Purification of a light-harvesting B880 complex from wild-type Rhodospirillum rubrum

The light-harvesting B880 complex of Rhodospirillum rubrum was purified by a new method which allowed recovery of 66% of the amount present in the crude solubilized extract. Electrophoretic analysis of the isolated complex, followed by either Coomassie brilliant blue or silver staining, revealed only two low-molecular-weight polypeptides. When compared to a previously described preparation, the stability of the complex was considerably increased. In addition, the new procedure yielded B880 of higher purity as evidenced both by the decreased protein to pigment ratio (A280/A880 = 0.4) and by the absence of contaminants previously detected by silver staining or by an immunochemical method in other preparations. The most prominent of those contaminants were identified in this work as lipopolysaccharides of the bacterial outer membrane.     

Glycolipid- and glycoprotein-based blood group A antigen expression in human thrombocytes. A1/A2 difference

Total non-acid glycolipid fractions and total sodium dodecylsulphate (SDS) solubilized protein fractions were isolated from human thrombocytes obtained from single human donors having different blood group A₁/A₂ phenotypes. The blood group A glycolipid antigens were characterized by immunostaining of thin layer plates with different monoclonal anti-A antibodies. The glycoproteins carrying blood group A epitopes were identified by SDS-PAGE and Western blot analysis using a monoclonal anti-A antibody. Blood group A glycolipid antigens were found in both A₁ and A₂ thrombocytes but the A₂ individuals expressed at least ten times less A glycolipids compared to the A₁ individuals. Expression of A type 3/4 chain and small amounts of A type 1 chain glycolipids were seen in thrombocytes of both A₁ and A₂ individuals, while the type 2 chain A glycolipids appeared to be missing from the A₂ thrombocytes. Blood group A reactive glycoproteins were only found in thrombocytes of A₁ individuals and could not be detected in A₂ individuals or a blood group O individual. The major blood group A glycoprotein were found as a double band migrating in the 130 kDa region.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPTLC high performance thin layer chromatography - CBB Coomassie brilliant blue - GVH graft versus host Part of this work was presented at the Xth International Symposium on Glycoconjugates, Jerusalem, Israel. September, 1989.In the short hand designation for glycolipids, the letter indicate blood group determinant, the first numeral, the number of sugar residues, and the second numeral, the type of carbohydrate chain. Thus, A-6-1 means a hexaglycosylceramide with a blood group A determinant based on the type 1 carbohydrate chain.     

Isolation, purification and characterization of levorin produced by Streptomyces levoris 99/23

Levorin produced by Streptomyces levoris 99/23 was isolated, purified and characterized. It was established that 80% of the levorin was localized in the mycelium and only 20% was in the cell-free supernatant. Amorphous yellow levorin with activity of 24 000 IU/mg and 96% purity was obtained. The preparation exhibited three absorption maxima: at λ 362, 382 and 404 nm. The antibiotic contained seven components: A₀, A₁, A₂, A₃, A₄, and two unidentified ones. According to its composition, the preparation corresponded to the levorin used for medicinal purposes. However, the levorin produced by S. levoris 99/23 contained half as much levorin A₂ and a more than 100 times larger quantity of the more active and less toxic component levorin A₃.     

Photochemical activities and photosynthetic ATP formation in membrane preparation from a facultative methylotroph,Protaminobacter ruber strain NR-1

Membrane preparation from the bacteriochlorophyll-containing cells of a facultative methylotroph, Protaminobacter ruber strain NR-1, contained reaction center bacteriochlorophyll similar to those in many species of purple bacteria and contained a few cytochrome species. -Peak of the reduced-minus-oxidized difference spectrum of one of the cytochromes was at 554 nm. The midpoint potential of the cytochrome at pH 7 (E_m7) was 350 mV. Two other cytochromes had the same reduced-minus-oxidized difference spectra with a split -band at 557 and 566 nm, but had two different E_m7s' of 130 mV and 0 mV.On flash or continuous light the reaction center bacteriochlorophyll and the cytochrome with -peak at 554 nm were reversibly oxidized. Redox titration of the light-induced cytochrome oxidation gave an E_m7 value of 356 mV. Under continuous illumination the membrane preparation reversibly took up protons, and formed ATP in the presence of ADP and inorganic phosphate. The ATP formation activity on the bacteriochlorophyll basis was one-third to one-fifth that in chromatophores from Rhodospirillum rubrum under similar experimental conditions. These results clearly indicated that the membrane preparation from P. ruber which contained bacteriochlorophyll had a cyclic photosynthetic electron transfer system and coupled ATP formation activity.Abbreviations Bchl (only in figure legends) bacteriochlorophyll - CCCP carbonylcyanide-m-chlorophenylhydrazone - E_h the ambient redox potential - E_m7 the midpoint potential at pH 7 - PMS N-methylphenazonium methosulfate - MES morpholinoethanesulfonic acid - MOPS morpholinopropanesulfonic acid     

