scholarly journals PARTIAL PURIFICATION AND PHOSPHOTUNGSTATE SOLUBILIZATION OF BASAL BODIES AND KINETODESMAL FIBERS FROM TETRAHYMENA PYRIFORMIS

1973 ◽  
Vol 57 (3) ◽  
pp. 601-612 ◽  
Author(s):  
Robert W. Rubin ◽  
William P. Cunningham
Keyword(s):  
Basal Body ◽  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Tetrahymena Pyriformis ◽  
Partial Purification ◽  
Basal Bodies ◽  
Thin Sectioning ◽  
Triton X

Previously devised methods for the isolation of basal bodies from ciliate protozoans were found to be inadequate for chemical analysis. We have modified and expanded these procedures and developed a method which gives preparations containing mainly basal bodies and kinetodesmal fibers. This procedure involved fixation of cells in 30% ETOH followed by digitonin or Triton X-100 solubilization and homogenization with a Brinkmann Polytron. This is followed by sucrose gradient centrifugation. Negative staining and thin sectioning revealed these preparations to be substantially more pure than those of previous workers. It was also found that neutralized phosphotungstate (PTA) solubilized many of the components present in fixed Tetrahymena. Neutralized 1.0% PTA solubilized axonemes, cortical, axonemal, and basal body microtubules as well as kinetodesmal fibers. These results have been confirmed by both electron microscope observations and gel electrophoresis of 100,000 g supernatants of the PTA extracts. A solution of 0.1% PTA did not affect the fibers but did solubilize basal bodies. Running 1.0% PTA extracts from our basal body fractions on sodium dodecyl sulfate (SDS) polyacrylamide gels allowed us to tentatively identify the peptides of basal bodies and kinetodesmal fibers. The latter structures appear to consist of a single 21,000 mol wt peptide. These results also suggest that great caution should be taken in interpreting PTA images, especially of microtubules and axonemes.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

Solubilization and immunoprecipitation of 125I-labelled antigens from Plasmodium knowlesi schizont-infected erythrocytes using non-ionic, anionic and zwitterionic detergents

Parasitology ◽  
1984 ◽  
Vol 88 (1) ◽  
pp. 27-36 ◽  
Author(s):  
R. J. Howard ◽  
J. W. Barnwell
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Plasmodium Knowlesi ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Ionic Detergents ◽  
Class A ◽  
Anionic Detergents ◽  
Triton X

SUMMARYPlasmodium knowlesi malaria-infected erythrocytes were radio-iodinated and several non-ionic, anionic and zwitterionic detergents were compared in their capacity to extract the labelled membrane proteins. The use of these detergents for antigen identification was tested by immunoprecipitation, after addition of Triton X-100 to some detergent extracts, using hyperimmune monkey antiserum and protein A-Sepharose. 125I-labelled antigens were specifically immunoprecipitated with all detergents tested, including the anionic detergents sodium dodecyl sulphate (SDS), deoxycholate and cholate; the zwitterions Zwittergent-312 and -314, CHAPS and Empigen BB, as well as several non-ionic detergents. The SDS-polyacrylamide gel electrophoresis patterns of 125I-labelled antigens varied after extraction with different detergents, there being no consistent pattern for detergents of a particular class. A total of 14 125I-labelled antigens were identified, 11 of them using Triton X-100. Some minor antigens identified with Triton X-100 were immunoprecipitated in greater amount after extraction in other detergents. Most importantly, two antigens Mr 200000 and 180000 were detected only after extraction with deoxycholate or SDS.

Structural Analysis of Basal Bodies of The Isolated Oral Apparatus of Tetrahymena Pyriformis

1970 ◽  
Vol 6 (3) ◽  
pp. 679-700
Author(s):  
J. WOLFE
Keyword(s):  
Structural Analysis ◽  
Cell Membrane ◽  
Basal Body ◽  
Structural Integrity ◽  
Tetrahymena Pyriformis ◽  
Basal Plate ◽  
Basal Bodies ◽  
The Third ◽  
Ionic Detergent

The oral apparatus of Tetrahymena pyriformis was isolated using a non-ionic detergent to disrupt the cell membrane. The mouth consists largely of basal bodies and microfilaments. Each basal body is attached to the mouth by a basal plate which is integrated into the meshwork of microfilaments that confers upon the oral apparatus its structural integrity. Each basal body is composed of 9 triplet microtubules. Two of the 3 tubules, subfibres ‘A’ and ‘B’ are composed of filamentous rows of globules with a spacing of 4.5nm. The third tubule, subfibre ‘C’, is only one-third the length of the basal body.

