集胞藻类金属硫蛋白的纯化,性质和溶液构象的研究

集胞藻Synechocysitissp.PCC6803光照培养,加入100μmol/LZnCl2诱导藻内类金属硫蛋白（MT-like）的表达。离心收集鲜藻,超声波破碎细胞,离心除沉淀。上清液72℃加热3min去杂蛋白,离心,上清液依次经过分子筛层析柱,阴离子交换柱和脱盐柱,可得蓝藻类金属硫蛋白。5L培养液收集鲜藻7.6g,一次上样1．52g鲜藻的裂解物,冻干后得15mg纯化的蛋白样,为鲜藻重的0．1％．经分析其等电点为pH4．5,分子量为6．986kD,由58个氨基酸残基组成。其序列中含有较多疏水氨基酸残基（36％）,但其半胱氨酸含量仅为5％。CD（圆二色性）图谱表明其二级结构主要为无规卷曲,不含α－helix和β-sheet,无哺乳动物的金属硫蛋白所具有的典型双结构域结构。紫外吸收光谱表明Zn结合的类金属硫蛋白在220nm也有较高的吸收值。红外光谱分析的结果表明,此类金属硫蛋白的吸收光谱与高等动物的金属硫蛋白的吸收光谱类似。     

聚球藻类金属硫蛋白的纯化及部分性质的研究

单细胞蓝藻以５０μｍｏｌ／Ｌ Ｚｎ＾２＋诱导８ｄ后收集,破碎取上清液经凝胶过滤,离子交换层主反相ＨＰＬＣ纯化得到类金属硫蛋白,产率为每升培养液收集１．５ｇ鲜藻,得２．５ｍｇ纯品。其单体分子量为８７５０,Ｎ末端测定为缬氨酸,氨基酸组成分析得每分子含１０个半胱氨酸,疏水氨酸较多,且含有芳香族氨基酸,原子吸收光谱测得每分子蛋白结合４个二价金属。     

酵母菌类金属硫蛋白的分离、纯化及性质鉴定

经过对酵母菌的金属抗性试验和培养条件试验,确定了高产类金属硫蛋白的酿酒酵母菌株Cu-21(Saccharomyces cerevisiae Cu-21)及其最佳培养条件。对该菌株的菌体蛋白进行SephadexG-100、DEAE－52结合的两次柱分离纯化,获得了类金属硫蛋白的三个亚型,蛋白性质鉴定结果表明含三个亚型、分子量小、富含半脱氨酸和金属元素,具有典型的巯基吸收特性。     

酵母菌类金属硫蛋白的分离、纯化及性质鉴定*

经过对酵母菌的金属抗性试验和培养条件试验,确定了高产类金属硫蛋白的酿酒酵母菌株Cu-21（Saccharomyces cerevisiae Cu-21）及其最佳培养条件。对该菌株的菌体蛋白进行SephadexG-100、DEAE-52结合的两次柱分离纯化,获得了类金属硫蛋白的三个亚型,蛋白性质鉴定结果表明含三个亚型、分子量小、富含半胱氨酸和金属元素,具有典型的巯基吸收特性。     

酵母菌类金属硫蛋白的分离、纯化及性质鉴定*

经过对酵母菌的金属抗性试验和培养条件试验,确定了高产类金属硫蛋白的酿酒酵母菌株Cu-21（Saccharomyces cerevisiae Cu-21）及其最佳培养条件。对该菌株的菌体蛋白进行SephadexG-100、DEAE-52结合的两次柱分离纯化,获得了类金属硫蛋白的三个亚型,蛋白性质鉴定结果表明含三个亚型、分子量小、富含半胱氨酸和金属元素,具有典型的巯基吸收特性。     

