Evidence for heterogeneity in the carbohydrate moieties of peroxidase isozymes from two environmentally induced flax genotrophs

1986 ◽  
Vol 64 (11) ◽  
pp. 2682-2687 ◽  
Author(s):  
Pierre-Richard Gaudreault ◽  
Hugh Tyson
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Concanavalin A ◽  
Posttranslational Modification ◽  
Stem Tissue ◽  
Carbohydrate Composition ◽  
Molecular Weights ◽  
Peroxidase Isozymes ◽  
Flax Genotrophs ◽  
Carbohydrate Chains

The corresponding isoperoxidases from the flax genotrophs L and S have different molecular weights. Utilizing affinity chromatography on Sepharose-bound concanavalin A, we have shown that this lectin has a stronger affinity for the isoperoxidases purified from S stem tissue than those from L. The presence of differences in the carbohydrate composition of L and S peroxidases was confirmed when it was observed that only S peroxidases were susceptible to digestion by endo-β-N-acetylglucosaminidase H. Glycoprotein-enriched fractions were then purified from L and S stem tissue. The results showed that most glycoproteins of S origin have higher molecular weights than their L counterparts. Certain glycoproteins were digested by endo-β-N-acetylglucosaminidase H only if they were of S origin, while others were digested regardless of their origin. In both cases, the original differences in molecular weight between L and S glycoproteins were eliminated. These results support our view that posttranslational modification at the level of the carbohydrate chains of the L and S peroxidases is the reason for their heterogeneity on polyacrylamide gels.

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.

scholarly journals Partial purification and characterization of chick-embryo prolyl 3-hydroxylase

1979 ◽  
Vol 183 (2) ◽  
pp. 303-307 ◽  
Author(s):  
K Tryggvason ◽  
K Majamaa ◽  
J Risteli ◽  
K I Kivirikko
Keyword(s):  
Molecular Weight ◽  
Chick Embryo ◽  
Ethylene Glycol ◽  
Affinity Chromatography ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Chick Embryo Extract

Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.

Molecular weight and net charge of peroxidase isozymes in F1 hybrids between L and S flax genotrophs

1982 ◽  
Vol 20 (9-10) ◽  
pp. 919-927 ◽  
Author(s):  
Hugh Tyson ◽  
Mary Ann Fieldes
Keyword(s):  
Molecular Weight ◽  
F1 Hybrids ◽  
Net Charge ◽  
Peroxidase Isozymes ◽  
Flax Genotrophs

scholarly journals Rabbit erythrocyte band 3: a receptor for staphylococcal alpha toxin

1980 ◽  
Vol 26 (4) ◽  
pp. 524-531 ◽  
Author(s):  
Indar Maharaj ◽  
Hugh B. Fackrell
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Affinity Chromatography ◽  
Concanavalin A ◽  
Hemolytic Activity ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Band 3 ◽  
Alpha Toxin ◽  
Rabbit Erythrocyte

Enzymes known to specifically cleave the band 3 component of the rabbit erythrocyte membrane were found to reduce both the hemolytic sensitivity to and the binding of the alpha toxin of Staphylococcus aureus. Lectins which bind to band 3 also inhibited the toxin. Lectins which do not bind to band 3 have no effect. Purified band 3, isolated by affinity chromatography on a concanavalin A column, was homogeneous by polyacrylamide gel electrophoresis, had a molecular weight of 100 000, and inhibited the hemolytic activity of alpha toxin. Antibodies to the toxin–toxoid receptor were serologically indistinguishable from antiband 3.

Affinity Chromatography of Human Factor VIII Using Human and Rabbit Antibodies to Factor VIII

1979 ◽  
Vol 42 (04) ◽  
pp. 1306-1315 ◽  
Author(s):  
Janet L Lane ◽  
H Ekert ◽  
A Vafiadis
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Affinity Chromatography ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Sds Page ◽  
Normal Human

SummaryFactor VIII, purified by gel filtration on Sepharose 2B, has an 8 band multiple subunit structure, with molecular weights ranging from 30,000 to 230,000, on reduction and SDS-PAGE at a protein concentration of 400 μg/gel. Affinity chromatography of this factor VIII preparation with insolubilized haemophilic antibody to factor VIII showed that 45-81% VIII:C and 0-33% VIILRag were attached to the column. Elution of the column with 0.25 M CaCl2 did not show VIII:C or VIILRag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed that 95 % of the protein bound by haemophilic antibody had a molecular weight similar to the low molecular weight subunits of the reduced factor VIII.In control experiments with normal Human IgG, 3% of VIII:C and 5% of VIILRag were attached to the column. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the protein, showed 2 faint bands with molecular weight consistent with heavy and light chains of IgG.Similar experiments with antibody to factor VIII showed that 67-83% of VIILC and 61-76% of VIII:Rag were attached to the column. Elution of the column with 0.25 M CaCl2 showed 10% of the applied VIII:C, but no VIII:Rag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed an 8 band subunit structure similar to the reduced factor VIII.

Aldosterone-induced proteins: characterization using lectin-affinity chromatography

1985 ◽  
Vol 249 (3) ◽  
pp. C215-C225 ◽  
Author(s):  
B. Blazer-Yost ◽  
M. Cox
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Concanavalin A ◽  
Specific Inhibitor ◽  
Partial Purification ◽  
Toad Urinary Bladder ◽  
Lectin Affinity Chromatography ◽  
Na Transport ◽  
Electrophoretic Polymorphism ◽  
Induced Proteins

Aldosterone-stimulated Na+ transport in toad urinary bladder is associated with the synthesis of a specific group of proteins whose induction appears to be related to the natriferic effect of the hormone. These aldosterone-induced proteins (AIPs) occur in two slightly different molecular weight classes (around 70 kDa), each class being composed of several proteins with discrete isoelectric points (range, 5.5-6.0). Because glycosylation is a common cause of such electrophoretic polymorphism and microheterogeneity, we examined whether these proteins are glycoproteins. Tunicamycin (a specific inhibitor of N-linked glycosylation) inhibited aldosterone-stimulated Na+ transport and AIP synthesis without affecting overall protein synthesis. The vast majority of epithelial cell proteins did not bind to the mannose-specific lectin, concanavalin A-sepharose. In contrast, both classes of AIPs bound to concanavalin A-sepharose, but the affinities of the higher and lower molecular weight proteins were markedly different: the former were readily eluted with 0.2 M alpha-methyl-D-mannoside alone, whereas the latter could only be eluted with 0.4 M alpha-methyl-D-mannoside in combination with high concentrations of NaCl (2.5-5.0 M). These studies indicate that 1) glycosylation is important in the natriferic response to aldosterone, 2) the AIPs are N-linked mannose-containing glycoproteins, and 3) the electrophoretic polymorphism of the AIPs is due, at least in part, to differences in glycosylation. Furthermore, concanavalin A-affinity chromatography provides a simple means for the partial purification of these putative "effectors" of the cellular action of aldosterone.

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.

scholarly journals Binding to antithrombin of heparin fractions with different molecular weights

1981 ◽  
Vol 193 (2) ◽  
pp. 427-433 ◽  
Author(s):  
Ȧke Danielsson ◽  
Ingemar Björk
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Binding Constants ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
High Affinity ◽  
Heparin Fractions ◽  
Fluorescence Titrations ◽  
In The Beginning ◽  
Difference Absorption

