Disulfide-bonded polymerization of plasma fibronectin in the presence of metal ions

Incubation of human plasma fibronectin in the presence of low concentrations of FeCl3 or CuSO4 led to the formation of disulfide-bonded multimers as revealed by analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing or reducing conditions. The polymers induced by FeCl3 did not enter the spacer gel, and those induced by CuSO4 migrated to the top of the running gel, indicating that the former polymers were larger than the latter, which in gel filtration experiments appeared to be larger than Mr 670,000. The polymerization occurred between pH 7 and 9 and more rapidly at 22 or 37 degrees C than at 4 degrees C and was inhibited by metal-chelating reagents. NaCl, heparin, spermine, urea, or guanidine hydrochloride did not appreciably affect the reaction, whereas dithioerythritol enhanced the CuSO4-induced polymerization of fibronectin. When incubated in the presence of FeCl3, the Mr 30,000 NH2-terminal, Mr 40,000 gelatin-binding, and the Mr 120,000-140,000 COOH-terminal fragments of fibronectin formed disulfide-bonded polymers, whereas only the Mr 140,000 fragment was polymerized in the presence of CuSO4. Disulfide-bonded polymers were also formed in the presence of FeCl3 but not CuSO4 when the free sulfhydryl groups of fibronectin were blocked by N-ethylmaleimide. The results suggest that in the presence of CuSO4, disulfide-bonded polymerization of fibronectin may involve predominantly the free sulfhydryl groups, whereas in the presence of FeCl3, also the intramolecular disulfides may exchange to form disulfides between separate fibronectin molecules. Thus, under different conditions, different parts of fibronectin may be susceptible to disulfide-bonded polymerization.     

Purification and properties of the membranal thiol oxidase from porcine kidney

A thiol oxidase was purified from porcine kidney cortex by chromatography of detergent-solubilized plasma membranes on cysteinylsuccinamidopropyl-glass beads, hydroxyapatite, and Sephacryl S-200. The oxidase was purified 2600-fold; 28% recovery of activity was obtained. With glutathione as substrate, the apparent Km was 0.73 mM and the V max was a 4.4 U/mg protein. The reaction catalyzed was 2 RSH + O2----RSSR + H2O2, and superoxide production was not detected during the reaction. Other low molecular weight thiols, including cysteine, dithiothreitol, N-acetylcysteine, and cysteamine, were substrates for the oxidase; 2-mercaptoethanol, reductively denatured ribonuclease A, and chymotrypsinogen A were not substrates. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band corresponding to 70 kDa; gel filtration on a Sephacryl column produced a single elution of activity with a protein corresponding to 120 kDa, indicating that the functional form is a dimer. On a high-pressure gel permeation column the protein eluted at 70 kDa under dilute conditions but at greater than 200 kDa at high concentrations, indicating that the protein also aggregates into larger multimers. Activity was inhibited by copper chelators, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), H2O2, and N-ethylmaleimide, suggesting the presence of copper and a sulfhydryl group at the active site. Following treatment with metal chelators, enzyme activity was reconstituted with CuSO4, but not with FeSO4. The purified enzyme contained 1 mol copper per subunit which was undetectable by electron paramagnetic resonance, suggesting that the copper is in a binuclear complex.     

Sulfhydryl groups in glycolipid transfer protein: formation of an intramolecular disulfide bond and oligomers by Cu2+-catalyzed oxidation