scholarly journals Improved Method for DNA Extraction and Purification from Tetrahymena pyriformis

2019 ◽  
Vol 2 (2) ◽  
pp. 40
Author(s):  
Ezzouhra El Maaiden ◽  
Youssef El Kharrassi ◽  
Abdel Khalid Essamadi ◽  
Khadija Moustaid ◽  
Boubker Nasser
Keyword(s):  
Dna Extraction ◽  
Sodium Dodecyl ◽  
Model Organism ◽  
Tetrahymena Pyriformis ◽  
Pyrrolidine Dithiocarbamate ◽  
Restriction Enzyme Digestion ◽  
Chelex 100 ◽  
Extraction And Purification ◽  
Ammonium Pyrrolidine Dithiocarbamate ◽  
Triton X

Tetrahymena pyriformis (protozoa) is intensely investigated as a model organism, offering numerous advantages in comprehensive and multidisciplinary studies using morphologic or molecular methods. Since DNA extraction is a vital step of any molecular experiment, here a new mixed surfactant (Sodium dodecyl sulfate (SDS) 20%/Triton X-100) was adopted for effective DNA extraction from Tetrahymena pyriformis under an easy, fast protocol. The efficiency of this technique was then compared with three widely-used alternative techniques, namely the Chelex 100 matrix, Ammonium pyrrolidine dithiocarbamate (APD) complex and SDS–chloroform methods. DNA extraction was analyzed by pulsed-field gel electrophoresis, spectral measurement, fluorometry (Qubit), restriction enzyme digestion, and polymerase chain reaction. Data analysis revealed that the quantity and quality of the recovered DNA varied depending on the applied DNA extraction method. The new method (SDS 20%/Triton X-100) was the most efficient for extracting DNA from Tetrahymena pyriformis with high integrity and purity, affordable cost, less time, and suitability for molecular applications.

Purification and Characterization of a 3,17 β-Hydroxysteroid Dehydrogenase from Streptomyces hydrogenans

1979 ◽  
Vol 34 (7-8) ◽  
pp. 533-540 ◽  
Author(s):  
Helmut Duchmann ◽  
Lothar Träger
Keyword(s):  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Sedimentation Coefficient ◽  
Hydroxysteroid Dehydrogenase ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization

3,17 β-Hydroxysteroid dehydrogenase has been enriched and purified from cytosol of Streptomyces hydrogenans. After ammonium sulfate precipitation and filtration on Sephadex G-100 the enzyme was finally purified by preparative gel electrophoresis and DEAE-Sephadex A-50 chro­matography. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate gave a single band of mobility corresponding to molecular weight of 70 200 ± 2 500. 3 β-. 17 β- as well as 20 β-hydroxy steroids were dehydrogenated by the enzyme in the presence of NAD+. The dehydrogenation proceeded faster than the reduction of the corresponding ketosteroids in the presence of NADH. The enzyme does not accent NADP+ or NADPH as co-substrates. The apparent Km values were calculated to be 11 μᴍ for 5 α-dihydrotestosterone, 20 μᴍ for testosterone ana 68 μᴍ for epiandrosterone in the NAD+-driven reaction, 1.8 x 10-4 m for NADH+ and 1.9 x 10-4 ᴍ for NADH. The catalytic activity was influenced by the ratio of NAD+/ATP. The inhibition by ATP appears to be of a competitive type with respect to NAD+ (Ki 1.15 x 10-3 ᴍ).After sucrose gradient centrifugation in a preparative ultracentrifuge the enzyme sediments with 4.1 ± 0.1 S as estimated in comparison to other proteins of known sedimentation coefficient. The isoelectric point was determined to be 3.9 with the LKB preparative isoelectric focusing col­umn (pH 2-11) and 4.1 with the analytical flat bed polyacrylamide isofocusing (pH 3 - 5). The number of SH groups was determined to be 2 mol/mol enzyme. In the presence of 6 M urea the fig­ure inceases to 3 mol SH/mol enzyme. In the presence of an excess of p-chloromercuribenzoate the enzyme activity decreases only partially.

scholarly journals Histone composition of a chromatin fraction containing ribosomal deoxyribonucleic acid isolated from the macronucleus of Tetrahymena pyriformis

1978 ◽  
Vol 173 (1) ◽  
pp. 155-164 ◽  
Author(s):  
R W Jones
Keyword(s):  
Ribosomal Dna ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Deoxyribonucleic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetrahymena Pyriformis ◽  
Active Chromatin ◽  
Chromatin Fraction ◽  
Inactive Chromatin