Development of radioimmunoassay for thromboxane B2

A simple method for the preparation of rat liver urate oxidase is described. The enzyme was purified from rat liver homogenate by cell fractionation, detergent treatment, alkali treatment, and affinity chromatography on 8-aminoxanthine-bound Sepharose 4B. This enzyme preparation had a specific activity of 9.1 U/mg of protein and was purified about 1000-fold from the liver homogenate. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue, this preparation yielded one protein band at a position corresponding to a molecular weight of 33,000.     

Cloning and expression of cellulase gene EG1 from Rhizopus stolonifer var. reflexus TP-02 in Escherichia coli

A novel gene, EG encoding enzymes involved in carboxymethyl cellulose (CMC) degradation was isolated, sequenced from the filamentous fungus Rhizopus stolonifer var. reflexus TP-02, and expressed in Escherichia coli BL21. The results showed that the gene amplified from the cDNA of the strain could be classified as the family of endoglucanase. During the fermentation process, the maximum endoglucanase activity (i.e. 0.715 IU/ml) of the recombinant bacteria was obtained at 36 h. The SDS–PAGE analysis on purified samples showed that a band with apparent molecular weight of about 40 kDa was detected after staining with Coomassie brilliant blue.     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

Sterilization mechanism of nitrogen gas plasma: induction of secondary structural change in protein

The mechanism of action on biomolecules of N₂ gas plasma, a novel sterilization technique, remains unclear. Here, the effect of N₂ gas plasma on protein structure was investigated. BSA, which was used as the model protein, was exposed to N₂ gas plasma generated by short‐time high voltage pulses from a static induction thyristor power supply. N₂ gas plasma‐treated BSA at 1.5 kilo pulses per second showed evidence of degradation and modification when assessed by Coomassie brilliant blue staining and ultraviolet spectroscopy at 280 nm. Fourier transform infrared spectroscopy analysis was used to determine the protein's secondary structure. When the amide I region was analyzed in the infrared spectra according to curve fitting and Fourier self‐deconvolution, N₂ gas plasma‐treated BSA showed increased α‐helix and decreased β‐turn content. Because heating decreased α‐helix and increased β‐sheet content, the structural changes induced by N₂ gas plasma‐treatment of BSA were not caused by high temperatures. Thus, the present results suggest that conformational changes induced by N₂ gas plasma are mediated by mechanisms distinct from heat denaturation.     

Identification of a novel sugar-H+ symport protein, FucP, for transport of L-fucose into Escherichia coli

l -Fucose (6-deoxy-l -galactose) is used as sole carbon source by many microorganisms, and its transport into Escherichia coli is mediated by An l -fucose-H⁺ symport activity, in order to determine the nature of a putative transporter encoded by the E. coli fucP gene and Identify its protein product it was cloned downstream of the inducible T7 RNA polymerase and lambda O_l P_l promoters, induction of the T7 promoter resulted in the expression of [¹⁴C]-l -fucose uptake activity and the concomitant expression of a [³⁵S]-Met-labelled 32 kDa protein at levels too tow for detection by staining with Coomassie briiiiant blue or for protein sequencing, induction of the lambda O_l P_l promoter caused the appearance of l -fucose-H₊ symport activity and of a Coomassie brilliant blue-stained 32 kDa membrane protein expressed at high levels sufficient for identification as FucP by N-terminal protein sequencing. The FucP protein is, therefore, a sugar-H₊ symporter different in amino acid sequence from any other known transporter. These and other results illustrate the general unpredictability of cloning strategies for attempting the amplified expression of membrane transport proteins.     

Identification of the polypeptides in the cytochromeb 6/f complex from spinach chloroplasts with redox-center-carrying subunits