Glycolipid transfer protein (GLTP) purified from pig brain facilitates the transfer of various glycolipids between lipid bilayers. Purified GLTP migrates as two bands of different mobility in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The slower component and the faster component constituted about 80% and about 15%, respectively, of purified GLTP. Treatment of GLTP with 45 microM CuSO4 resulted in a decrease in the slower component, an increase in the faster component, and the formation of oligomeric components. The faster and oligomeric components were quantitatively converted to the slower component by reduction with 2% 2-mercaptoethanol in the presence of 1% SDS. The formation of oligomeric components was enhanced by increasing the concentration of CuSO4 to 450 microM and 4.5 mM. Oxidation of GLTP catalyzed by CuSO4 resulted in a decrease in the transfer activity and an increase in the apparent binding affinity of GLTP to 1-O-(beta-D-galactopyranosyl)-N-[10-(1-pyrenyl)decanoyl]-D-erythro- sphingosine (PyrGalCer). The oligomeric components and the monomeric components were isolated by chromatography on a Sephadex G-75 column. It was found that GLTP in fractions enriched with the monomeric components had very high transfer activity and is responsible for most of the transfer activity in the oxidized GLTP. Treatment of GLTP with 1.27 mM HgCl2 resulted in a formation of components unresolvable on SDS-PAGE and also resulted in a reduction of the transfer activity to one-third. However, no obvious change in the binding affinity of GLTP to PyrGalCer was observed by HgCl2 treatment. Treatment with 2-mercaptoethanol restored the activity of GLTP inactivated by HgCl2, whereas the activity inactivated by CuSO4 was not restored by treatment with 2-mercaptoethanol. These results suggest that the transfer activity depends on the turnover rate of the GLTP-PyrGalCer complex which is affected by modification of sulfhydryl groups of GLTP. The sulfhydryl group content of GLTP was estimated by the use of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). A value of 2.2 mol sulfhydryl groups per mol of GLTP was found in the presence of 0.5% SDS and one sulfhydryl group in a GLTP molecule was very rapidly oxidized in the native state, from which it is assumed that the slower component contains three sulfhydryl groups per GLTP molecule and the faster component contains one sulfhydryl group and one disulfide bond per GLTP molecule.     

Enhanced proteomic analysis of Streptomyces peucetius cytosolic protein using optimized protein solubilization protocol

Improvements in the dissolution of proteins in two-dimensional gel electrophoresis have greatly advanced the ability to analyze the proteomes of microorganisms under a wide variety of physiological conditions. This study examined the effect of various combinations of chaotropic agents, a reducing agent, and a detergent on the dissolution of the Streptomyces peucetius cytosolic proteins. The use of urea alone in a rehydration buffer as a chaotropic agent gave the proteome a higher solubility than any of the urea and thiourea combinations, and produced the highest resolution and clearest background in two-dimensional gel electrophoresis. Two % CHAPS, as a detergent in a rehydration buffer, improved the protein solubility. After examining the effect of several concentrations of reducing agent, 50 mM DTT in a rehydration buffer was found to be an optimal condition for the proteome analysis of Streptomyces. Using this optimized buffer condition, more than 2,000 distinct and differentially expressed soluble proteins could be resolved using two-dimensional gel electrophoresis with a pI ranging from 4-7. Under this optimized condition, 15 novel small proteins with low-level expression, which could not be analyzed under the non-optimized conditions, were identified. Overall, the optimized condition helped produce a better reference gel for Streptomyces peucetius.     

Isolation from Micrococcus sp. n. of a homogeneous heme-containing catalase and a crystalline protein with catalase activity

A method for isolation and purification of catalases from the culture of Micrococcus sp. n. grown under aeration conditions is described. Heme-containing catalase (I) and the protein possessing a catalase activity (II) were separated by fractionation with ammonium sulfate. The specific activity of the highly purified protein causing degradation of H2O2 is 200 times less than that of the heme-containing enzyme. The molecular weights of catalases I and II as determined by electrophoresis in polyacrylamide gel gradient 4/30% are 240000 and 130000, respectively. The method described is designed at rapid isolation of preparative amounts of catalases from Micrococcus sp. n.     

小麦地下茎双向电泳技术体系的优化

以东农冬麦1号为材料,对苗期地下茎处的蛋白提取方法、蛋白溶解、上样量、胶条的转移等方面进行试验,结果表明：在蛋白提取方面,TCA/丙酮法（T法）和尿素/硫脲法（N法）相比T法能减少低丰度蛋白的损失得到蛋白点数更多的图谱;在蛋白溶解方面,经过两次水化液溶解的蛋白纯度较高,在等电聚焦时能保持8000伏较高电压;上样量方面,10mg粗蛋白溶于两次水化液能得到清晰、分离效果好、蛋白点数较多的图像;胶条转移方面,先向胶面中加入400μl 0.3％普通琼脂糖溶液后,用200μl的电极缓冲液冲洗胶条的支撑膜会使胶条顺利转移到第二向胶面上且胶条与胶面间不会产生气泡。     

Effects of copper sulfate toxicity on cercariae and metacercariae of Echinostoma caproni and Echinostoma trivolvis and on the survival of Biomphalaria glabrata snails