脱金属硫蛋白与二价汞离子的络合作用及构象研究

用圆二色谱研究兔肝锐金属硫蛋白的两个亚型ａｐｏ－ＭＴ１、ａｐｏ－ＭＴ２与Ｈｇ＾２＋在不同分子数比和ｐＨ值下的络合规律。发现：（１）在ｐＨ２下与Ｈｇ＾２＋重组ＭＴｓ的ＣＤ谱特征峰是３０４ｎｍ（＋）,２６０ｎｍ（－）,２５０ｎｍ（＋）。络合Ｈｇ＾２＋的数目ｎ远远超过７。任何ｐＨ值下过量Ｈｇ＾２＋的存在都将破坏ＭＴｓ的正常构象。ｐＨ值对形成和维持ＭＴｓ稳定构象的影响很大,当ｐＨ≥４．９时,ａｐｏ－ＭＴｓ     

雪松聚球藻金属硫蛋白的纯化及其性质的研究

在雪松聚球藻的培养基中逐渐增加氯化镉的浓度以诱导藻细胞内金属硫蛋白的合成,经Ｓｅｐｈａｄｅｘ Ｇ－５０,ＤＥＡＥ－ｃｅｌｌｕｌｏｓｅ和Ｓｅｐｈａｄｅｘ Ｇ－２５柱层析,获得的ＭＴ没有亚型,经ＳＤＳ－ＰＡＧＥ分析是高度均一的。每个蛋白分子约含５个Ｃｄ原子,１０个巯基,分子量约为７．６ＫＤ,等电位ｐＨ４．５左右,半胱氨酸含量约占总氨基酸量的１５．５％,还含有少量的芳香族氨基酸,ＭＴ的最大紫外吸收在２５     

脱金属硫蛋白与镉离子的络合作用及构象研究

用圆二色（ＣＤ）谱地研究兔肝脱金属硫属蛋白的两个亚型与Ｃｄ＾２＋的络合作用及对重组ＭＴ构象的影响。观测了ａｐｏ－ＭＴ垢巯基在空气和室温下的稳定性。在ＰＨ４．７１,镉重组ＭＴ１的ＣＤ谱特征峰在２５７ｎｍ（＋）,２３８ｎｍ（－）,２２６ｎｍ（＋）与镉诱导的天然ＭＴ１相同。在空气存在和ＰＨ７．９０的ＣＤ谱只有２４３ｎｍ（＋）一个峰。向两亚型分别加入７ｅｑＣｄ＾２＋测定ＣＤ谱随ＰＨ值的变化,发现在ＰＨ２．     

镉诱导蚯蚓金属硫蛋白的分离纯化及其溶液构象的研究

镉诱导蚯蚓匀浆液经热沉淀,乙醇沉淀后,经凝胶过滤ＳｅｐｈａｃｒｙＳ－２００柱层析,得三个镉结合蛋白峰,分子量依次为４０,１７及９ｋＤ。这三个组份再分别经ＤＥＡＥＳｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ柱层析,分别得三、三、二个镉结合蛋白峰。根据光谱学特征、巯基含量及氨基酸组成等分析,认为凝胶过滤第二峰为金属硫蛋白（ＭＴ）,其经ＤＥＡＥ柱层析所得三个峰为ＭＴ三个亚型。对蚯蚓ＭＴ三个亚型在不同ＰＨ条件下     

