scholarly journals Partial purification of a dicyclohexylcarbodi-imide-sensitive membrane adenosine triphosphatase complex from the obligately anaerobic bacterium Clostridium Pasteurianum

1976 ◽  
Vol 154 (3) ◽  
pp. 725-729 ◽  
Author(s):  
D J. Clarke ◽  
J. G Morris
Keyword(s):  
Adenosine Triphosphatase ◽  
Anaerobic Bacterium ◽  
Partial Purification ◽  
Clostridium Pasteurianum ◽  
Obligately Anaerobic ◽  
Triton X ◽  
Sensitive Membrane

The membrane adenosine triphosphatase complex of vegetatively growing Clostridium pasteurianum, solublized with Triton X-100, has been recovered as a significantly purified particulate preparation that is still sensitive to inhibition by dicyclohexylcarbodiimide and butyricin 7423.

scholarly journals Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum.

1977 ◽  
Vol 75 (1) ◽  
pp. 135-147 ◽  
Author(s):  
A L Blitz ◽  
R E Fine ◽  
P A Toselli
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Ions ◽  
Adenosine Triphosphatase ◽  
Sodium Dodecyl ◽  
Maximal Activity ◽  
Coated Vesicles ◽  
Potassium Oxalate ◽  
Triton X ◽  
Trapping Agent ◽  
The Brain

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.

Evolution of vacuolar H+-ATPases: immunological relationships of the nucleotide-binding subunits

1989 ◽  
Vol 67 (6) ◽  
pp. 306-310 ◽  
Author(s):  
Morris F. Manolson ◽  
Judith M. Percy ◽  
David K. Apps ◽  
Xiao-Song Xie ◽  
Dennis K. Stone ◽  
...  
Keyword(s):  
Anaerobic Bacterium ◽  
Common Ancestor ◽  
Sequence Data ◽  
Structural Features ◽  
Eukaryotic Cells ◽  
Clostridium Pasteurianum ◽  
Chromaffin Granules ◽  
Α Subunit ◽  
Evolutionary Origins ◽  
Vacuolar Membranes

The evolution of the endomembrane systems of eukaryotic cells can be examined by exploring the evolutionary origins of the endomembrane H+-ATPases. Recent studies suggest that certain polypeptides are common to all H+ pumps of this type. Tonoplast H+ -ATPase from Beta vulgaris L. was purified and antibodies raised to two of its subunits. Each of these antisera reacted with a polypeptide of the corresponding size in bovine chromaffin granules, bovine clathrincoated vesicles, and yeast vacuolar membranes, suggesting common structural features and a common ancestor for endomembrane H+-ATPases of different organelles and different kingdoms. The antiserum raised against the 57-kDa polypeptide of plant tonoplast H+ -ATPase also reacted with subunit "a" of the H+-ATPase from the obligately anaerobic bacterium Clostridium pasteurianum and to the α subunit of the H+ -ATPase from Escherichia coli. There was no reactivity with chloroplast or mitochondrial ATPases. These results are discussed in relation to recent sequence data which suggest that endomembrane H+-ATPases may be evolutionarily related to the F0F1 ATPases.Key words: H+ -ATPase, evolution, immunology, vacuole, endomembrane.

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.

Partial purification and properties of the TSH receptors from human thyroid plasma membranes

1983 ◽  
Vol 103 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Yasuhiro Iida ◽  
Junji Konishi ◽  
Kanji Kasagi ◽  
Keigo Endo ◽  
Takashi Misaki ◽  
...  
Keyword(s):  
Sodium Acetate ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Human Thyroid ◽  
Partial Purification ◽  
Affinity Column ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Con A ◽  
Triton X

Abstract. Human thyroid plasma membranes were solubilized with 0.5% Triton X-100 and TSH receptors were purified by using DEAE-Sephadex, Con A and TSH affinity chromatography. A TSH binding activity was bound to DEAE-Sephadex equilibrated with 0.05 m sodium acetate, pH 6.3, 0.2% Triton X-100 and was eluted by a linear gradient of 0.1 m to 1.0 m ammonium acetate, pH 6.3. Eighty-five per cent of the activity was absorbed to Con A Sepharose and was eluted with 0.5 m α-methyl-D-mannoside, 0.05 m sodium acetate, pH 6.0. Seventy-five per cent of the TSH binding capacity could be absorbed to TSH-affinity column and was eluted with 0.1 m glycine-HCl, pH 3.0. By sequential application of the above procedures, more than 100-fold purification of the receptor activity was attainable. [125I]TSH binding of this fraction was inhibited by addition of unlabelled TSH in a dose-dependent manner. Scatchard analysis gave a curvilinear plot with a high affinity association constant of 0.72 × 109 m−1. By using Ultrogel AcA 34 gel filtration, the molecular size of the hormonereceptor complex was estimated to be 180 000.

scholarly journals Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

1980 ◽  
Vol 191 (1) ◽  
pp. 117-124 ◽  
Author(s):  
R Zecher ◽  
H U Wolf
Keyword(s):  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Half Maximum ◽  
Purification And Characterization ◽  
Dimeric Molecule ◽  
Maximum Inhibition ◽  
Optimum Ph ◽  
Triton X

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

scholarly journals Ring-and N-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system

1975 ◽  
Vol 150 (3) ◽  
pp. 561-564 ◽  
Author(s):  
P D Lotlikar ◽  
K Zaleski
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Enzyme System ◽  
Partial Purification ◽  
Hydroxylated Metabolites ◽  
Triton X ◽  
Cytochrome P 450 ◽  
Ring Hydroxylation ◽  
Hydroxylation Activity ◽  
Nadph Cytochrome C Reductase

The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions.

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