Copper in the form of copper sulfate (CuSO4) decreases the survival of Biomphalaria glabrata snails, but the effects of this molluscicide on Echinostoma caproni and Echinostoma trivolvis, 2 species of digeneans that use B. glabrata as intermediate hosts, are not known. Studies were done on the effects of various concentrations of CuSO4 in artificial spring water (ASW) on the survival and infectivity of E. caproni and E. trivolvis cercariae. Solutions containing 1.0, 0.1, and 0.01% CuSO4 were 100% lethal within 2 hr of exposure for both species. Time to 50% mortality in 0.001% CuSO4 was 8 hr for E. caproni and 16 hr for E. trivolvis; at 24 hr, the controls showed 50 and 65% mortality, respectively. Treatment of cercariae of both species for 0.5 hr in 0.001% CuSO4 had no effect on the ability of cercariae to form normal cysts in juvenile B. glabrata snails. However, treatment with 0.01% CuSO4 for 0.5 hr caused a significant reduction in the ability of cercariae of both species to encyst in snails. Treatment of encysted metacercariae of both species in 0.001% CuSO4 for I hr had no effect on subsequent excystation of these echinostomes in a trypsin-bile salts medium, whereas concentrations of 1.0, 0.1, and 0.01% CuSO4 and 1.0 and 0.1% CuSO4 decreased chemical excystation of E. caproni and E. trivolvis cysts, respectively. Survival studies on the effects of CuSO4 in Locke's solution on chemically excysted metacercariae of both species were also done. Excysted metacercariae of both species were killed by 2 hr in either 0.1 or 0.01% CuSO4 in Locke's solution. However, time to 50% mortality for both species of excysted metacercariae in 0.001% CuSO4 was approximately 5 hr. Time to 50% mortality for the controls was about 12 hr. Survival of juvenile B. glabrata snails was also examined. All B. glabrata snails were dead by 6 hr in 1 and 0.1% CuSO4 in ASW. Biomphalaria glabrata snails showed 50% mortality by about 6 hr in 0.01% CuSO4 and about 80% were still alive at 24 hr in 0.001% CuSO4. All controls were alive at 24 hr, at which time the experiment was terminated. Concentrations greater than 0.001% CuSO4 increased snail mortality, as well as that of the cercariae and excysted metacercariae of E. caproni and E. trivolvis. Our findings suggest that concentrations of copper sufficient to eliminate juvenile B. glabratta snails are also sufficient to kill the cercariae and excysted metacercariae of these digeneans but not the encysted metacercariae, which may be protected by their cyst walls.     

Peptide mapping and microsequencing of proteins separated by SDS-PAGE after limited in situ acid hydrolysis.

A method is described for the isolation of peptide fragments from proteins separated by polyacrylamide gel electrophoresis. After completion of the electrophoresis step, gels are stained with Ponceau S or Coomassie Blue. Gel portions containing protein stained with Ponceau S are excised and transferred to borosilicate glass digestion tubes containing 0.9 ml of 1 mM NaOH or 5 mM Na2HPO4. After complete dissociation of the dye from the protein, 0.1 ml of 20% formic acid is added and the protein is hydrolyzed in situ at 112 degrees C for four hours. Subsequently the acid solution is made 10% in acetonitrile and chromatographed as such on a C18 (C4) reversed-phase column using an appropriate large-volume sample loading syringe and injection loop. Proteins stained with Coomassie Blue can be hydrolyzed in situ after complete removal of the dye with an aqueous solution containing 40% acetone, 10% triethylamine and 5% acetic acid. The gel slices are next washed with HPLC-grade water and protein is hydrolyzed in 2% formic acid under standard conditions. Gel-related contaminants do not interfere with the peptide separation under the proper conditions of HPLC analysis.     

Immunoenzymic studies on testicular hyaluronidase from rhesus monkeys (Macaca mulatta)

Hyaluronidase from rhesus monkey testes was purified by detergent extraction, ammonium sulphate fractionation, Sephadex G-200 column chromatography and concanavalin A-Sepharose affinity chromatography. The purified hyaluronidase showed one protein band on acrylamide gel electrophoresis. Antibodies to the purified hyaluronidase were raised in rabbits and showed a single precipitin line by Ouchterlony gel diffusion. The enzyme had a molecular weight of 62,000. The Km was 0.5 mg/ml for hydrolysis of hyaluronic acid at 37 degrees C. The optimum pH for the enzyme was 5.0 but activity was present over a broad pH range. The hyaluronidase was inhibited by HgCl2, CuSO4, FeSO4 and p-chloromercuribenzoate all at a concentration of 2 x 10(-4) M. Cysteine protected the enzyme against HgCl2 inhibition.     