An improved procedure for the isolation of the cytochromeb ₆/f complex from spinach chloroplasts is reported. With this preparation up to tenfold higher plastoquinol-plastocyanin oxidoreductase activities were observed. Like the complex obtained by our previous procedure, the complex prepared by the modified way consisted of five polypeptides with apparent molecular masses of 34, 33, 23, 20, and 17 kD, which we call Ia, Ib, II, III, and IV, respectively. In addition, one to three small components with molecular masses below 6 kD were now found to be present. These polypeptides can be extracted with acidic acetone. Cytochromef, cytochromeb ₆, and the Rieske Fe-S protein could be purified from the isolated complex and were shown to be represented by subunits Ia + Ib, II, and III, respectively. The heterogeneity of cytochromef is not understood at present. Estimations of the stoichiometry derived from relative staining intensities with Coomassie blue and amido black gave 1:1:1:1 for the subunits Ia + Ib/II/III/IV, which is interesting in of the presence of two cytochromesb ₆ per cytochromef. Cytochromef titrated as a single-electron acceptor with a pH-independent midpoint potential of +339 mV between pH 6.5 and 8.3, while cytochromeb ₆ was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withE _m(1)=–40 mV andE _m(2)=–172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.Abbreviation Tris tris(hydroxymethyl)aminomethane - SDS sodium dodecylsulfate - SDS-PAGE SDS polyacrylamide gel electrophoresis - MES 2-(N-morpholino)ethanesulfonic acid     

荧光素钠和考马斯亮蓝应用于小麦白粉病菌染色效果比较*

比较了荧光素钠和考马斯亮蓝应用于小麦白粉病菌染色的效果。荧光素钠法中样品处理只需20min.左右,具有直接、快速的特点;荧光指示剂对病菌分生孢子萌发及菌丝生长无抑制作用,主要沉集于活菌体的隔膜和细胞质部位,使病菌产生明显的亮绿荧光和清晰的细胞轮廓,亮绿荧光衰退期为7min.;借助荧光显微镜可以观察病菌在小麦叶表的发展过程,区别活菌体和失活菌体。考马斯亮蓝法包括传统的组织学染色步骤,经过改进后的样品处理过程需要40min.左右;染色后使寄主组织呈现淡蓝色,病菌菌体染成深蓝色;该方法可以观察病菌在小麦叶表和被侵染细胞内部发育形成的结构,包括孢子发育形成的初生芽管、附着胞芽管、成熟附着胞以及在寄主细胞内形成的初生吸器原体、成熟的指状体吸器和次生吸器。     

Influence of blue light on the structure stability of antenna complexes from Allochromatium minutissimum with different content of carotenoids

Effects of photooxidation of bacteriochlorophyll (absorbtion at 850 nm) from the light-harvesting complex LH2 of Alc. minutissimum membranes on the LH2 complex structure have been studied. Photooxidation was induced by blue light that is absorbed by carotenoids. Four samples with different levels (from 100% to 3–5%) and composition of carotenoids were obtained by inhibiting the carotenoid biosynthesis in bacteria with diphenylamine. Electrophoresis in polyacrylamide gel showed that after illumination LH2 complex contained all the oxidized bacteriochlorophyll. The carotenoid composition did not change after the oxidation of the main part of bacteriochlorophyll in the LH2 complex. The results suggest that oxidation takes place in the bacteriochlorophyll part, which is essential for the molecule optical properties (the system of double conjugated bonds is changed), but does not influence the stability of the structure of the LH2 complex.     

Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica

R-phycoerythrin is one of the three phycobiliproteins which are extensively employed as fluorescent probes, and it is prepared from red macroalgae. Phycobiliproteins in the marine red macroalga Heterosiphonia japonica were extracted in 50 mM phosphate buffer (pH 7.0) and precipitated by salting-out. The R-phycoerythrin was isolated by gel filtration with Sepharose CL-4B and Sephadex G-200. Then it was purified by ion exchange chromatography on DEAE Sepharose Fast Flow which was developed by linear ionic strength gradients. The purified R-phycoerythrin gave a ratio of A₅₆₅ to A₂₈₀ of 4.89. It showed a single band and a pI of 4.8 on the examination by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. The polypeptide analysis of the purified R-phycoerythrin by SDS–PAGE demonstrated that it contains four chromophore-carrying subunits and no colorless polypeptide and has two hexameric aggregates. The preparative procedures of the R-phycoerythrin purification established based on the experiments exhibit advantages and can offer a reference for R-phycoerythrin preparation from other marine red macroalga.     

Sequential synthesis of some proteins in cultured carrot explant (Daucus carota) cells during callus induction

Freshly isolated explants of the secondary phloem of carrot roots were exposed to ¹⁴C-leucine for various periods from t₀—to 18 h and the ¹⁴C labelling of protein was studied by 2-dimensional PAGE followed by fluorograph. The labelling pattern of proteins indicated a sequential activation of synthesis of about 130 proteins during the 18 h experimental period prior to the onset of cell division activity.Abbreviations IAA indole acetic acid - 2iP 2-isopentenyladenine - PVP polyvinylpyrrolidone - CBB Coomassie brilliant blue - RuBPCase ribulosebisphosphate carboxylase - LSC liquid scintillation counter - spec.act. specific radioactivity - u.l. uniformly labelled     