中华鳖肝金属硫蛋白的分离、纯化及鉴定

金属硫蛋白metallothionein,MT)是一类低分子量、富含半胱氨酸的金属结合蛋白。MT几乎广泛分布于所有生物,包括哺乳动物、两栖动物、鱼、植物、真菌和蓝细菌,不同生物金属硫蛋白理化特性和其氨基酸序列及中心片段的比较研究,对研究MT的结构和生物功能及生物的分子进化提供重要依据。哺乳动物MT研究较多,爬行动物鳖MT的研究尚属空白,本文报道鳖肝的金属硫蛋白,中华鳖（Pelodiscus sinensis)分别经皮下注射ZnSO4,CuSO4和CdCl2溶液诱导后,取乙醇沉淀的肝脏无细胞提取液再经SephadexG-50、DEAE-SepharoseCL-6B及SephadexG-25凝胶过滤和离子交换柱层析分离,自鳖肝脏中分别获得Zn-MT、Cu-MT和Cd-MT,未经诱导的鳖肝脏中无MT,质谱和HPLC分析其分子量约为6300dalton。根据氨基酸组成分析。鳖肝脏MT含61个氨基酸残基,其中MT的典型氨基酸Cys含量占17%。Lys,Glu和Asp含量较高,而芳香族氨基酸和组氨酸含量极低,从紫外光谱特性分析,Zn-MT、Cu-MT、Cd-MT紫外吸收肩分别在220nm、270nm和250nm。表明确为鳖肝脏MT。从氨基酸残基数和分子量看,鳖肝脏MT与哺乳动物MT类似;而从氨基酸组成和结合金属离子的量看,又与低等生物蚯蚓酵母菌的MT类似。鳖MT的特性介于哺乳动物MT与低等生物MT之间,体现了鳖这种生物进化的特点。     

Purification of glutamyl-tRNA reductase from Synechocystis sp. PCC 6803

delta-Aminolevulinic acid is the universal precursor for all tetrapyrroles including hemes, chlorophylls, and bilins. In plants, algae, cyanobacteria, and many other bacteria, delta-aminolevulinic acid is synthesized from glutamate in a reaction sequence that requires three enzymes, ATP, NADPH, and tRNA(Glu). The three enzymes have been characterized as glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. All three enzymes have been separated and partially characterized from plants and algae. In prokaryotic phototrophs, only the glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase have been decribed. We report here the purification and some properties of the glutamyl-tRNA reductase from extracts of the unicellular cyanobacterium, Synechocystis sp. PCC 6803. The glutamyl-tRNA reductase has been purified over 370-fold to apparent homogeneity. Its native molecular mass was determined to be 350 kDa by glycerol density gradient centrifugation, and its subunit size was estimated to be 39 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was determined for 42 residues. Much higher activity occurred with NADPH than with NADH as the reduced pyridine nucleotide substrate. Half-maximal rates occurred at 5 microM NADPH, whereas saturation was not reached even at 10 mM NADH. Purified Synechocystis glutamyl-tRNA reductase was inhibited 50% by 5 microM heme. Activity was unaffected by 10 microM 3-amino-2,3-dihydrobenzoic acid. No flavin, pyridine nucleotide, or other light-absorbing prosthetic group was detected on the purified enzyme. The catalytic turnover number of purified Synechocystis glutamyl-tRNA reductase is comparable to those of prokaryotic and plastidic glutamyl-tRNA synthetases.     

Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803. Purification, assembly, and quaternary structure.

The phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 forms holoprotein adducts with close spectral similarity to plant phytochromes when autoassembled in vitro with bilin chromophores. Cph1 is a 85-kDa protein that acts as a light-regulated histidine kinase seemingly involved in 'two-component' signalling. This paper describes the improvement of Cph1 purification, estimation of the extinction coefficient of holo-Cph1, spectral analyses of the assembly procedure and studies on quaternary structure. During assembly with the natural chromophore phycocyanobilin (PCB), a red-shifted intermediate is observed. A similar result was obtained when phycoerythrobilin was used as chromophore. As shown by SDS/PAGE and Zn2+ fluorescence, the covalent attachment of PCB is blocked by 1 mM iodoacetamide, a cysteine-derivatizing agent. When PCB was incubated with blocked apo-Cph1, again a shoulder at longer wavelengths appeared. It is therefore proposed that the long-wavelength-absorbing form represents the protonated, noncovalently bound bilin. Biliverdin, which is neither protonated nor covalently attached, undergoes spectral changes in its blue-absorbing band upon incubation with apo-Cph1. On the basis of these data we therefore propose a three-step model for phytochrome autoassembly. Size-exclusion chromatography revealed different mobilities for the apoprotein, red-absorbing Cph1-PCB and far-red-absorbing Cph1-PCB. The major peaks of both holoprotein adducts had apparent molecular masses approximately 200 kDa, a result in agreement with the notion that autophosphorylation in sensory histidine kinases requires dimerization. When Cph1-PCB was further purified by preparative native electrophoresis, the mobility on size-exclusion chromatography was approximately 100 kDa, and it was found to have lost its kinase activity, results implying that the material had lost its capacity to dimerize.     