Changes in antioxidant and lignifying enzyme activities in sunflower roots (Helianthus annuus L.) stressed with copper excess

Treatment with 50 microM CuSO4 for five days caused significant decrease in dry-matter production and protein level of ten-day-old sunflower seedling roots. An increase of lipoperoxidation product rate was also observed. The involvement of some enzyme activities in the sunflower root defence against Cu-induced oxidative stress was studied. Copper treatment induced several changes in antioxidant enzymes. SOD (superoxide dismutase, EC 1.15.1.1) activity was reduced but CAT (catalase, EC 1.11.1.6) and GPX (guaiacol peroxidase, EC 1.11.1.7) activities were significantly enhanced. The lignifying peroxidase activities, assayed using coniferyl alcohol and syringaldazine, were also stimulated. Analysis by native gel electrophoresis of syringaldazine peroxidase activity showed the stimulation of an isoform (A2) and the induction of another one (A1) under cupric stress conditions. On the other hand, the activity of PAL (phenylalanine ammonia lyase, EC 4.3.1.5), which plays an important role in plant defence, was also activated. The possible mechanisms by which Cu-induced growth delay and changes in enzymatic activities involved in plant defence processes are discussed.     

The low temperature,rapid dissolution of gellan away from root cultures

Summary A buffer system consisting of 50 mM Tris-HCl-TRIZMA base plus 10 mM EDTA was used to rapidly dissolve gellan gels used for maintaining transformed carrot root cultures. The optimum conditions of pH 7.5 in the presence of 10 mM EDTA for dissolving gellan were first worked out on a model test system containing 0.4% gellan, 0.025% MgSO₄·7H₂O, and blue dye. The conditions were then tested on gellan gels (0.2% gellan plus nutrients) containing carrot roots. This gel dissolution system was rapid (18 to 20 min), did not require heating, and could also be efficiently performed at 4 °C. Furthermore, the buffer system used for gel dissolution is a standard one used for plant cell fractionation studies.     

One-step purification of recombinant yeast 6-phosphofructo-2-kinase after the identification of contaminants by MALDI-TOF MS

His-tagged yeast 6-phosphofructo-2-kinase was overexpressed in the yeast strain DFY658 under the control of the Gal1 promoter. Here we describe a simple and fast purification protocol for the recombinant enzyme under native conditions using a HiTrap affinity column loaded with CuSO(4). The use of MALDI-TOF MS after in-gel-digestion enabled us to identify a critical contamination of the end product as yeast alcohol dehydrogenase1 (Adh1p). After identification this contaminant could be efficiently removed by carrying out the washing steps at 25 degrees C instead of at 4 degrees C. To reduce the cellular proteolytic activities a low phosphate concentration in the growth medium was applied. This simple modification of the yeast cell growth conditions increased significantly the yield of the recombinant protein.     

Swelling and dissolution of beta-lactoglobulin gels in alkali

It is well documented in the literature that during the dissolution of whey protein gels in alkali, the gels swell to a great extent. However, the relevance of the swelling step in the dissolution process of the protein gel remains unknown. In the present article we present a systematic study on the swelling of beta-lactoglobulin gels at different alkaline pH and ionic strengths. The equilibrium swelling degree at different conditions has been modeled using a simple model developed for polyelectrolyte gels, modified to take into account the ionization of the residues in a protein. The model can describe the swelling behavior of the gels over a wide range of conditions, but it underpredicts the equilibrium swelling under conditions close to those when dissolution is observed. Dissolution is only noticeable above pH 11.5-12 and only for those gels that are swollen over a minimum degree, suggesting the existence of a dissolution threshold.     

