Purification and some properties of the glutamate dehydrogenase of Salmonella typhimurium

1973 ◽  
Vol 19 (4) ◽  
pp. 427-438 ◽  
Author(s):  
J. W. Coulton ◽  
M. Kapoor
Keyword(s):  
Salmonella Typhimurium ◽  
Glutamate Dehydrogenase ◽  
Ammonium Sulfate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ammonium Sulfate Fractionation ◽  
Sulfate Fractionation

NADP-specific glutamate dehydrogenase (GDH) from Salmonella typhimurium was purified 190-fold by heat treatment, ammonium sulfate fractionation, DEAE-Sephadex chromatography, reverse ammonium sulfate fractionation, and gel filtration. The enzyme proved to be stable to 55 °C, and displayed a pH optimum at 8.6 in the amination reaction. The sedimentation coefficient of GDH, as determined by sucrose density gradient centrifugation, was about 10.3 S. From gel filtration chromatography, the molecular weight and Stokes' radius for the enzyme were estimated at 280 000 daltons and 54 × 10−8 cm, respectively. Unusual resistance was displayed by the enzyme to high concentrations of the protein denaturants, urea, SDS, and guanidine hydrochloride.

scholarly journals BACTERICIDAL SUBSTANCE FROM STAPHYLOCOCCUS AUREUS

1970 ◽  
Vol 131 (5) ◽  
pp. 1004-1015 ◽  
Author(s):  
Adnan S. Dajani ◽  
Ernest D. Gray ◽  
Lewis W. Wannamaker
Keyword(s):  
Staphylococcus Aureus ◽  
Ammonium Sulfate ◽  
Phage Type ◽  
Gel Filtration ◽  
Considerable Degree ◽  
Ammonium Sulfate Fractionation ◽  
Sulfate Fractionation ◽  
Extracellular Products ◽  
Group Ii ◽  
Degree Of Purification

A bactericidal substance previously isolated from phage type 71 Slaphylococcus aureus has been further identified and characterized. Staphylococci belonging to phage type 71 produce the substance in higher titers than staphylococci lysed by other phages in group II in addition to phage 71. Other staphylococci do not produce the bactericidal substance. The bactericidal substance shares several of the properties of bacteriocins but differs from this group of antibiotic substances in some respects. A combination of ammonium sulfate fractionation and gel filtration on a Sephadex G-100 column resulted in considerable degree of purification of the bactericidal substance. The substance is a previously unrecognized product of S. aureus and is distinct from other extracellular products of this organism.

Purification and characterization of a lytic peptidase produced by Bdellovibrio bacteriovorus 6-5-S

1973 ◽  
Vol 19 (5) ◽  
pp. 659-666 ◽  
Author(s):  
H. B. Fackrell ◽  
J. Robinson
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Culture Fluid ◽  
Diaminopimelic Acid ◽  
Lytic Enzyme ◽  
Bdellovibrio Bacteriovorus ◽  
Ammonium Sulfate Fractionation ◽  
Sulfate Fractionation

Bdellovibrio bacteriovorus 6-5-S releases at least two enzymes into the culture fluid when grown on autoclaved cells of Spirillum serpens VHL. One of the enzymes is bacteriolytic and the other is proteolytic. The lytic enzyme was purified by a factor of 3000 using ammonium sulfate fractionation, Sephadex G-100 gel filtration, and the anion exchanger DEAE-Sephadex. The lytic enzyme degrades peptidoglycan of S. serpens by hydrolyzing the diaminopimelic acid – alanine bond in the tetrapeptide chain. Ca2+ or Mg2+ exerted little or no effect on the activity of the lytic enzyme. The enzyme had a molecular weight of 40 000 as indicated by gel filtration. Crude preparations were unstable at 4C. The second enzyme, a protease that digested Azocoll, was purified by a factor of 7 by ammonium sulfate fractionation followed by gel filtration with Sephadex G-100. The protease was eluted in the void volume from a Sephadex G-100 column and therefore may have a molecular weight of at least 100 000. Its activity was enhanced by additions of Ca2+ and Mg2+. The enzyme was stable at 4C.

Molecular characteristics of cytostatic factors in amphibian egg cytosols

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 799-808
Author(s):  
E.K. Shibuya ◽  
Y. Masui
Keyword(s):  
Density Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Molecular Characteristics ◽  
Small Peptides ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytostatic Factor

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated ‘cytostatic factor, (CSF)’. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20–30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 × 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.

scholarly journals Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes

1975 ◽  
Vol 145 (2) ◽  
pp. 225-232 ◽  
Author(s):  
R K Craig ◽  
H M Keir
Keyword(s):  
Dna Polymerase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Dna Polymerase I ◽  
Sucrose Density Gradient Centrifugation ◽  
Molecular Weights ◽  
Molecular Asymmetry ◽  
Polymerase I ◽  
Dna Polymerase Ii