Characterization of the Cph1 holo-phytochrome from Synechocystis sp. PCC 6803.

The cph1 gene from the unicellular cyanobacterium Synechoycstis sp. PCC 6803 encodes a protein with the characteristics of plant phytochromes and histidine kinases of two-component phospho-relay systems. Spectral and biochemical properties of Cph1 have been intensely studied in vitro using protein from recombinant systems, but virtually nothing is known about the situation in the natural host. In the present study, His6-tagged Cph1 was isolated from Synechocystis cells. The cph1-his gene was expressed either under the control of the natural cph1 promoter or over-expressed using the strong promoter of the psbA2 gene. Upon purification with nickel affinity chromatography, the presence of Cph1 in extracts was confirmed by immunoblotting and Zn2+-induced fluorescence. The Cph1 extracts exhibited a red/far-red photoactivity characteristic of phytochromes. Difference spectra were identical with those of the phycocyanobilin adduct of recombinant Cph1, implying that phycocyanobilin is the chromophore of Cph1 in Synechocystis.     

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Lit-tle is known regarding the biochemical properties and functions of the deamidating enzyme glutami-nase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Es-cherichia coli. The purified protein possessed glutaminase activity, validating the functional assign-ment of the genomic annotation. The apparent Km value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na . Moreover, the Km value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na . These data demon-strate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na through in-creasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synecho-cystis by targeted mutagenesis and the △slr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between △slr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, △slr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosyn-thetic oxygen evolution rate of △slr2079 cells was higher than that of the wild-type. To further charac-terize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in △slr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimi-lation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in △slr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress re-sponse by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.     

Characterization of a mutant lacking carboxysomal carbonic anhydrase from the cyanobacterium Synechocystis PCC6803

A fully-segregated mutant (ccaA::kanR) defective in the ccaA gene, encoding a carboxysome-associated beta-carbonic anhydrase (CA), was generated in the cyanobacterium Synechocystis sp. PCC6803 by insertional mutagenesis. Immunoblot analysis indicated that the CcaA polypeptide was absent from the carboxysome-enriched fraction obtained from ccaA::kanR, but was present in wild-type (WT) cells. The carboxysome-enriched fraction isolated from WT cells catalyzed 18O exchange between 13C18O2 and H2O, indicative of CA activity, while ccaA::kanR carboxysomes did not. Transmission and immunogold electron microscopy revealed that carboxysomes of WT and ccaA::kanR were of similar size, shape and cellular distribution, and contained most of the cellular complement of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The ccaA::kanR cells were substantially smaller than WT and were unable to grow autotrophically at air levels of CO2. However, cell division occurred at near-WT rates when ccaA::kanR was supplied with 5% CO2 (v/v) in air. The apparent photosynthetic affinity of the mutant for inorganic carbon (Ci) was 500-fold lower than that of WT cells although intracellular Ci accumulation was comparable to WT measurements. Mass spectrometric analysis revealed that the CA-like activity associated with the active CO2 transport system was retained by ccaA::kanR cells and was inhibited by H2S, indicating that CO2 transport was distinct from the CcaA-mediated dehydration of intracellular HCO3-. The data suggest that the ccaA mutant was unable to efficiently utilize the internal Ci pool for carbon fixation and that the high-CO2-requiring phenotype of ccaA::kanR was due primarily to an inability to generate enough CO2 in the carboxysomes to sustain normal rates of photosynthesis.     

