Reduction of castor-seed 2S albumin protein by thioredoxin

The 2S albumin from the endosperm of castor seed (Ricinus communis L.) seed was reduced by thioredoxin from either wheat germ or Escherichia coli. The 2S protein is made up of a large (approx. 7 kDa) subunit that contains two intramolecular disulfides and a small (approx. 4 kDa) subunit that lacks intramolecular disulfides. The two subunits are joined by at least one intermolecular disulfide bond. Thioredoxin could be reduced either enzymically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol. Reduced glutathione and glutaredoxin (from E. coli) were without effect. The ability of the 2S protein to undergo reduction by thioredoxin was demonstrated by a direct reduction procedure based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by an enzymatic procedure in which reduction is linked to activation of chloroplast NADP-malate dehydrogenase. Analyses indicated that thioredoxin actively reduced the intramolecular disulfides of the 2S large subunit, but was ineffective in reducing the intermolecular disulfide(s) that connect the large to the small subunit. These findings extend the role of thioredoxin to the reduction of a seed protein that is widely distributed in oil producing plants.Abbreviations DDT dithiothreitol - mBBr monobromobimane - NTR NADP-thioredoxin reductase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by a grant from the National Science Foundation.     

臭鼩血清蛋白和六种组织乳酸脱氢酶同工酶的研究

本实验对臭鼩的血清蛋白及心肌、骨骼肌、肾脏、脾脏、肝脏,睾丸6种组织器官的乳酸脱氢酶(LDH)同工酶进行了聚丙烯酰胺凝胶盘状电泳的分析研究。臭鼩血清蛋白存在15—17条带,各组织的LDH同工酶均由5条带构成,其中心肌LDH-1、LDH-2和肾脏LDH-1各出现1条亚带。     

水稻种子贮藏谷蛋白的改良电泳分析法

利用15％－25％丙烯酰胺梯度凝胶的SDS－PAGE分析法可将水稻种子贮藏谷蛋白分离为3个酸性（α）亚基和3个碱性（β）亚基,通过调节两性电解质比例对现有等用点了和焦电泳分析法进行改良,可将谷蛋白酸性亚基和碱性亚基分别分划为13和14条多肽带,将上述两种方法结合起来的双向电泳分析法可以高清晰度地离析谷蛋白并获得单一多肽,此改良的电泳分析系统有助于确定水稻谷蛋白变异及谷蛋白的生化研究。     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

A polymorphic antigen displaying different molecular weights in sheep and cattle sera

The polymorphic antigen Bl is present, exhibiting different molecular weights, in both sheep and cattle sera. The molecular weight of the antigen, purified from both sera by a specific immunoabsorbent, was analysed by SDS polyacrylamide gel electrophoresis and calibrated gel filtration. Results indicate that only one subunit, 35 000 MW, forms cattle Bl while a further subunit, 60 000 MW, is present in sheep Bl. The antigen activity is localized on the 35 000 MW subunit, which appears to be the same in both species.     

Demonstration of calmodulin-stimulated phosphatase isozymes by monoclonal antibodies

A procedure combining immunoprecipitation and immunotransblot employing subunit-specific monoclonal antibodies of the brain phosphatase, VJ6 and VA1, was used on tissues including heart, muscle, lung, spleen, pancreas, uterus, and liver. The various tissue extracts were subjected to immunoprecipitation by the beta subunit-specific VA1-immunoabsorbant, the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunotransblot, using both the alpha and beta subunit-specific antibodies VJ6 and VA1, respectively. Protein bands corresponding to alpha and beta subunits and the immunostain of beta subunit were detected in all samples, whereas alpha subunit was strongly stained only in the brain extract, weakly in heart and muscle extracts, and essentially negatively in all the other samples. In contrast, a polyclonal antiserum of bovine brain calmodulin-stimulated phosphatase could immunostain both alpha and beta subunits from all tissues. Calmodulin-binding protein fractions from a number of bovine tissues were all shown to contain the immunoprecipitable alpha subunit, as well as calmodulin-stimulated p-nitrophenylphosphatase activity. Micropeptide mapping showed that alpha subunits of bovine brain and bovine lung calmodulin-stimulated phosphatase isozymes were distinct molecular species. These results provide direct evidences for the existence of calmodulin-stimulated phosphatase isozymes in mammalian tissues.     

