quantified during the growth cycle with an alcian blue dye-binding assay. Mucus in the culture increased during growth and became high when the culture entered the stationary phase. Cultures were grown under conditions which uniformly labeled all sulfur containing compounds with ³⁵S. A purification scheme was devised using 0.15 M NaCl and 0.10 M EDTA at pH 8 (Marmur's solution) to separate the mucus from the cells without cell breakage. The isolated purified mucus was fractionated with sodium dodecyl sulfate (SDS) at 100° C into soluble and insoluble components. The soluble fraction was separated by SDS polyacrylamide gel electrophoresis into 18 polypeptide bands ranging from 22 to 320 kdaltons that stained with Coomassie blue; 16 of these bands also stained for carbohydrates using periodic acid-Schiffs reagent, indicating their glycoprotein nature. On hydrolysis, the SDS soluble fraction yielded xylose, fucose, rhamnose, and hexose. The SDS insoluble fraction contained no ³⁵S label, and, therefore, presumably no protein or bound sulfate; this gelatinous material does not contain the same sugar residues as the glycoproteins in the SDS soluble fraction. Its staining properties with alcian blue and its resistance to hydrolysis suggested the presence of uronic acids. Comparison with other Euglena fractions showed that bands comigrating with the mucus glycoproteins were not detectable in the fractions containing the whole cells or the culture medium. Although the mucus of Euglena yielded appreciable sulfate during mild acid treatment, most if not all of this sulfate appears to have come from the oxidation of reduced sulfur rather than from the hydrolysis of covalently bound sulfate. An infrared spectrum of the mucus showed only minor peaks in the correct regions for the S-O linkage. Thus, the mucus of Euglena is composed of glycoproteins and polysaccharides which contain little or no ester sulfate.     

Changes in the sarcoplasmic reticulum ATPase protein under denaturing conditions used for sodium dodecyl sulfate--polyacrylamide gel electrophoresis

The electrophoretic pattern of the sarcoplasmic reticulum (SR) ATPase protein was found to change, depending on the conditions used to denature the SR vesicles in sodium dodecyl sulfate (SDS), prior to SDS-polyacrylamide gel electrophoresis. A smearing of the gel pattern above the ATPase protein was observed when the SR vesicles were denatured at 100 °C in SDS, in the absence of β-mercaptoethanol (β-ME). Denaturation of the SR vesicles in SDS at 100 °C in the presence and the absence of β-ME reduced the amount of SR ATPase protein by half. More high-molecular-weight aggregates were formed in the presence than in the absence of β-ME. The other proteins of the SR as well as myofibrils and bovine serum albumin were found to be relatively unaffected by these treatments. It is concluded that, for the study of SR ATPase protein by SDS-polyacrylamide gel electrophoresis, denaturation at 100 °C should be avoided.     

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins

Electrophoresis of the high-molecular-mass proteins (greater than 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.     

Isolation and characterization of thrombomodulin from bovine lung

Bovine thrombomodulin was isolated from the lung by Triton X extraction, affinity chromatography on diisopropyl phosphate-thrombin-agarose, and gel filtration on Ultrogel AcA-44. The final preparation was purified 6000-fold from the membrane extract with a yield of 21%. It showed apparent Mr of 78,000 and 105,000, before and after reduction, respectively, on polyacrylamide gel electrophoresis in SDS. The activity of the thrombomodulin was stable under the conditions of 1% SDS, 8 M urea, pH 2 and 10, and heat treatment at 60 degrees C for 30 min, but was unstable against treatment with 2-mercaptoethanol. Activation of protein C by thrombin in the presence of the thrombomodulin depended on Ca2+, and an equimolar complex formation between thrombin and thrombomodulin was required for the maximum rate activation. The rate of protein C activation by thrombin was increased 900-fold by thrombomodulin. Thrombomodulin inhibited the thrombin-induced fibrinogen clotting and platelet activation. However, it did not affect the inhibition of thrombin by antithrombin III with or without heparin, a protein C inhibitor or several synthetic inhibitors. These properties of bovine thrombomodulin were similar to those of rabbit thrombomodulin reported earlier.     

Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The K_m and V_max values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)⁻¹ h⁻¹, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba²⁺, Cu²⁺, Mn²⁺ and Hg²⁺. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.     

Isolation of corticotropin-beta-lipotropin common precursor from ovine pituitary glands

Ovine corticotropin-beta-lipotropin common precursor was purified to homogeneity from commercial frozen ovine pituitary glands. A crude preparation was obtained following a procedure published elsewhere (Lee, T.H. and Lee, M.S. (1977) Biochemistry 16, 2824-2829) and was purified by gel filtration on Sephadex G-200 in the presence of 0.5% SDS and 0.1% 2-mercaptoethanol, and under an atmosphere of nitrogen. The gel filtration was repeated once. The partially purified preparation obtained from the second Sephadex G-200 gel filtration was further fractionated by preparative SDS-acrylamide gel electrophoresis, using immunoprecipitated and electrophoretically purified [125I]corticotropin-beta-lipotropin common precursor as a marker. The preparation was judged homogeneous by the appearance of a single protein band in analytical SDS-acrylamide gel electrophoresis, which exhibited both corticotropin and beta-lipotropin immunoreactivities, and a single symmetrical peak in high-pressure liquid chromatography on a reverse phase C18 column. The isolated ovine corticotropin-beta-lipotropin common precursor possessed specific activities of 116 micrograms of immunoreactive corticotropin and 210 micrograms of immunoreactive beta-lipotropin per mg of protein, equivalent to 89 and 62% of theoretical values, respectively. The amino acid composition of the homogeneous preparation was determined.