Nuclear and cytoplasmic fractions were prepared from exponentially-growing BHK-21/C13 cells; DNA polymerase was extracted from them and analysed by gel filtration and sucrose-density-gradient centrifugation. DNA polymerase I is heterogeneous comprising species covering a considerable range of molecular weights. These have been tentatively identified as four subspecies of apparent molecular weights 900000-1000000 (IA), 460000-560000 (IB), 270000-320000 (IC) and 140000-200000 (ID), as assessed by gel filtration through Sepharose 6B. DNA polymerase II has a mol.wt. of 46000 +/- 4000 as assessed by gel filtration on Sepharose 6B, and 48000 +/- 2000 as assessed by gel filtration on Sephadex G-100. Sedimentation analyses on sucrose density gradients showed that the DNA polymerase I species had sedimentation coefficients predominantly in the range 6-8 S. DNA polymerase II had predominantly a sedimentation coefficient of 3.2 S although a component with lower sedimentation coefficient was found. The lack of correlation between the molecular weights derived from gel filtration and the sedimentation coefficients is attributed to molecular asymmetry. DNA polymerase I was found to be associated predominantly with the cytoplasm although certain types of nuclear preparation contained large amounts of it. DNA polymerase II was found to be mostly if not exclusively in nuclear preparations.

PRELIMINARY CHARACTERIZATION OF AN ANDROGEN-MACROMOLECULAR COMPLEX FROM THE RAT VENTRAL PROSTATE

1969 ◽  
Vol 62 (1) ◽  
pp. 153-164 ◽  
Author(s):  
Olav Unhjem ◽  
Kjell J. Tveter ◽  
Asbjørn Aakvaag
Keyword(s):  
Proteolytic Enzymes ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Ventral Prostate ◽  
Macromolecular Complex ◽  
Sucrose Density Gradient Centrifugation

ABSTRACT Following administration of (1,2-3H)-testosterone to castrated rats or incubation of prostatic tissue with the same steroid, a gel filtration technique has been used for the isolation of a soluble steroid-macromolecular complex from the tissues. Subsequent steroid analyses revealed that 5α-androstan-17β-ol-3-one was the major component associated with the macromolecules both in the in vivo and by in vitro experiments. The complex is destroyed by proteolytic enzymes like trypsin and pronase, but is unaffected by DNase and RNase. The complex is excluded from G-200 as well as P-300 gel beds. By sucrose density gradient centrifugation two macromolecular components were found associated with radioactivity. The largest component had a sedimentation coefficient of 9.3 S and probably corresponds to the macromolecular complex demonstrated by gel filtration, whereas the smaller component had a sedimentation coefficient of 4.5 S and might represent an association of steroids with serum albumin.

scholarly journals Chemical cross-linking of chick oviduct progesterone-receptor subunits by using a reversible bifunctional cross-linking agent

1979 ◽  
Vol 181 (1) ◽  
pp. 201-213 ◽  
Author(s):  
M E Birnbaumer ◽  
W T Schrader ◽  
B W O'Malley
Keyword(s):  
Progesterone Receptor ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Cross Linking ◽  
Sucrose Density Gradient Centrifugation ◽  
Receptor Proteins ◽  
The Cross

Chick oviduct progesterone-receptor proteins were treated in cytosol with the reversible cross-linking reagent methyl 4-mercaptobutyrimidate. The product of the reaction was a 7S complex that could be detected and recovered after sucrose-density-gradient centrifugation in 0.3M-KCl. The extent of the reaction was dependent on the concentration of methyl 4-mercaptobutyrimidate and independent of the presence of bound hormone, since unlabelled receptors could also be cross-linked. The cross-linking reaction required conditions in which the cytosol 6S complex was preserved. A Stokes radius of 7.3 nm was determined by gel filtration in Agarose A-1.5 m in 0.3 M-KCl. The sedimentation coefficient, which was also determined in 0.3 M-KCl, allowed us to calculate a mol. wt. of 228,000. We were also able to cross-link partially purified receptor forms isolated by using an Agarose A-15 m column. On reduction with beta-mercaptoethanol the complex broke down to 4S monomers that were identified by DEAE-cellulose and phosphocellulose chromatography, adsorption on DNA-cellulose and gel filtration in an Agarose A-1.5 m column. In most cases, A and B receptor proteins were released in equivalent amounts, implying that the cross-linked form was an A-B complex.

scholarly journals Physical measurements of the liver glucocorticoid receptor

1978 ◽  
Vol 169 (2) ◽  
pp. 445-448 ◽  
Author(s):  
G Litwack ◽  
M H Cake ◽  
R Filler ◽  
K Taylor
Keyword(s):  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Axial Ratio ◽  
Steroid Receptor ◽  
Sucrose Density Gradient Centrifugation ◽  
Uniform Size ◽  
Receptor Complexes ◽  
Physical Measurements

Physical measurements were made on the cytosolic form of the liver [3H]dexamethasone receptor. These include a Stokes radius of 3.5 nm, determined by gel filtration, and sedimentation coefficients of 5.1 and 7-8S, by sucrose-density-gradient centrifugation. From these measurements, the following physical properties were calculated: apparent mol. wt. 78000 (the 5.1 S form); D app. 6.1 × 10(-7) cm2-S-1; f/fo 1.25; axial ratio 4.7; these indicate a globular protein. Measurements of sedimentation coefficient of cytosol steroid-receptor complexes previously subjected to various activating conditions gave different values and lead to the conclusion that the mechanism of activation in vitro enabling the steroid-receptor complex to bind to DNA is more complex than simple disaggregation to a uniform size.

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