不吸水链霉菌葡萄糖异构酶的物化表征

葡萄糖异构酶是一种催化葡萄糖异构为果糖的酶。本文用紫外吸收光谱、红外光谱、氨基酸组分分析、聚丙烯酰胺凝胶梯度电泳、SDS-聚丙烯酰胺凝胶电泳、超薄 层聚丙烯酰胺凝胶等电聚焦电泳技术研究了不吸水链霉菌嗜热亚种M1033菌株产生的葡萄糖异构酶的一些物化性质。结果表明由本实验室制备的均一葡萄糖异构酶的A₂₈₀与A₂₆₀的比值是1.76。它是由一个亚单位组成的酶分子。最小分子量是49000。pI值是5.2。氨基酸组分与其它来源的葡萄糖异构酶的氨基酸组分相比较有一些差异,其中Glu,Gly,ALa和Leu的含量都此其它异构酶的高,而Met,Trp,Asp,Thr则比其它葡萄糖异构酶的低。     

Isolation and properties of a lectin from the seeds ofMimosa invisa L.

A lectin has been purified from the seeds ofMimosa invisa L. by gel filtration and preparative Polyacrylamide gel electrophoresis. The purified lectin was homogeneous as judged by analytical Polyacrylamide gel electrophoresis, immunodiffusion and Immunoelectrophoresis. The apparent molecular weight is 100,000; the protein is a tetramer with two types of subunits (molecular weight 35,000 and 15,000). The lectin is a glycoprotein with approximately 21% carbohydrate and interacts with Sephadex and concanavalin A-Sepharose. It agglutinates erthrocytes non-specifically, does not agglutinate leucocytes and is not mitogenic, agglutinates Mimosa-nodulatingRhizobium and is a panagglutinin; the agglutination is not inhibited by several simple sugars. It is thermo-stable and has no metal ions.     

Lac repressor — Lac operator complexes

Complexes between the Lac repressor and a small DNA operator fragment (29 base pairs) were investigated using polyacrylamide gel electrophoresis and solution X-ray scattering. Titration of the DNA fragment with the repressor, followed by gel electrophoresis showed that only two types of complexes are formed with repressor/operator ratios of 0.5 and 2. Radii of gyration and forward scattered intensities were obtained from Guinier plots for repressor/operator ratios ranging from 0.3 to 2. They demonstrated that the first complex contains one repressor and two operators, whereas the second one contains four repressors and two operators. Mixing operator and repressor in equimolar concentrations leads to a mixture of both complexes. A possible model for the four repressor/two operator complex is proposed.     

Purification of an autocatalytic protein-glycosylating enzyme from cell suspensions of Daucus carota L.

A glycosyltransferase was identified in the 174 000 · g membrane pellet and the supernatant from extracts of cell suspensions of Daucus carota L. The enzyme from the supernatant was enriched 475-fold, and sodium dodecyl sulfate-gel electrophoresis and fluorography of this purified sample showed that the only enriched protein band (40 000 Da) was simultaneously an enzyme and a glucose-acceptor. Gel filtration and electrophoresis under non-denaturing conditions proved that in vivo this protein provides the subunits for a very large molecule. Radio-gas-liquid chromatography demonstrated that only one glucosyl moiety was transferred from UDP-glucose to the protein.Abbreviations DEAE diethylaminoethyl - GT IsU glycosyltransferase I, soluble, substrate UDPglucose - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Bean lectins IV: genetic variation in the non-denatured structure of lectins from different Phaseolus vulgaris L. cultivars

Summary Variation in the native conformation of bean lectins was examined using electrophoresis of non-denatured total protein extracts and purified albumin and globulin lectin. The observed variation was related to the genetic variation reported previously for lectin polypeptide composition as revealed by two-dimensional isoelectricfocusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE). When eleven cultivars with different IEF-SDS/PAGE lectin polypeptide compositions were compared, eight had unique non-denatured lectin patterns and three had identical patterns. For some cultivars differences in non-denatured lectin patterns were observed between the purified albumin and globulin lectin preparations.     

Genetic polymorphism in Norway spruce,Picea abies (L.) Karst,assessed by two-dimensional gel electrophoresis of needle proteins

Summary A comparative analysis of three Norway spruce genotypes by two-dimensional gel electrophoresis is presented. This study has led to the identification of approximately 970 gene products for each genotype. Significant qualitative and quantitative differences have been identified, and qualitative and quantitative divergence indices between genotypes have been computed. Two-dimensional electrophoresis appears to be an efficient tool for studying modifications of gene expression in Norway spruce in response to climatic and pollution stresses.     

Large-scale mapping using high-molecular-weight DNA fromP. vulgaris

The application of megabase methods is essential for positional cloning of genes for which no products have been characterized. We have set about the isolation of genes conferring resistance to pathogens in the french beanPhaseolus vulgaris. We describe here the isolation of high-molecular-weight DNA, pulse-field gel electrophoresis and large-scale mapping of thePal2 locus inP. vulgaris. We describe the construction of such a map by embedding DNA isolated from leaf nuclei in agarose plugs, restricting it with rare cutters and subjecting the DNA to pulse-field gel electrophoresis.     

Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b ₆ f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.     

A Proteomics Approach to Characterizing Tick Salivary Secretions

The saliva of ticks contains a complex mixture of bioactive molecules including proteins that modulate host responses ensuring successful feeding. The limited amount of saliva that can be obtained from ticks has hampered characterization of salivary proteins using traditional protein chemistry. Recent improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize small amounts of protein. These methods were employed to characterize salivary proteins from Amblyomma americanum and Amblyomma maculatum. Salivation was induced by injection of dopamine and theophylline. It was necessary to desalt and concentrate saliva before analysis by 2-D electrophoresis. Comparison of 1-D and 2-D gel patterns revealed that the major protein component of saliva did not appear on 2-D gels. Characterization of this protein showed that it was identical to the major protein present in the hemolymph of both tick species. Protein profiles obtained by 1-D and 2-D gel electrophoresis were similar for both tick species, however, higher concentrations of lower molecular weight proteins were present in A. maculatum. Protein analysis by MALDI-TOF mass spectrometry and western blot analysis showed that except for the most abundant protein with a molecular weight of 95 kDa, all of the proteins detected were of host origin. It is not known if this is an artifact of the collection method or has physiological significance. In either case, in these species of ticks, host proteins will have to be removed from saliva samples prior to 2-D analysis in order to characterize lower abundance proteins of tick origin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of sucrose-hydrolyzing enzymes of Zymomonas mobilis

Extracellular proteins of Zymomonas mobilis were analyzed by two-dimensional gel electrophoresis and protein maps drawn up. One of these proteins showed sucrose-hydrolyzing activity, as indicated by activity staining after polyacrylamide gel electrophoresis. It was purified from the extracellular extract of a glucose fermentation by polyacrylamide gel electrophoresis, using a two-step procedure. The molecular mass of the protein was 46 kDa and its isoelectric point 5.0. A rabbit antiserum was raised against this protein. As shown by immunoblotting, the same protein was present in extracellular extracts obtained from glucose, fructose and sucrose fermentations. A cross-reaction was also detected by immunoblotting, with a cellular protein of molecular mass 46 kDa present on the three carbon sources studied. However, activity staining was unsuccessful on gels after electrophoresis of these cellular extracts. The extracellular protein extract obtained from a fermentation run on glucose contained another sucrose-hydrolyzing protein of molecular mass 51 kDa and with an isoelectric point of 4.8. This protein was absent in fructose and sucrose fermentations but showed a positive reaction with the antiserum raised against the 46 kDa extracellular protein. Partially purified sucrose-hydrolyzing proteins also catalyzed transfructosylation reactions, suggesting that they could be of the levansucrase type.     

Purification of rosmarinic acid synthase from cell cultures of Coleus blumei Benth

Rosmarinic acid synthase from cell cultures of Coleus blumei Benth. was purified to apparent homogeneity by fractionated ammonium sulfate precipitation (60–80% saturation), hydrophobic interaction chromatography, affinity chromatography and gel filtration. This purification procedure resulted in a 225-fold-enriched specific enzyme activity with a yield of 9%. The protein preparation was apparently pure according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis. The apparent molecular mass determined by gel filtration and SDS-PAGE was 77 kDa, indicating that rosmarinic acid synthase is a monomeric enzyme.Abbreviations DTT dithiothreitol - HIC hydrophobic interaction chromatography - RA rosmarinic acid - RAS rosmarinic acid synthase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis The financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged. Two-dimensional gel electrophoresis was done with the help of Dr. Guy Bauw, University of Gent, Belgium.     

Methylation strongly enhances DNA bending in the replication origin region of the Escherichia coli chromosome

Summary Two-dimensional gel electrophoresis, at high and low temperatures, and gel mobilities of circularly permuted DNA segments showed a large bending locus about 50 bp downstream from the right border of the 245 by oriC box, a minimal essential region of autonomous replication on the Escherichia coli chromosome. Bending was strongly enhanced by Dam methylation. In DNA from a Dam^– strain, the mobility anomaly arising from altered conformation was much reduced, but was raised to the original level by methylation in vivo or in vitro. Enhancement of the mobility anomaly was also observed by hybrid formation of the Dam^– strand with the Dam⁺ strand. Near the bending center, GATC, the target of Dam methylase, occurs seven times arranged essentially on the same face of the helix with 10.5 by per turn. We concluded that small bends at each Dam site added up to the large bending detectable by gel electrophoresis.     

Determination of GA3 in Chara vulgaris by capillary electrophoresis system

Method for simultaneous measurement of gibberellic acid was applied using capillary zone electrophoresis. Gibberellic acid was identified in extracts of apical part of thallus of Chara vulgaris L. The amount of gibberellins measured on the basis of activity determined by the micro-drop bioassay (59.8 mg·kg⁻¹; with gibberellic acid as a standard) was comparable with that estimated by capillary electrophoresis (54.9 mg·kg⁻¹).     

Floral-specific polypeptides of the Japanese morning glory

Polypeptides from stems, leaves, sepals, corollas, stamens and pistils of the Japanese morning glory (Ipomoea nil Roth (Pharbitis nil Chois.)) were separated by one- and two-dimensional gel electrophoresis and visualized by silver staining. The majority of polypeptides were expressed in two or more organs, while those specific to only one organ were comparatively rate. Among the polypeptides of the former class were two which appeared to be floral-specific. A 46-kDa (kilodalton) polypeptide was expressed in corollas, stamens and pistils, whereas a 32-kDa polypeptide was observed only in extracts prepared from reproductive organs. Polypeptide spots from the various organs were compared with those from leaves, and it was found that sepals and stems shared 40–50% of their polypeptides with leaves, whereas corollas, stamens and pistils shared 20% or less. The latter organs shared 120 polypeptides or roughly 15% of those identified in the floral extracts. Floralorgan-specific polypeptides comprised nearly 10% of the total floral polypeptides identified.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Diverse<Emphasis Type="Italic"> Mesorhizobium plurifarium</Emphasis> populations native to Mexican soils

Forty-six Mesorhizobium strains associated with the leguminous plants Leucaena leucocephala and Sesbania herbacea in an uncultivated Mexican field were characterized using a polyphasic approach. The strains were identified as Mesorhizobium plurifarium based upon the close relationships with the reference strains for this species in PCR-based restriction fragment length polymorphism analyses, sequencing of 16S rRNA genes, multilocus enzyme electrophoresis, and DNA-DNA hybridization. Although the strains isolated from both plants formed the same group in multilocus enzyme electrophoresis and cross-nodulations were observed in the laboratory, different electrophoretic types were obtained from the two plants grown in natural soils, indicating the existence of a preferable association between the plants and the rhizobia. The M. plurifarium strains from Mexico and the reference strains from Africa and Brazil formed different phenotypic clusters in a numerical taxonomy. The Mexican strains did not grow at 37 °C and were sensitive to salty-alkaline conditions, while the reference strains from Africa and Brazil grew at 42 °C and were more resistant to salty-alkaline conditions. These results demonstrate that both the plants and environmental factors affected the evolution of rhizobia and that the Mexican strains had adapted to the neutral soils and the cool climate where they were isolated.     

Molecular evolution among someDrosophila species groups as indicated by two-dimensional electrophoresis

Summary The evolutionary and phylogenetic relationships of sevenDrosophila species groups (represented byD. melanogaster, D. mulleri, D. mercatorum, D. robusta, D. virilis, D. immigrans, D. funebris, andD. melanica) were investigated by the use of two-dimensional electrophoresis. The resulting phylogeny is congruent with the current views of evolution among these groups based on morphological characters and immunological distances. Previous studies indicated that the ability of one-dimensional electrophoresis to resolve relationships between distantly related taxa extended to about the Miocene [25 million years (Myr) ago], but the present study demonstrates that two-dimensional electrophoresis is a useful indicator of phylogeny even back to the Paleocene (65 Myr ago). In addition, two-dimensional electrophoresis is shown to be a useful technique for detecting slowly evolving structural proteins such as actins and tropomyosins.     

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described.Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and E_m was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form.Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c ₂, although the physico-chemical properties of these two cytochromes were similar to each other.Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.Abbreviations E_m Midpoint redox potential - PAGE Polyacrylamide ge electrophoresis - SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMAO Trimethylamine N-oxide     

Tamm-horsfall glycoprotein: a lectin binding study

Preparations of Tamm-Horsfall glycoprotein were examined by SDS-polyacrylamide gel electrophoresis and detection using various¹²⁵I-lectins as well as Coomassie Brilliant Blue. Considerable heterogeneity of electrophoretic pattern was seen. This was not due to a genetic polymorphism. Variation in binding by Soy-bean agglutinin was also seen. This was correlated with the Sd^a phenotype of the individual.     

Gene expression during fruit ripening in avocado

The poly(A) ⁺RNA populations from avocado fruit (Persea americana Mill cv. Hass) at four stages of ripening were isolated by two cycles of oligo-dT-cellulose chromatography and examined by invitro translation, using the rabbit reticulocyte lysate system, followed by two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of the resulting translation products. Three mRNAs increased dramatically with the climacteric rise in respiration and ethylene production. The molecular weights of the corresponding translation products from the ripening-related mRNAs are 80,000, 36,000, and 16,500. These results indicate that ripening may be linked to the expression of specific genes.     

A zymogram method to detect endoglucanases fromBacillus subtilis,Myrothecium verrucaria andTrichoderma reesei

Summary A purified endoglucanase (Eg) fromBacillus subtilis and crude Eg preparations fromMyrothecium verrucaria andTrichoderma reesei were denatured by heat in the presence of SDS and separated by using SDS polyacrylamide gel electrophoresis. Eg zymogram activity bands were detected directly in carboxymethylcellulose-laden separating gels after enzyme renaturation. Denaturing zymogram analysis was a simple method for the separation and detection of Egs.