Photoaffinity cross-linked A1 adenosine receptor-binding subunits. Homologous glycoprotein expression by different tissues

Mammalian A1 adenosine receptor-binding peptides can be visualized by covalently labeling them with the photoaffinity cross-linking ligand N6-2-(4-amino-3-[125I] iodophenyl)ethyladenosine followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. The proteins comprising the A1 adenosine receptor-binding subunit of rat brain and fat migrate with Mr 38,000. In this study, the glycoproteins representing the radiolabeled A1 adenosine receptor-binding subunit expressed in each of these tissues (brain and fat) were compared through the use of peptide mapping and exo- and endoglycosidase treatments. Peptide mapping studies with several enzymes demonstrate that the protein component of the radiolabeled A1 adenosine receptor-binding subunit is conserved between different tissues. Both labeled receptor peptides demonstrate a sensitivity to neuraminidase as evidenced by increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the receptors contain complex-type carbohydrate chains. Insensitivity to alpha-mannosidase suggests a lack of high mannose-type carbohydrate chains. Deglycosylation of the labeled receptor-binding subunits with endoglycosidase F results in a single labeled polypeptide of Mr 32,000 for both systems. These data suggest that the A1 adenosine receptor-binding subunits expressed in the rat brain and fat are similar glycoproteins as evidenced by similar overall molecular weights, identical peptide maps, and equivalent responses to endo- and exoglycosidase treatment.     

Studies on muscle differentiation. IV. Electrophoretic and immunochemical analyses of tropomyosin from adult and embryonic skeletal muscles

Tropomyosin preparations from skeletal muscles of the adult frog, chick and rabbit were resolved in 8 M urea-polyacrylamide gel electrophoresis into at least 5 to 6 components. Of these, main components clearly reacted with anti-frog tropomyosin antiserum in agar diffusion test. Especially, main components of the frog tropomyosin preparation contained both genus- and organ-specific and genus- and organ-nonspecific antigens without being differentiated into separate entities. The chick tropomyosin preparation formed a single band when thioglycolic acid was included in 8 M urea-polyacrylamide gel electrophoresis. This single component was revealed in an SDS-polyacrylamide gel electrophoresis to be monomeric tropomyosin subunit with a molecular weight of 34,000. Both adult and embryonic chick tropomyosin preparations in their course of purification were observed in 8 M urea-polyacrylamide gel electrophoresis to decrease in amount of the monomeric component with a concomitant increase in number and in amount of polymerized components. It was concluded that the monomeric subunit was the major form of tropomyosin molecules in both adult and embryonic skeletal muscle extracts of the chick and that the polymerized components with inter-subunit disulfide bonds were formed in the course of purification of the preparations.     

Immunoreactivity and ouabain-dependent phosphorylation of (Na+ + K+)-adenosinetriphosphatase catalytic subunit doublets

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 6% polyacrylamide was used to resolve the 100-kDa catalytic (alpha subunit) polypeptide of (Na+ + K+)-adenosinetriphosphatase from various tissues. The catalytic subunit was identified on immunoblots with antisera against mouse brain catalytic subunit and lamb kidney holoenzyme. Immunoblots and Coomassie Blue-stained companion gels showed double species of the 100-kDa subunit in sucrose gradient fractions of mouse brain and kidney, bovine grey and white matter, purified lamb kidney and duck salt gland holoenzyme, electroplax microsomes, and NaI-extracted microsomes of goldfish and rat brain. The apparent molecular mass differences between the two species in each tissue all ranged between 5 and 8 kDa. Both forms in rat brain and lamb kidney enzyme contain common epitopes reactive with antibodies immunoaffinity-purified on either species from mouse brain. In addition, ouabain-dependent acid-stable inorganic phosphate incorporation was tested with mouse brain, lamb kidney, and electroplax enzyme. Ouabain-dependent phosphorylation was demonstrated in both species in lamb kidney and electroplax and in the larger of the two forms in mouse brain. These results suggest that double species of the phosphorylatable subunit are present generally in epithelial as well as excitable tissues and in fish and avian as well as mammalian species. Work is needed to elucidate their qualitative and quantitative characteristics in different tissues.     