Resistance of reaction centers from Rhodobacter sphaeroides against UV-B radiation. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by ultraviolet-B (UV-B, 280–320 nm) radiation have been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides R26. UV-B irradiation results in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm reflecting the formation of the P⁺(Q_AQ_B)^– state. In addition to this effect, the charge recombination accelerates and the damping of the semiquinone oscillation increases in the UV-B irradiated reaction centers. A further effect of UV-B is a 2 fold increase in the half- inhibitory concentration of o-phenanthroline. Some damage to the protein subunits of the RC is also observed as a consequence of UV-B irradiation. This effect is manifested as loss of the L, M and H subunits on Coomassie stained gels, but not accompanied with specific degradation products. The damaging effects of UV-B radiation enhanced in reaction centers where the quinone was semireduced (Q_B ^–) during UV-B irradiation, but decreased in reaction centers which lacked quinone at the Q_B binding site. In comparison with Photosystem II of green plant photosynthesis, the bacterial reaction center shows about 40 times lower sensitivity to UV-B radiation concerning the activity loss and 10 times lower sensitivity concerning the extent of reaction center protein damage. It is concluded that the main effect of UV-B radiation in the purple bacterial reaction center occurs at the Q_AQ_B quinone acceptor complex by decreasing the binding affinity of Q_B and shifting the electron equilibration from Q_AQ_B ^– to Q_A ^–Q_B. The inhibitory effect is likely to be caused by modification of the protein environment around the Q_B binding pocket and mediated by the semiquinone form of Q_B. The UV-resistance of the bacterial reaction center compared to Photosystem II indicates that either the Q_AQ_B acceptor complex, which is present in both types of reaction centers with similar structure and function, is much less susceptible to UV damage in purple bacteria, or, more likely, that Photosystem II contains UV-B targets which are more sensitive than its quinone complex.Abbreviations Bchl bacteriochlorophyll - P Bchl dimer - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - RC reaction center - UV-B ultraviolet-B     

Application of coomassie brilliant blue staining to cultured hepatocytes

Coomassie brilliant blue staining developed by Pena (1980) was applied to cultured hepatocytes of adult rats with some modifications. Many of organelles in the cytoplasms were clearly visible as blue granules by this method. Various cytoskeletal elements were also visualized clearly. Because of its simplicity, Coomassie blue staining proved to be a very powerful tool for study of morphological changes of cell organelles and cytoskeletal systems of cultured hepatocytes.     

Partial purification and characterization of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas

This study reports the presence of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas, and its partial purification and some properties. Crude enzyme preparation was obtained by extraction from acetone-dried powder of the pancreas at pH 7.6. For solubilization of enzyme, freezing and thawing were carried out. Crude enzyme extract was fractionated with ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and ion-exchange chromatography on DEAE-cellulose. Partially purified enzyme showed 2897-folds purification. The enzyme activity on polyacrylamide gel electrophoresis showed good agreement with a main protein band stained with Coomassie brilliant blue. Molecular weight of this enzyme from the pancreas was estimated to be 300 000 by gel filtration on Sephacryl S-300 column. Optimum pH was between 8.5 and 9.0, and K_m value for glycylproline-p-nitroanilide tosilate was 0.33 mM. This enzyme from the pancreas was a serine enzyme and was relatively stable to heat at 60°C for 10 min.     

Occurrence of cytochrome c-554 in a facultative methylotroph,Protaminobacter ruber strain NR-1, during bacteriochlorophyll formation

A facultative methylotroph, Protaminobacter ruber was grown under two different conditions (aerobically grown under light, and aerobically in the dark after a light period). Bacteriochlorophyll was synthesized inducibly in the cells which were initially grown in the ligt and then grown in the dark, while bacteriochlorophyll was not found in the cells cultured under continuous light. Cytochrome c-554 was solely synthesized parallel to bacteriochlorophyll after switching from light to dark conditions. Both cytochrome c-554 and bacteriochlorophyll levels in the membrane preparation reached to a plateau in 24 h after switching from light and dark conditions. This cytochrome was membrane-bound and its M _r was 45,000 by sodium dodecylsulfate polyacrylamide gel electrophoresis. The midpoint potential was 358 mV at pH 7. Other major membrane-bound cytochromes and two soluble cytochromes were present in both types of cells and their content did not change irrespective of growth conditions.Abbreviations SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Bchl bacteriochlorophyll     

Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD

Magnesium chelatase catalyses the insertion of Mg²⁺ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg²⁺ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg²⁺. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f, -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272 000 × g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A₂₆₀:A₂₈₀ ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively. Received: 9 September 1996 / Accepted: 22 October 1996     

Purification and Characterization of an NAD(P)H:Quinone Oxidoreductase from Glycine Max Seedlings

Abstract

An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.