Effects of metal ions on the antioxidant enzyme activities, protein contents and lipid peroxidation of carp tissues

1. Studies were performed regarding the effects of CuSO4 in concentrations of 5, 10, 25 and 50 ppm and ZnSO4 in concentrations of 10 and 100 ppm on the antioxidant enzyme activities, lipid peroxidation and protein contents of tissues of common carp (Cyprinus carpio morpha L.) exposed to these pollutants for 24 hr. 2. The results demonstrated that CuSO4 was more toxic than ZnSO4 and that both treatments brought about significant changes in these parameters in carp hepatopancreas (liver), gill and white muscle. 3. An increase of the CuSO4 concentration led to significant decreases in the antioxidant enzyme activities, except that of glutathione peroxidase, which was increased significantly, and significant increases in the lipid peroxidation and protein contents. 4. An increase of the ZnSO4 concentration led to slight changes in the antioxidant enzyme activities, lipid peroxidation and protein contents of carp tissues.     

Biochemical Characterization of Soluble Acid and Alkaline Invertases from Shoots of Etiolated Pea Seedlings

Soluble invertase was purified from pea(Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation,DEAE-Sepharose column,Con-A-and Green 19-Sepharose affinity columns,hydroxyapatite column,ultra-filtration,and Sephacryl 300 gel filtration.The purified soluble acid(SAC) and alkaline(SALK) invertases had a pH optimum of 5.3 and 7.3,respectively.The temperature optimum of two invertases was 37 ℃.The effects of various concentrations of Tris-HCl,HgCl2,and CuSO4 on the activities of the two purified enzymes were examined.Tris-HCl and HgCl2 did not affect SAC activity,whereas 10 mM Tris-HCl and 0.05 mM HgCl2 inhibited SALK activity by about 50%.SAC and SALK were inhibited by 4.8 mM and 0.6 mM CuSO4 by 50%,respectively.The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis.The Kms of SAC and SALK were determined to be 1.8 and 38.6 mM,respectively.The molecular masses of SAC shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were 22 kDa and 45 kDa.The molecular mass of SALK was 30 kDa.Iso-electric points of the SAC and SALK were estimated to be about pH 7.0 and pH 5.7,respectively.     

Purification and properties of a carbonyl reductase involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate from Cylindrocarpon sclerotigenum IFO 31855

A NADPH-dependent carbonyl reductase (CSCR1) was purified to homogeneity from Cylindrocarpon sclerotigenum IFO 31855. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (S)-alcohol with a >99% enantiomer excess. The relative molecular mass of the enzyme was estimated to be 68,000 by gel filtration chromatography and 24,800 on SDS polyacrylamide gel electrophoresis. The enzyme had an extremely narrow substrate specificity and it highly reduced conjugated diketone, 2,3-butanedion, in addition to ethyl 4-chloro-3-oxobutanoate. The enzyme activity was inhibited by HgCl(2) (100%), 5,5'-dithiobis(2-nitrobenzoic acid) (56%), dicoumarol (42%), and CuSO(4) (46%). The N-terminal amino acid sequence of the enzyme (P-Q-G-I-P-T-A-S-R-L) showed no apparent similarity with those of other oxidoreductases.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by the Ionic Detergent Sodium-Lauryl Sarcosinate

The sensitivity of the outer and cytoplasmic membranes of Escherichia coli to detergent was examined by isopycnic sucrose density gradient centrifugation. Sodium lauryl sarcosinate (Sarkosyl) was found to disrupt the cytoplasmic membrane selectively under conditions in which Triton X-100 and dodecyl sodium sulfate solubilized all membrane protein. These results were verified by gel electrophoresis; membrane proteins solubilized by Sarkosyl were identical to those of the cytoplasmic membrane. The presence of Mg(2+) during treatment with Sarkosyl was found to afford partial protection of the cytoplasmic membrane from dissolution.     

Yeast copper-thionein can reconstitute the Japanese-lacquer-tree (Rhus vernicifera) laccase from the Type 2-copper-depleted enzyme via a direct copper(I)-transfer mechanism.

The Type 2-Cu-depleted laccase from the Japanese lacquer tree (Rhus vernicifera) can be reconstituted with CuSO4 aerobically and much more rapidly and efficiently under anaerobic reducing conditions. This is to be related to a more favourable conformation of a laccase in the reduced state, rather than to reduction of the metal ion. In fact, reconstitution with Cu(I)-thionein from baker's yeast (Saccharomyces cerevisiae) only proceeds under anaerobic reducing conditions, via a direct transfer of Cu(I).     

ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN

—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing.