scholarly journals Isolation of peroxidase isozymes from two flax genotypes by column chromatography

1976 ◽  
Vol 54 (11) ◽  
pp. 1180-1188 ◽  
Author(s):  
M. A. Fieldes ◽  
N. Bashour ◽  
C. L. Deal ◽  
H. Tyson
Keyword(s):  
Molecular Weight ◽  
Horseradish Peroxidase ◽  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Relative Mobility ◽  
Peroxidase Isozymes ◽  
Chromatographic Elution

Isolation of the four major peroxidase isozymes (isozymes 1, 2, 3, and 4) of two flax genotypes was achieved by modifying the procedure used by Shannon et al. (1966) for the isolation of horseradish peroxidase isozymes. The net positive and net negative charges of isozymes 1, 2, and 4 were different. Isozyme 3 resembled isozyme 4 in charge but differed in apparent molecular weight. The chromatographic elution profiles of both genotypes were the same. Anionic gel electrophoresis demonstrated that after isolation and repurification, relative mobility differences existed between the corresponding isozymes of the two genotypes for all four isozymes.

DIFFERENCES IN MOLECULAR WEIGHT BETWEEN CORRESPONDING ANIONIC PEROXIDASE ISOZYMES FROM TWO FLAX GENOTROPHS

1980 ◽  
Vol 22 (4) ◽  
pp. 529-534 ◽  
Author(s):  
H. Tyson ◽  
M. A. Fieldes
Keyword(s):  
Molecular Weight ◽  
Linear Regression ◽  
Linum Usitatissimum ◽  
Gel Electrophoresis ◽  
Relative Mobility ◽  
Anionic Peroxidase ◽  
Linum Usitatissimum L ◽  
Peroxidase Isozymes ◽  
Flax Genotrophs ◽  
Adult Plants

Anionic peroxidase isozymes from main stem tissues of adult plants of two flax (Linum usitatissimum L.) genotrophs were separated using acrylamide gel electrophoresis. A range of seven acrylamide concentrations was used for the gels, enabling the effect of gel concentration on relative mobility (Rm) to be examined. The regression of log (Rm) on gel concentration was linear for two of the four main isozymes found. Differences in linear regression slope between the L and S flax genotroph isozymes suggested genotroph differences in molecular weight.

Variation in goat fibre protein

1989 ◽  
Vol 40 (3) ◽  
pp. 675 ◽  
Author(s):  
DJ Tucker ◽  
AHF Hudson ◽  
A Laudani ◽  
RC Marshall ◽  
DE Rivett
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Wool Fibre ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Cashmere Goats

The proteins from a range of cashmere, mohair, angoratcashmere crossbred and wool fibre samples were extracted at pH 8 with 8 M urea containing dithiothreitol, and were then radiolabelled by S-carboxymethylation using iodo(2-14C) acetate. The proteins from each sample were examined by two dimensional polyacrylamide gel electrophoresis in which the separation in the first dimension was according to charge at pH 8.9 and in the second dimension according to apparent molecular weight in the presence of sodium dodecyl sulfate. After electrophoresis the proteins were detected by fluorography. Protein differences in keratin samples from some individual goats existed, although the overall protein patterns were similar. None of the differences were consistent with any one goat fibre type. The protein patterns obtained for fibre samples from individual cashmere goats showed some differences when compared to those found for commercial blends from the same country of origin, indicating that blending can mask any animal-to-animal variation. While the electrophoretic technique does not unequivocally distinguish between cashmere, mohair and angora/cashmere crossbred fibres it does differentiate between wool and goat fibres.

Zur Strahleninaktivierung von Ribonuclease

1966 ◽  
Vol 21 (3) ◽  
pp. 224-231 ◽  
Author(s):  
Horst Jung ◽  
Helga Schüssler
Keyword(s):  
Molecular Weight ◽  
Gamma Radiation ◽  
Enzymatic Activity ◽  
Column Chromatography ◽  
Apparent Molecular Weight ◽  
Ribonuclease A ◽  
Nitrogen Atmosphere ◽  
The Other ◽  
Radiation Action ◽  
Higher Doses