A New Metabolite from Aspergillus versicolor

Neurospora sitophila produced extracellular and cell wall-associated lectins. The addition of l-sorbose to a culture resulted in a decrease in the production of the former lectin and complete abolition of the latter. The lectin in the culture filtrate was purified by bovine submaxillary mucin-conjugated Sepharose chromatography. The molecular weight of the lectin was calculated to be approx. 40,000 by Sephacryl S-200 gel filtration, and that of the subunit to be approx. 22,000 by SDS/polyacrylamide- gel electrophoresis. The lectin was not inhibited by simple sugars or their homopolymers. It was inhibited strongly by glycoproteins from human erythrocyte membrane and bovine submaxillary mucin, and moderately by α1-acid glycoprotein from human plasma, human IgA and IgM, and fetal calf fetuin. The lectin agglutinated human type A, B and O erythrocytes to the same degree. Erythrocytes from chick, horse, rabbit and sheep were more efficiently agglutinated.     

Chalcone synthases from spinach (Spinacia oleracea L.)

The two chalcone-synthase forms from leaves ofSpinacia oleracea L. were purified to apparent homogeneity. Antibodies were raised against both proteins in rabbits. The specificity of the antibodies was tested using immunotitration, immunoblotting, and immunoelectrophoresis techniques. The antibodies exhibited exclusive specificity for chalcone synthase and did not discriminate between the two antigens. The homodimeric chalcone synthases had the same subunit molecular weight but differed in their apparent native molecular weights. The peptide maps indicated extensive homology between the proteins. Chalcone-synthase activity was not detected in isolated spinach chloroplasts. Both enzyme forms were present in spinach cell-suspension cultures in which they were induced by light.Abbreviations DEAE diethylaminoethyl - DTE 1,4-dithioerythritol - EDTA ethylenediaminetetraacetic acid - HPLC high-performance liquid chromatography - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Parts of the results were presented at the 14^th International Botanical Congress at Berlin in July 1987     

Site specific phosphorylation of cytochrome c oxidase subunits I, IVi1 and Vb in rabbit hearts subjected to ischemia/reperfusion

We have mapped the sites of ischemia/reperfusion-induced phosphorylation of cytochrome c oxidase (CcO) subunits in rabbit hearts by using a combination of Blue Native gel/Tricine gel electrophoresis and nano-LC-MS/MS approaches. We used precursor ion scanning combined with neutral loss scanning and found that mature CcO subunit I was phosphorylated at tandem Ser115/Ser116 positions, subunit IVi1 at Thr52 and subunit Vb at Ser40. These sites are highly conserved in mammalian species. Molecular modeling suggests that phosphorylation sites of subunit I face the inter membrane space while those of subunits IVi1 and Vb face the matrix side.     

Isolation and characterization of flavin-linked glycerol-3-phosphate dehydrogenase from rabbit skeletal muscle mitochondria and comparison with the enzyme from rabbit brain

Mitochondrial glycerol-3-P dehydrogenase (EC 1.1.99.5) has been purified in 20% yield from both rabbit skeletal muscle and brain using a four step procedure involving osmotic shock, solubilization with Triton X-100, hydrophobic chromatography, gel filtration, and preparative column isoelectrofocusing. The active muscle and brain enzymes were found to be 95% and 80% homogeneous, respectively. Final purification was performed on the denatured subunit. The active enzyme from each of the tissues focused at pH 5.25 +/- 0.12 and each produced similar biphasic thermal inactivation plots at 50 degrees C. Mixtures of the purified brain and muscle enzymes co-migrated in discontinuous electrophoresis gels and each enzyme exhibited a single polypeptide component on sodium dodecyl sulfate (SDS) gels either when run separately or in mixtures. The subunit molecular weight was shown to be 76,000 +/- 3,000 by SDS-gel electrophoresis and gel filtration in 6 M guanidine HCl. One mole of noncovalently bound FAD and 1 mole of iron were measured per Mr = 100,000. The amino acid composition was determined based on the assumption of 70 aspartate residues per subunit to give a Mr = 76,000. The absorption spectrum has a maximum at 416 nm and a shoulder at 450 to 460 nm which is bleached on treatment with sodium dithionite. The maximum at 416 nm is removed by treatment with mersalyl.     

