Purification and characterization of 8 of the pathogenesis-related proteins in tobacco leaves reacting hypersensitively to tobacco mosaic virus

Summary Leaves of tobacco plants (Nicotiana tabacum cv. Samsun NN) which are reacting hypersensitively to infection with tobacco mosaic virus contain 10 major pathogenesis-related (PR) proteins which are absent, or present in small amounts in uninfected leaves. We describe here a preparative procedure of purification of the tobacco PR-proteins which involves a combination of conventional and high-performance liquid chromatography. The separation and isolation of the proteins were based on differences in net charge at different pH values, in isoelectric point and in apparent molecular weight. This procedure led to the purification to homogeneity of 8 PR-proteins, as shown by polyacrylamide slab gel electrophoresis (PAGE) of the purified proteins under denaturing and non-denaturing conditions. These were the 3 well-known proteins PR-1a,-1b and-1c, and 5 other major PR-proteins, called PR-2,-N,-O,-P and-Q, according to the nomenclature of Van Loon (39). None of the purified PR-proteins gave a positive Schiff reaction for carbohydrate content. Molecular weight determinations from gel permeation chromatography and from sodium dodecyl sulphate (SDS)-PAGE indicated that all 8 PR-proteins were monomers and that three groups could be distinguished among them. The first group is the PR-1 group containing PR-1a,-1b and-1c (12000 MW), the second consists of PR-P and PR-Q (14000 MW) and the third of PR-2, PR-N and PR-O (25000 MW). In the PR-1 group, PR-1a can be distinguished clearly from the two other members on denaturing slab gels containing both SDS and urea.     

Proteoglycans of human umbilical cord arteries

Proteoglycans (PGs) were dissociatively extracted from human umbilical cord arteries (UCAs) with 4 M guanidine hydrochloride containing Triton X-100 and protease inhibitors, purified by Q-Sepharose anion exchange chromatography and lyophilized. They were analysed by gel filtration, SDS/PAGE and agarose gel electrophoresis before and after treatment with chondroitinase ABC. It was found that the PG preparation was especially enriched in chondroitin/dermatan sulphate PGs. The predominant PG fraction included small PGs that emerged from Sepharose CL-2B with Kav = 0.74. Their molecular mass, estimated by SDS/PAGE, was 160-200 kDa and 90-150 kDa, i.e. it was typical for biglycan and decorin, respectively. Treatment with chondroitinase ABC yielded the core proteins of 45 and 47 kDa, characteristic for both small PGs. Remarkable amounts of the 45 kDa protein were detected in non-treated PG samples, suggesting the presence of free core proteins of biglycan and decorin. Large PGs were present in lower amounts. In intact form they were eluted from Sepharose CL-2B with Kav = 0.17 and 0.43. Digestion with chondroitinase ABC yielded the core proteins with a molecular mass within the range of 180-360 kDa but predominant were the bands of 200, 250 and 360 kDa. The large PGs probably represent various forms of versican or perlecan bearing chondroitin sulphate chains.     

NADH-细胞色素b5还原酶在大肠杆菌中的表达及其产物的纯化

为了探讨 NADH-细胞色素 b5还原酶基因突变引起遗传性高铁血红蛋白血症的分子病理机制 ,研究突变型 ( b5R)蛋白结构和功能的关系 ,用基因重组技术将野生型和突变型 ( C2 0 3Y) b5Rc DNA克隆于 p GEX- 2 T载体 ,在大肠杆菌 BL2 1中诱导表达 .Western印迹鉴定所表达的蛋白为GST- b5R融合蛋白 .应用谷胱甘肽 - Sepharose 4B亲和层析 ,还原型谷胱甘肽洗脱得到纯化的GST- b5R和 GST- b5RC2 0 3Y融合蛋白 .比较 GST- b5R和 GST- b5RC2 0 3Y酶活性及稳定性 ,发现野生型和突变型的酶活性基本相同 .但与野生型酶相比 ,突变型酶对热的稳定性较差 ,对胰蛋白酶更加敏感 .结果提示 ,C2 0 3Y突变可引起蛋白质二级结构改变而导致酶的稳定性下降 .     

Rat hepatic microsomal cytochrome b5. A simple large-scale purification procedure and antibody production by antigen-containing liposomes.

Cytochrome b5 from rat liver microsomes (microsomal fractions) was purified in its native form. The procedure described has great capacity, is fast, and the final product is pure as judged from SDS/polyacrylamide-gel electrophoresis. Antibodies to cytochrome b5 are obtained after administration of the antigen inserted into small unilamellar lipid vesicles.     