Characterization of a naturally occurring trans-splicing intein from Synechocystis sp. PCC6803

A naturally occurring trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) was used to characterize the intein-catalyzed splicing reaction. Trans-splicing/cleavage reactions were initiated by combining the N-terminal splicing domain of the Ssp DnaE intein containing five native N-extein residues and maltose binding protein as the N-extein with the C-terminal Ssp DnaE intein splicing domain (E(C)) with or without thioredoxin fused in-frame to its carboxy terminus. Observed rate constants (k(obs)) for dithiothreitol-induced N-terminal cleavage, C-terminal cleavage, and trans-splicing were (1.0 +/- 0.5) x 10(-3), (1.9 +/- 0.9) x 10(-4), and (6.6 +/- 1.3) x 10(-5) s(-1), respectively. Preincubation of the intein fragments showed no change in k(obs), indicating association of the two splicing domains is rapid relative to the subsequent steps. Interestingly, when E(C) concentrations were substoichiometric with respect to the N-terminal splicing domain, the levels of N-terminal cleavage were equivalent to the amount of E(C), even over a 24 h period. Activation energies for N-terminal cleavage and trans-splicing were determined by Arrhenius plots to be 12.5 and 8.9 kcal/mol, respectively. Trans-splicing occurred maximally at pH 7.0, while a slight increase in the extent of N-terminal cleavage was observed at higher pH values. This work describes an in-depth kinetic analysis of the splicing and cleavage activity of an intein, and provides insight for the use of the split intein as an affinity domain.     

Characterization of a Highly pH Stable Chi-Class Glutathione S-Transferase from Synechocystis PCC 6803

Glutathione S-transferases (GSTs) are multifunctional enzymes present in virtually all organisms. Besides having an essential role in cellular detoxification, they also perform various other functions, including responses in stress conditions and signaling. GSTs are highly studied in plants and animals; however, the knowledge regarding GSTs in cyanobacteria seems rudimentary. In this study, we report the characterization of a highly pH stable GST from the model cyanobacterium- Synechocystis PCC 6803. The gene sll0067 was expressed in Escherichia coli (E. coli), and the protein was purified to homogeneity. The expressed protein exists as a homo-dimer, which is composed of about 20 kDa subunit. The results of the steady-state enzyme kinetics displayed protein’s glutathione conjugation activity towards its class specific substrate- isothiocyanate, having the maximal activity with phenethyl isothiocyanate. Contrary to the poor catalytic activity and low specificity towards standard GST substrates such as 1-chloro-2,4-dinitrobenzene by bacterial GSTs, PmGST B1-1 from Proteus mirabilis, and E. coli GST, sll0067 has broad substrate degradation capability like most of the mammalian GST. Moreover, we have shown that cyanobacterial GST sll0067 is catalytically efficient compared to the best mammalian enzymes. The structural stability of GST was studied as a function of pH. The fluorescence and CD spectroscopy in combination with size exclusion chromatography showed a highly stable nature of the protein over a broad pH range from 2.0 to 11.0. To the best of our knowledge, this is the first GST with such a wide range of pH related structural stability. Furthermore, the presence of conserved Proline-53, structural motifs such as N-capping box and hydrophobic staple further aid in the stability and proper folding of cyanobacterial GST- sll0067.     

Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp. PCC 6803

The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the psbAII promoter was inserted upstream of the hoxE gene. In addition, the accessory genes (hypF, C, D, E, A, and B) from Nostoc sp. PCC 7120 were expressed under control of the psbAII promoter. The respective strains show higher hydrogenase activities compared with the wild type. For the first time a Fourier transform infrared (FTIR) spectroscopic characterization of a [NiFe] hydrogenase from an oxygenic phototroph is presented, revealing that two cyanides and one carbon monoxide coordinate the iron of the active site. At least four different redox states of the active site were detected during the reversible activation/inactivation. Although these states appear similar to those observed in standard [NiFe] hydrogenases, no paramagnetic nickel state could be detected in the fully oxidized and reduced forms. Electron paramagnetic resonance spectroscopy confirms the presence of several iron-sulfur clusters after reductive activation. One [4Fe4S]⁺ and at least one [2Fe2S]⁺ cluster could be identified. Catalytic amounts of NADH or NADPH are sufficient to activate the reaction of this enzyme with hydrogen.