Subunit structure of γ-conglycinin in soybean seeds

Dissociated subunits of purified γ-conglycinin were isolated on a DEAE-Sephadex A-50 column. A single band was seen on two kinds of gel electrophoresis and isoleucine was shown as the only N-terminal amino acid. The isolated subunit reacted with antisera to the native γ-conglycinin. The M_r of the subunit was 51 000–51 500 estimated by urea-acetic acid and SDS-urea gel electrophoresis. A value of 50 000 was obtained by gel filtration with guanidine-hydrochloric acid on Sepharose CL-6B. The γ-conglycinin molecule was found to be made up of three subunits. This was determined by cross-linking the subunits and then submitting them to gel electrophoresis. Differences and similarities of subunit structure among γ-conglycinin, β-conglycinin and glycinin are discussed.     

The Preparation of a Cellulose-like Polymer from 2,3,6-Tri-O (N-pheny1carbamyl)-D-glucopyranose by the Action of Phosphorus Pentoxide in Dimethyl Sulfoxide

Starch phosphorylase was purified from either freshly harvested or stored roots of sweet potato (.Ipomoea batatas (L.) Lam. cv Tain on 65). Both enzyme preparations in their native state showed on polyacrylamide gel electrophoresis a cluster of about six closely located activity bands, which had common antigenic determinants as they were simultaneously probed by monoclonal antibodies. The molecules of enzymes from stored roots were smaller than those from fresh roots. However, the two enzyme preparations had completely fused precipitin lines in double diffusion assays with an antiserum raised against the fresh root preparation. One large subunit and several small ones were found for both enzyme preparations. The small subunits appeared to be the degradation products of the large ones as revealed by peptide mapping and immunoblotting. Immunofluorescence microscopy showed that the enzyme was present in the amyloplasts and cell walls of root storage parenchyma.     

Identification of different quaternary structures of beef heart cytochrome-c oxidase by two-dimensional polyacrylamide gel electrophoresis

A two-dimensional gel electrophoresis is described to identify different quaternary structures of the heart cytochrome-c oxidase. Bovine enzyme was purified and separated by discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions in the 1st dimension into several discrete complexes and thereupon shown to be heterodisperse in Triton X-100 and dodecyl maltoside. A discontinuous SDS-polyacrylamide gel electrophoresis in the 2nd dimension was used to determine the subunit composition of the isolated complexes. One of these represents the intact enzyme with 12 different polypeptides while the others have an incomplete subunit composition.     

Activation of turkey erythrocyte adenylate cyclase and blocking of the catecholamine-stimulated GTPase by guanosine 5'-(gamma-thio) triphosphate.

By SDS-polyacrylamide gel electrophoresis, mitochondrial proteins having covalently-bound flavin were analyzed. Mitochondria were prepared from the liver of rat injected with radioactive riboflavin. Radioactivity was found to be associated with four protein components. Their subunit molecular weights were 91,000, 72,000, 60,000 and 44,000. The first two components exhibited yellowish fluorescence on a gel under ultraviolet illumination. The component of the highest molecular weight seems to be a new protein containing covalently-bound flavin.     

聚合酶链反应技术检测禽网状内皮组织增殖病病毒

目的建立聚合酶链反应(PCR)技术检测禽网状内皮组织增殖病病毒(REV)的方法。方法提取感染REV-T和脾坏死病毒(SNV)的SPF鸡胚成纤维细胞DNA为模板,利用前病毒长末端重复序列(LTR)区引物进行扩增。采集肿瘤病鸡,以及人工感染REV 28 d后鸡肝脏、脾脏、肾脏、心脏、胸腺、法氏囊等器官,进行扩增。同时将采集的脏器组织,进行HE染色和免疫组化试验(IHC)。结果REV-T感染的组织未检测出电泳条带,而SNV感染的细胞中检测到了一条300bp特异而清晰的电泳条带,而且SNV感染的鸡组织中,PCR方法检测到了特异的条带。通过HE染色和免疫组化技术观察到了肿瘤组织,肿瘤细胞的形态、分布。结论PCR检测REV更快捷,特异更好。     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.