Separation of proteins by ammonium sulfate gradient solubilization

Tp, King

doi:10.1021/bi00753a010

Cited by 141 publications

(41 citation statements)

References 4 publications

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“…The precipitate was mixed with a suspension of Celite 545 in buffer-A containing 95% saturation of ammonium sulfate solution (buffer-B), and was subjected to subsequent salting-out column chromatography. 21) The suspension was gently packed into a column (4:7 Â 25 cm), and then washed with buffer-B. The enzyme was eluted by reverse gradient of 95-0% saturation of ammonium formed by buffer-B and buffer-A.…”

Section: Resultsmentioning

confidence: 99%

Purification and Characterization of α-Glucosidase I from Japanese Honeybee (Apis cerana japonica) and Molecular Cloning of Its cDNA

Wongchawalit

Yamamoto

Nakai

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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α-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 °C and in a pH-range of 4.5–10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl α-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of α-amylase family enzymes. Western honeybees (Apis mellifera) had three α-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I

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Section: Resultsmentioning

confidence: 99%

Purification and Characterization of α-Glucosidase I from Japanese Honeybee (Apis cerana japonica) and Molecular Cloning of Its cDNA

Wongchawalit

Yamamoto

Nakai

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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“…The pooled fractions (55 1) were concentrated by ultrafiltration to a final volume of 1 1. To the concentrated pool 200 g of dry celite were added, and the suspension was made 80% saturated in ammonium sulfate (reverse ammonium sulfate chromatography according to Kind [18]). After 30 min of equilibration the suspension was filled into a column ( 9 x 9 cm), washed with a column volume of 8Oo/l, saturated ammonium sulfate solution and eluted with 100 1 of a linear gradient falling from 80 to 0% ammonium sulfate.…”

Section: Purijcatinnmentioning

confidence: 99%

The Malate Dehydrogenase Isoenzymes of Saccharomyces cerevisiae. Purification, Characterisation and Studies on Their Regulation

1978

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1. One mitochondrial and one cytoplasmic malate dehydrogenase isoenzyme could be purified from acetate grown cells of the yeast Saccharomyces cerevisiae. 2. The purification procedure uses chromatography on dextran blue columns as an essential step for enrichment, and reverse ammonium sulfate chromatography on celite for isoenzyme separation. 3. The homogeneity of the preparations was established by gel electrophoreses in the presence of sodium dodecylsulfate and by a sedimentation run in the analytical ultracentrifuge. 4. Both enzymes are dimers with a molecular weight of 75 000 for the cytoplasmic and of 68 000 for the mitochondrial enzyme. 5. Amino acid analysis and peptide mapping showed that both enzymes are closely related, but genetically different (true isoenzymes). 6. The cytoplasmic enzyme shows electrophoretic splitting. This is most likely due to post‐translational deamination in vivo. 7. Antibodies to both isoenzymes could be obtained in rabbits. The antisera to cytoplasmic malate dehydrogenase were specific for this enzyme. Antisera to mitochondrial malate dehydrogenase react with both isoenzymes. Neither type of antisera precipitated an inactive protein after the glucose‐dependent inactivation of cytoplasmic malate dehydrogenase in vivo.

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“…The instructions given in the instruction manual of the supplier of the device were followed, with the exception that the upperelectrode solution was omitted. The King (1972). Concentration of protein solutions was carried out with the Amicon ultrafiltration system TCF-10 (Amicon B.V., Oesterhout N.B., Holland) with UM 20E membranes.…”

Section: Column Chromatographymentioning

confidence: 99%

Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues

Mäkinen¹,

Mäkinen²

1978

Biochemical Journal

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Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex C-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAEcellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphthylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl-concentration (0.15M). The molecular weights (by gel filtration) of enzymes I and II were 85000 and 52500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2'+ which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl--activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

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Separation of proteins by ammonium sulfate gradient solubilization

Cited by 141 publications

References 4 publications

Purification and Characterization of α-Glucosidase I from Japanese Honeybee (Apis cerana japonica) and Molecular Cloning of Its cDNA

Purification and Characterization of α-Glucosidase I from Japanese Honeybee (Apis cerana japonica) and Molecular Cloning of Its cDNA

The Malate Dehydrogenase Isoenzymes of Saccharomyces cerevisiae. Purification, Characterisation and Studies on Their Regulation

Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues

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