Properties of acid phosphatase from scutella of germinating maize seeds

One acid phosphatase (optimum pH at 5.4) was purified from maize scutellum after 96 hr of germination. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulfate (SDS). The enzyme has a MW of 65 000 ± 4000 as determined by Sephadex G-200 gel filtration and SDS-PAGE. The enzyme contained 16% neutral sugars, and cations are not required for activity. The purified enzyme was not inactivated by DTNB at pH 8. The hydrolysis of glucose-6-phosphate in the presence of 4 mM fluoride and 4 mm EDTA, at pH 6.7 (optimum pH), seems to be catalysed by this acid phosphatase.     

Proteomic profiles of thylakoid membranes and changes in response to iron deficiency

The proteomic profile of thylakoid membranes and the changes induced in that proteome by iron deficiency have been studied by using thylakoid preparations from Beta vulgaris plants grown in hydroponics. Two different 2-D electrophoresis approaches have been used to study these proteomes: isoelectrical focusing followed by SDS PAGE (IEF-SDS PAGE) and blue-native polyacrylamide gel electrophoresis followed by SDS PAGE (BN-SDS PAGE). These techniques resolved approximately 110–140 and 40 polypeptides, respectively. Iron deficiency induced significant changes in the thylakoid sugar beet proteome profiles: the relative amounts of electron transfer protein complexes were reduced, whereas those of proteins participating in leaf carbon fixation-linked reactions were increased. A set of polypeptides, which includes several enzymes related to metabolism, was detected in thylakoid preparations from Fe-deficient Beta vulgaris leaves by using BN-SDS PAGE, suggesting that they may be associated with these thylakoids in vivo. The BN-SDS PAGE technique has been proven to be a better method than IEF-SDS PAGE to resolve highly hydrophobic integral membrane proteins from thylakoid preparations, allowing for the identification of complexes and determination of their polypeptidic components.     

SDS聚丙烯酰胺凝胶电泳快速染色新方法的研究

通过几种金融盐溶液对SDS聚丙烯酰胺凝胶电泳染色的实验表明,0.25mol/L的CaCl2和MgCl2溶液能够对蛋白质进行有效的染色,经这2种溶液染色的蛋白质都能够从凝胶中洗脱回收。尤其是CaCl2法灵敏度更高,而且蛋白质条带形成之后也十分稳定,所以在运用制备电泳纯化蛋白质时这种新的染色方法较适用。     

兔阑尾中一种新的21kD的钙结合蛋白的纯化与鉴定

纯化与鉴定了Ｂ淋巴细胞中一种新的分子量为２１ｋＤ的钙结合蛋白（ＣａＢＰ２１）。兔阑尾淋巴细胞匀浆经热变性,Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ与ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱层析,自每１ｋｇ细胞沉积物中获得ＳＤＳ－ＰＡＧＥ均一的ＣａＢＰ２１５．３ｍｇ。ＨＣｌ水解后的酸性氨基酸（Ａｓｐ＋Ｇｌｕ）含量为２６％。如同大多数钙结合蛋白一样,Ｎ末端封闭阻止其进行Ｅｄｍａｎ降解。ＣａＢＰ２１中疏水性氨基酸（计Ｇｌｙ,不计Ｔｒｐ）约占４６％,碱性氨基酸１０％,酸性氨基酸与极性氨基酸约４４％。ＣａＢＰ２１有较高的Ｓｅｒ、Ｔｙｒ含量。肽谱分析等确证ＣａＢＰ２１为２个相同或相似亚基二聚体。以ＡｒｓｅｎａｚｏⅢ作Ｃａ２＋结合分析表明每分子ＣａＢＰ２１可结合４分子Ｃａ２＋,对Ｃａ２＋的结合常数约为１０－５ｍｏｌ／Ｌ。各种性质表明ＣａＢＰ２１是一种不同于其他已知钙结合蛋白的新钙结合蛋白。     

Purification and evidence for heterogeneity of acid phosphatase from Saccharomycescerevisiae

The protoplast-secreted acid phosphatase of yeast $\text{Saccharomyces}\text{cerevisiae}$ was purified about 60 fold by ultrafiltration, gel filtration and chromatography on DEAE-Sephadex A-25. It was established that the enzyme is free of inactive proteins as well as polysaccharides and contains 48% of neutral sugars. The failure to separate the protein from the carbohydrates by several procedures indicates that the carbohydrate part is covalently linked to the protein. A pronounced heterogeneity of the enzyme with respect to charge as well as to molecular weight was found. The data obtained by gel filtration indicated enzyme heterogeneity in respect to carbohydrate content.     