The interaction between bovine antithrombin, a plasma proteinase inhibitor, and heparin species of different molecular weights was studied. A commercial heparin preparation was divided by gel chromatography into a number of fractions with average molecular weights ranging from 6000 to 34700. Each of these fractions was further fractionated by affinity chromatography on matrix-bound antithrombin. In the latter procedure, those heparin fractions that had molecular weights lower than about 14000 were separated into three peaks. The material in the first of these was not adsorbed on the column, and the other two peaks corresponded to the low-affinity and high-affinity peaks described previously. In contrast, high-molecular-weight heparin samples gave only the low-affinity and high-affinity fractions. U.v. difference absorption studies showed that the non-adsorbed heparin fraction bound to antithrombin in solution with a binding constant at physiological ionic strength only slightly lower than that of low-affinity heparin. The division between the two fractions thus is arbitrary and only dependent on the conditions selected for the affinity-chromatography experiment. Stoicheiometries and binding constants for the binding of several high-affinity heparin species to antithrombin were determined by fluorescence titrations. High-affinity heparin fractions of equal elution positions in the beginning of the peaks of the affinity chromatographies, but with different molecular weights, showed stoicheiometries that were not experimentally distinguishable from 1:1 and also had no appreciable differences in binding constants. However, the anticoagulant activities, calculated on a molar basis, of these fractions increased markedly with molecular weight, a behaviour that thus cannot be explained by differences in the binding of the fractions to antithrombin. In contrast, high-affinity samples of similar molecular weights, which were eluted at increasing ionic strengths from matrix-linked antithrombin, were found to have an increasing proportion of chains with two binding sites for antithrombin and also to have progressively higher binding constants. These binding properties at least partly explain the increasing anticoagulant activities that were observed for these fractions.

An Inactive, Prorenin-like Substance in Human Kidney and Plasma

1980 ◽  
Vol 59 (s6) ◽  
pp. 29s-33s ◽  
Author(s):  
S. A. Atlas ◽  
J. H. Laragh ◽  
Jean E. Sealey ◽  
T. E. Hesson
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Plasma ◽  
Concanavalin A ◽  
Binding Protein ◽  
Human Kidney ◽  
Renin Activity ◽  
Cibacron Blue ◽  
Active Renin ◽  
Inactive Renin

1. Plasma prorenin (inactive renin), which accounts for about 70% of the total renin in human plasma, was almost completely separated from active renin by affinity chromatography on Cibacron blue F3G-A-agarose. The slight residual renin activity present in the prorenin peak can be removed on concanavalin A-Sepharose, demonstrating that prorenin is completely inactive. 2. The renin activity of both human renal cortical extract and renal perfusate increased after incubation with trypsin. This trypsin-activable renin accounted for 15 and 40% of the total renin in extract and perfusate respectively. 3. Trypsin-activable renin from both renal extract and renal perfusate was, like plasma prorenin, almost completely separated from active renin on Cibacron blue F3G-A-agarose. After additional chromatographic steps, the trypsin-activable renin from renal cortical extract was found to be completely inactive. 4. We conclude that human kidney contains, and is able to release, a trypsin-activable renin that resembles plasma prorenin. It may differ from many of the 60 000 molecular-weight forms of renin previously identified in renal extracts, since these possess considerable intrinsic renin activity and probably represent a complex of renin with a binding protein.

COMPARATIVE THERMAL STABILITY OF PEROXIDASE ISOZYMES FROM TWO FLAX GENOTROPHS

1982 ◽  
Vol 24 (4) ◽  
pp. 427-435 ◽  
Author(s):  
Mary Ann Fieldes ◽  
Hugh Tyson
Keyword(s):  
Thermal Stability ◽  
Molecular Weight ◽  
Prolonged Treatment ◽  
Linum Usitatissimum L ◽  
Peroxidase Isozymes ◽  
Heat Treated ◽  
Peak Areas ◽  
Flax Genotrophs ◽  
Thermal Stability Of

The thermal stability of peroxidase isozymes was examined in vitro in Linum usitatissimum L. Extracts of main stem tissues of the L and S genotrophs produced by Durrant were heat treated over a range of temperatures and times. Isozymes in treated extracts were separated electrophoretically, and peak areas for the four main anionic isozymes, together with their relative mobilities (Rms), were recorded. Peak areas supplied estimates of relative activities. Short duration treatments at 60° and 70 °C demonstrated differences in thermal stability between isozymes and produced changes in Rm. With prolonged treatment at 40 °C, the thermal stability of one isozyme differed from those of the other three. This isozyme was known to have a higher molecular weight than the others. In addition, prolonged treatment at 40 °C demonstrated increased thermal stability of the three lower molecular weight isozymes of genotroph S compared to those of L.

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