Ribonuclease A was irradiated in water with 60Co gamma radiation, and the products formed were separated according to their molecular weight by column chromatography on Sephadex G-50. When the irradiations are carried out in nitrogen atmosphere aggregation to dimers and higher polymers is observed. An appreciable fraction of this aggregated component retains enzymatic activity. At higher doses the enzymatic activity of the aggregates is inactivated at the same rate as monomer ribonuclease A. Irradiation in air yields two components; one is equivalent to that found in nitrogen atmosphere, the other has an apparent molecular weight of about 20 000 but contains no enzymatic activity. This last component is not observed when methionine is present during irradiation. In methionine containing solutions all inactivited ribonuclease molecules exist in the form of dimers and polymers. Irradiation in the dry state leads to the same result. Consequently, every model designed to describe the radiation action on ribonuclease has to consider the fact that in solution, in the presence of a protecting agent, and in the dry state the loss of enzymatic activity is always accompanied by aggregation to products with increased molecular weight.

scholarly journals Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes

1975 ◽  
Vol 151 (3) ◽  
pp. 685-697 ◽  
Author(s):  
M Letarte-Muirhead ◽  
A N Barclay ◽  
A F Williams
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Lentil Lectin ◽  
Polypeptide Chains

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.

scholarly journals Anomalous migration of leghaemoglobin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis

1978 ◽  
Vol 169 (1) ◽  
pp. 251-253 ◽  
Author(s):  
P Lehtovaara
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Relative Mobility ◽  
Concomitant Decrease ◽  
Helical Content

The estimate of the molecular weight of leghaemoglobin by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis is about 20% too low. This is due to an anomalously high limiting relative mobility. Leghaemoglobin binds 1.4 g of sodium dodecyl sulphate/g of protein with a concomitant decrease in the helical content from 71-72% to 49-51%.

scholarly journals Factor XI antigen and activity in human platelets

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1148-1156 ◽  
Author(s):  
GP Tuszynski ◽  
SJ Bevacqua ◽  
AH Schmaier ◽  
RW Colman ◽  
PN Walsh
Keyword(s):  
Molecular Weight ◽  
Electrophoretic Mobility ◽  
Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Single Band ◽  
Factor Xi ◽  
Specific Staining ◽  
Coagulant Activity ◽  
Plasma Factor

Washed platelets, contaminated with less than 0.20% plasma factor XI, were examined for the presence of factor XI antigen and activity. These platelets contained a factor-XI-like coagulant activity (0.67 +/- 0.11 U/10(11) platelets) that remained constant after successive washes. By means of indirect immunofluorescence, a monospecific antibody to factor XI showed specific staining of both normal platelets and platelets from patients deficient in plasma factor XI. Radiolabeled Triton extracts of washed platelets and labeled purified factor XI solutions were analyzed for factor XI antigen by Staph A immunoprecipitation analysis using antibody to purified plasma factor XI followed by SDS gel electrophoresis. On unreduced gels, the platelet material ran as a single band having an apparent molecular weight of 220,000 daltons, whereas purified plasma factor XI gave a single band at 160,000 daltons. On reduced gels, the platelet material analyzed as a single band at 52,000 daltons, whereas purified factor XI gave a single band of 80,000 daltons. Analysis of a partially purified factor XI preparation from platelets by immunoelectrophoresis revealed that the platelet preparation displayed a slightly lower cathodal electrophoretic mobility at pH 8.6 than did plasma factor XI and yet appeared to possess complete antigenic identity with plasma factor XI. These results indicate that platelets possess a form of factor XI that exists as a disulfide-linked 52,000-dalton tetramer in contrast to the plasma form that circulates as a 80,000-dalton disulfide-linked dimer.

scholarly journals Variability in the Apparent Molecular Weight of Inhibin

1985 ◽  
Vol 38 (3) ◽  
pp. 327 ◽  
Author(s):  
H WG Baker ◽  
LW Eddie ◽  
B Hudson ◽  
HD Niall
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Polyacrylamide Gel ◽  
Pituitary Cells ◽  
Response Curves

An in vitro bioassay based on suppression of GnRH-stimulated FSH secretion by pituitary cells in culture was used to monitor inhibin activity after dialysis, gel filtration or polyacrylamide gel electrophoresis of protein preparations from a variety of gonadal secretions and extracts under native and dissociating conditions. The suggestion that inhibin is a peptide of molecular weight less than 5000 was not confirmed. Although some fractions of low molecular weight suppressed FSH secretion, the amount of activity was low and the dose response curves were not parallel with a standard preparation of inhibin. Under most conditions, inhibin eluted with an apparent molecular weight of about 90 000. However, gel filtration of rete testis fluid protein in I M acetic acid resulted in elution of inhibin activity with a lower apparent molecular weight and with polyacrylamide gel electrophoresis in o� 1% (w/v) sodium dodecylsulfate, the apparent molecular weight was 30 000. It is concluded that inhibin is a protein which tends to aggregate and coelute with larger molecules.