Partial purification of Cytochrome P450 from Helicoverpa armigera (Hübner)

Abstract The cytochrome P450 (Cyt‐P450) proteins from the fat body and midgut of the cotton bollworm, Helicoverpa armigera, were respectively partially purified by a set of purification procedures including differential centrifugation, solubilization of CHAPS, protein precipitation by PEG precipitation and DE‐32 column chromatography. The Cyt‐P450 was detected by methods of CO difference spectrum and SDS‐PAGE. Fraction of detergent solubilized microsomes from the fat body of H. armigera was purified more than 17‐fold. Three protein bands were detected by SDS‐PAGE with molecular masses of 70 600, 63 300 and 571 200Da. It is possible that the proteins with molecular mass of 63 300 and 571 200Da were the isozymes of Cyt‐P450.     

On the glycosylation state of five rat hepatic microsomal cytochrome P-450 isozymes

The glycosylation states of five rat hepatic microsomal cytochrome P-450 isozymes (cytochromes P-450a, P-450b, P-450c, P-450d, and P-450e) were examined by quantitative carbohydrate analysis. Carbohydrate content of the purified enzymes as determined by acid hydrolysis, reduction, and gas chromatography of the alditol acetates revealed only trace amounts of neutral and amino hexoses in each of the five isozymes. Levels of mannose ranged from 0.3 to 1.7 mol/mol of cytochrome P-450 whereas levels of galactose were less than or equal to 0.2 mol/mol of cytochrome P-450 for the five hemoproteins. The amino sugars glucosamine and galactosamine were usually present at levels less than or equal to 0.2 mol/mol of cytochrome P-450, although one preparation of cytochrome P-450b had as much as 0.5 mol of glucosamine/mol of cytochrome P-450. Other carbohydrate residues (xylose and arabinose) were not detected in significant quantities. Since N- and O-glycosylation of proteins occurs primarily through N-acetylglucosaminyl and N-acetylgalactosaminyl residues, respectively, the lack of significant amounts of these amino sugars indicates that these five cytochrome P-450 isozymes are not normally glycosylated in the native state. Purified NADPH-cytochrome c reductase, which functions as an electron donor for microsomal cytochrome P-450, contained no detectable quantities of hexose sugars.     

羊血铜锌超氧化物歧化酶(Cu-ZnSOD)的分离纯化

目的:对羊血进行提取SOD。方法:采用有机溶剂去除血红蛋白、热变性、丙酮沉淀、DEAE-32离子交换柱层析的方法,对羊红细胞Cu,Zn-SOD进行分离纯化。利用非变性凝胶电泳NBT活性染色鉴定SOD,SDS-PAGE测定分子量。结果:表明1 000ml羊血中得到SOD干粉1 257mg,总活力为79 453U,比活力为3 871.4U/mg。活性染色结果证明羊血SOD有2条带,表明已达到电泳纯。SDS-PAGE测得SOD亚基分子量分别为16.71kDa、15.97kDa。H2O2对羊血CuZn-SOD活性有抑制作用,通过紫外光谱扫描,羊血SOD在231nm处有最大吸收峰。     

Immunoaffinity purification and characterisation of p29--an estrogen receptor related protein

p29, a 29 kDa protein recognised by D5, a monoclonal antibody prepared against partially purified cytosolic estrogen receptor (ER), has been purified to homogeneity from ZR-75-1, a human breast cancer cell line. Ammonium sulphate fractionation followed by immunoaffinity chromatography on a three column system using Protein A-Sepharose coupled D5, produced purified p29. Silver stained SDS one-dimensional polyacrylamide gel electrophoresis (PAGE) and two-dimensional PAGE showed p29 to have been purified to homogeneity. Amino acid analysis showed no unusual characteristics. Partial N-terminal sequencing studies showed that purified p29 shared a 100% homology with the sequence of a pp89, murine cytomegaloviral protein.     

Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells.

The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-450(25] from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253, 549-552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol.mg of protein-1, and the protein showed a single spot with an apparent isoelectric point of 7.4 and an Mr of 50,500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124 pmol.min-1.nmol of cytochrome P-450-1 and towards 1 alpha-hydroxyvitamin D3 it was 1375 pmol.min-1.nmol-1. The preparation also catalysed the 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol at a rate of 1000 pmol.min-1.nmol of cytochrome P-450-1 and omega-1 hydroxylation of lauric acid at a rate of 200 pmol.min-1.nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(25) from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5 beta-cholestane-3 alpha,7 alpha-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-450(25) was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-450(25) from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.     

Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome-c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit.

Cytochrome-c reductase was isolated from Saccharomyces cerevisiae GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE. Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 center (Q, ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax was 50% higher. Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.     

Age-Related Changes of Myelin Proteins in the Rat Peripheral Nervous System

Age-related changes in amounts of myelin proteins from rat sciatic nerve or spinal root were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In the aged peripheral nerve myelin, the relative amounts of band 105K and proteins X and Y increased, whereas those of proteins P0 and P1 and band 190K decreased. Band 105K purified by preparative SDS-PAGE exhibited three bands of 105K, 28K, and 21K at the second electrophoresis. A repeated SDS-PAGE did not improve the purity of bank 105K, but increased the ratio of 21K to 28K. Compared with P0 protein, band 105K has a very similar peptide map pattern and amino acid composition, as well as the identical NH2 terminal residue, isoleucine. These findings suggest that band 105K is an aggregate form of P0 protein and its fragment, 21K. The 21K protein is a distinct entity from X protein. The quantitative and qualitative alterations in myelin proteins, as we report here, may reflect continuing demyelination and remyelination in aged peripheral nerves.     

Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52） and Sephacryl S-200 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.     

亲和层析法分离鉴定人类端粒酶复合体及其蛋白质组分分析

为分离纯化人类端粒酶复合体并对其蛋白质组分进行分析 ,自行设计一套特异的以端粒重复序列为配体 ,以亲和磁珠为介质 ,以竞争性碱基序列为洗脱原则的寡核苷酸亲和纯化法 ,对He La细胞端粒酶复合体进行分离纯化 .并采用近年发展起来的主要用于检测序列特异性 DNA结合蛋白的凝胶迁移阻滞分析法对纯化产物进行鉴定分析 .应用 SDS- PAGE对纯化蛋白质亚基组分进行分析与鉴定 ,并采用电泳迁移率和已知蛋白质分子质量标准的对数作图分析测得所得蛋白质亚基成分的相对分子质量 .结果表明 ,纯化产物以 TRAP法检测酶活性可见典型的梯形条带 ;比活性为每 mg蛋白质的 cpm值为 1 80× 1 0 9,纯化倍数 1 80 0 ,得率 90 % .凝胶迁移阻滞法分析显示特异的凝胶迁移阻滞性电泳条带 ;以 SDS- PAGE检测得到 4种蛋白质亚基成分 ,其蛋白质相对分子质量分别为 2 2 0 ku、2 1 2 ku、1 1 6ku和 43ku.由上述可见 ,采用自行设计的寡核苷酸亲和纯化法获得了人端粒酶复合体及 4种蛋白质亚基组分 .     

Studies on algal cytochromes VI: some properties and amino acid sequence of cytochrome c6 from a green alga, Bryopsis maxima

A photosynthetic c-type cytochrome, cytochrome c6, was extracted from a green alga, Bryopsis maxima, by cutting and immersing the frozen thalli in phosphate buffer, pH 7.0, and purified by acrinol treatment, ammonium sulfate fractionation, DEAE-Sephacel chromatography and Bio-Gel P-10 gel filtration. The ferrcytochrome c6 has absorption maxima at 553.5 (alpha), 523 (beta), 417 (gamma), 318 (delta), and 275 nm, and the ferricytochrome at 695, 528, and 411 (gamma). The molecular weight was estimated to be about 10,000 from Sephadex G-75 gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The midpoint redox potential for the cytochrome was determined by equilibrium titration with a ferro- and ferricyanide system to be 0.385 volt at pH 7.0. Isoelectric points for ferro- and ferricytochromes were determined by density gradient isoelectric focusing electrophoresis to be at pH 3.91 and 4.02, respectively. The complete amino acid sequence of the cytochrome was determined by Edman degradation and by carboxypeptidase digestions of the Cm-cytochrome, 6 staphylococcal protease peptides and 5 lysyl endopeptidase peptides. The cytochrome contained 88 amino acid residues, giving a molecular weight of 9,904 including 1 mol of heme c. The sequence is as follows: GGDLEIGADVFTGNCAACHAGGANSVEPLKTLNKEDVTKYLDGGLSIEAITSQVRNGKGAMPAWSDRLD DEEIDGVVAYVFKNINEGW. A phylogenetic tree of 13 algal cytochromes c6 was constructed by comparing the amino acid differences.