The histone compositions of a chromatin fraction containing ribosomal DNA and of the remaining macronuclear chromatin of Tetrahymena pyriformis was analysed by gel electrophoresis. These chromatin fractions were used as models for transcriptionally active and inactive chromatin respectively. The extent of histone modification, as indicated by the distribution of histone between differently charged subspecies in acid-urea gels, is not grossly different in the two chromatin fractions. However, histone H1 is present, but may be differently modified in the two chromatin fractions. The histone/DNA ratio in ribosomal chromatin, measured after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of samples of chromatin, was found to be the same whether chromatin was extracted from growing or stationary organisms, and to be approx. 40% of this ratio in the remaining macronuclear chromatin. The implications of these results for the possible structure of transcriptionally active chromatin are discussed.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

scholarly journals The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans

1977 ◽  
Vol 167 (2) ◽  
pp. 509-512 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Partial Specific Volume ◽  
Triton X

N-Methylglutamate dehydrogenase, purified to a specific activity of 0.29 unit/mg of protein, gave one band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a molecular weight of 130 000. Enzyme-Triton complexes were found to have a partial specific volume of 0.73 cm3/g, suggesting that the protein binds less than 0.1 g of Triton/g of protein. A molecular weight for the intact enzyme in the presence of 1% (w/v) Triton X-100 of 550 000 suggested that the enzyme may be a tetramer.

scholarly journals Partial isolation and translation in vitro of messenger ribonucleic acid for phosphoenolpyruvate carboxykinase (guanosine triphosphate)

1976 ◽  
Vol 156 (3) ◽  
pp. 619-626 ◽  
Author(s):  
S M Tilghman ◽  
L M Fisher ◽  
L Reshef ◽  
F J Ballard ◽  
R W Hanson
Keyword(s):  
Gel Electrophoresis ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Messenger Ribonucleic Acid ◽  
Partial Isolation ◽  
Heterologous Cell ◽  
Rabbit Reticulocytes

1. mRNA was extracted from the livers of starved rats and incubated in a heterologous cell-free protein-synthesizing system derived from rabbit reticulocytes. The presence of newly synthesized phosphoenolpyruvate carboxykinase (GTP) was detected by immunoprecipitation with a specific antibody to the enzyme and analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The synthesis of the enzyme was dependent on the addition of rat liver RNA, whereas RNA isolated from rat spleen was inactive. If ovalbumin and anti-ovalbumin were used to form the immunoprecipitates, no radioactivity that migrated as phosphoenolpyruvate carboxykinase was detected. 3. The optimal concentrations of magnesium acetate and KCl for phosphoenolpyruvate carboxykinase synthesis were determined. 4. When polyribosomal RNA was separated by sucrose-gradient centrifugation, phosphoenolpyruvate carboxykinase mRNA migrated between 20 and 26 S in keeping with the high mol. wt. of the protein (85 000). 5. The presence of poly(A) in phosphoenolpyruvate carboxykinase mRNA was suggested by retention of mRNA activity on oligo(dT)-cellulose columns. 6. It was concluded that the cell-free synthesis of phosphoenolpyruvate carboxykinase can serve as a bioassay for intracellular phosphoenolpyruvate carboxykinase mRNA.

Xanthacin. A bacteriocin of Myxococcus xanthus f b

1974 ◽  
Vol 20 (2) ◽  
pp. 131-135 ◽  
Author(s):  
Howard D. McCurdy Jr. ◽  
Thomas H. MacRae
Keyword(s):  
Molecular Sieve ◽  
Density Gradient Centrifugation ◽  
High Titer ◽  
Gradient Centrifugation ◽  
Myxococcus Xanthus ◽  
Sodium Dodecyl ◽  
Uranyl Acetate ◽  
Partial Purification ◽  
Differential Centrifugation ◽  
Varied Size

An extremely stable, particulate, bacteriocin, namely xanthacin, has been found in Myxococcus xanthus f b after mitomycin C induction. Xanthacin is resistant to trypsin, protease type VI, RNase, DNase, sodium dodecyl sulfate (SDS), acetone, ether, ultraviolet irradiation, and autoclaving. A high-titer preparation was obtained after partial purification by pervaporative concentration, differential centrifugation, molecular sieve chromatography, and density gradient centrifugation. Electron microscopy of platinum-shadowed and uranyl acetate stained preparations revealed the presence of circular bodies of varied size which resembled membrane fragments.

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