scholarly journals The separation and characterization of marmoset kidney β-d-galactosidase and β-d-glucosidase

1977 ◽  
Vol 167 (3) ◽  
pp. 765-773 ◽  
Author(s):  
R J Pierce ◽  
R G Price
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Starch Gel Electrophoresis ◽  
Lysosomal Fraction ◽  
Glucosidase Activity ◽  
Starch Gel

beta-D-Galactosidase and beta-D-glucosidase activities were determined in homogenates of marmoset kidney by using the appropriate 4-methylumbelliferyl glycoside, beta-D-Galactosidase activity was separated into two main components by ion-exchange chromatography on DEAE-cellulose, starch-gel electrophoresis, isoelectric focusing and gel filtration on Sephadex G-200. One form designated A had a pI of 5.1, was loosely bound to DEAE-cellulose at pH7.0, remained near the origin on starch-gel electrophoresis at pH 7.0 and had an apparent molecular weight of 160000. The second beta-D-galactosidase component, designated B, was associated with the total beta-D-glucosidase activity, had a pI of 4.3, was firmly bound to DEAE-cellulose, migrated rapidly towards the anode on starch-gel electrophoresis and had an apparent molecular weight of 50000. The optimum pH values of beta-D-galactosidase A and B were 4.5 and 6.0 respectively. beta-D-Galactosidase A was activated by 0.1 M-NaC1 but the activity of the B form was inhibited by 1 M-NaC1 at pH 4.5. beta-D-galactosidase had a bimodal distribution, the A form being recovered in the lysosomal fraction whereas the B form was present in the soluble fraction, as was the major portion of the beta-D-glucosidase activity. The lysosomal and soluble forms were further characterized by DEAE-cellulose chromatography.

scholarly journals Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity

1976 ◽  
Vol 155 (3) ◽  
pp. 679-687 ◽  
Author(s):  
M J Crabbe ◽  
R D Waight ◽  
W G Bardsley ◽  
R W Barker ◽  
I D Kelly ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Substrate Specificity ◽  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Diamine Oxidase ◽  
Amine Oxidase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Lysine Vasopressin

1. Isoelectric focusing studies of human placental diamine oxidase showed the pI value of the active enzyme to be 6.5. This information was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH gradient elution and this, together with affinity chromatography on concanavalin A-Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration of diamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultracentrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx. 1.0 and 1.2g-atom respectively/70000mol.wt. unit. 5. The e.s.r. spectral intensity did not decrease nor did the spectral line shape change when excess of p-dimethylaminomethylbenzylamine was added to the enzyme. 6. Addition of K13CN to the enzyme eliminated the copper e.s.r. signal without affecting the manganese signal. 7. The placental enzyme therefore appears to differ from other amine oxidases in terms of its metal cofactor requirement, molecular weight and substrate specificity, and possible roles in vivo for this enzyme are discussed.

scholarly journals Preliminary characterization of peroxidase isozymes isolated from two flax genotrophs

1977 ◽  
Vol 55 (11) ◽  
pp. 1465-1473 ◽  
Author(s):  
M. A. Fieldes ◽  
C. L. Deal ◽  
H. Tyson
Keyword(s):  
Molecular Weight ◽  
Oxidase Activity ◽  
Indoleacetic Acid ◽  
Relative Mobility ◽  
Fresh Weight ◽  
Iaa Oxidase ◽  
Peroxidase Isozymes ◽  
Flax Genotrophs ◽  
Very High

Four peroxidase (EC 1.11.1.7) isozymes were isolated from each of two flax genotrophs. All four isozymes were glycoproteins and all exhibited indoleacetic acid (IAA) oxidase activity. The percentage purity of two of the isozymes was very high; these isozymes differed in percentage carbohydrate and in peroxidase and IAA oxidase specific activities. Three of the isozymes displayed molecular weight values of about 43 000; for the fourth, molecular weight was considerably higher. Corresponding isozymes from the genotrophs and from two other flax genotypes displayed molecular weight differences which corresponded to electrophoretic relative mobility differences. Enzyme yield per unit fresh weight was higher for one genotroph than the other, and the balance between peroxidase activity and IAA oxidase activity between the genotrophs was different.

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