Acetolactate Synthase from Barley (Hordeum vulgare L.): Purification and Partial Characterization

1988 ◽  
Vol 43 (11-12) ◽  
pp. 850-856 ◽  
Author(s):  
Jörg Durner ◽  
Peter Böger
Keyword(s):  
Hordeum Vulgare ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Acetolactate Synthase ◽  
Active Species ◽  
Basic Unit ◽  
Hordeum Vulgare L ◽  
Molecular Weights ◽  
Sds Page ◽  
Branched Chain

Abstract Acetolactate synthase (EC 4.1.3.18; ALS) has been extracted from etiolated barley shoots (Hordeum vulgare L.) and purified to near homogeneity. Purification was made possible by a fivestep procedure using hydrophobic interaction, gel filtration, anion-exchange and hydroxylapatite chromatography, the last two steps performed with an HPLC- and FPLC-system, respectively. A 300-fold purification was achieved representing 13% of the initial activity in the crude extract; only small amounts of pure acetolactate synthase could be isolated. Although the enzyme was found labile during the chromatographic steps, purified ALS maintained its activity for several hours and could be stored at 70 K for weeks with a 15-30% loss. The apparent molecular weights of the enzymatically active species as determined by gel filtration were about 440 kDa and 200 kDa, respectively. We assume these species are no isozymes but different polymeric forms of a basic unit of ALS. SDS-PAGE analysis showed one polypeptide with an apparent molecular weight of 58 kDa. Preliminary enzymatic characterization of the purified enzyme confirms a marked synergism in the feedback control by branched-chain amino acids. The combination of valine plus leucine exhibited the most co-operative inhibition.

scholarly journals Hordein variation in Brazilian barley varieties (Hordeum vulgare L.) and wild barley (H. euclaston Steud. and H. stenostachys Godr.)

2000 ◽  
Vol 23 (2) ◽  
pp. 425-433 ◽  
Author(s):  
Cinara Echart-Almeida ◽  
Suzana Cavalli-Molina
Keyword(s):  
Hordeum Vulgare ◽  
Native Species ◽  
Wild Barley ◽  
Operational Taxonomic Unit ◽  
Southern Brazil ◽  
Hordeum Vulgare L ◽  
Molecular Weights ◽  
Polypeptide Patterns ◽  
Sds Page ◽  
Cultivated Species

SDS-PAGE was used to analyze the hordein polypeptide patterns of Brazilian barley varieties (Hordeum vulgare L.) and of two native species of Hordeum from southern Brazil (H. euclaston Steud. and H. stenostachys Godr.). Forty different hordein polypeptide bands with molecular weights ranging from 30 to 94 kDa were found in the seeds of the three species studied. Twelve of the 14 varieties examined showed intravarietal polymorphism. The number of bands ranged from 10 to 17, depending on the variety, and from 3 to 13 among individual seeds, with a total of 26 bands in H. vulgare. Phenograms using each seed as an operational taxonomic unit (OTU) showed that the seeds from most varieties did not form distinct clusters. Seeds from different plants of the native species varied considerably. The molecular weights of the hordein polypeptides of the two native species were quite different from those of H. vulgare. There was a greater similarity between the native species than with H. vulgare, although H. stenostachys was slightly closer to the cultivated species than H. euclaston.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

A PLASMINOGEN ACTIVATOR INHIBITOR (PAI-2) CIRCULATES IN TWO HIGH MOLECULAR WEIGHT FORMS IN PREGNANCY

1987 ◽  
Author(s):  
N A Booth ◽  
A Reith ◽  
B Bennett
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Plasminogen Activator ◽  
Apparent Molecular Weight ◽  
Late Pregnancy ◽  
Plasminogen Activator Inhibitor ◽  
Molecular Weights ◽  
Histiocytic Cell ◽  
Sds Page ◽  
Pai 1

Normal vascular endothelium and platelet α-granules contain an inhibitor of plasminogen activator (PAI-1) of about 48000 molecular weight, which is released by stimuli such as thrombin. An immunologically distinct inhibitor (PAI-2) of about 47000 molecular weight has been purified from placenta and from a histiocytic cell line U-937. The level of PA-inhibition in plasma is raised in late pregnancy and this may be due to increases in PAI-1 or in PAI-2 or in both.Using SDS-PAGE and zymography on fibrin/plasminogen /u-PA detector gels, we have found that normal plasma contains a band of inhibition of apparent molecular weight 40000, which can be neutralised by antiserum raised against PAI-1. Pregnancy plasma contained this band as well as additional inhibitor bands of apparent molecular weights 75000 and 130000. The novel high molecular weight PA-inhibitors were detectable by zymography at about 12 weeks gestation. They were specific for plasminogen activator and did not inhibit plasmin. They were inhibited by antiserum raised against PAI-2 from U-937 cells (a gift from Dr EKO Kruithof) and thus are immunologically related to PAI-2. They may represent circulating complexes of PAI-2 with another protein or aggregates of PAI-2, which retain inhibitory activity after SDS-PAGE. PAI-2 appears to represent a pregnancy associated protein that circulates in a number of different molecular weight forms.

scholarly journals Analysis of a rare recombination event within the multigenic Hor 2 locus of barley (Hordeum vulgare L.)

1990 ◽  
Vol 55 (3) ◽  
pp. 171-176 ◽  
Author(s):  
Peter R. Shewry ◽  
Saroj Parmar ◽  
Julian Franklin ◽  
Shirley R. Burgess
Keyword(s):  
Hordeum Vulgare ◽  
Recombination Event ◽  
Rflp Analysis ◽  
Genomic Clone ◽  
Class I ◽  
Class Iii ◽  
Hordeum Vulgare L ◽  
Ancestral Gene ◽  
Sds Page ◽  
High Degree

SummaryA rare recombinant within the multigenic Hor 2 locus of barley was detected by SD-PAGE of hordein fractions from F2 grain from the cross Bomi × P12/3. Analysis of a homozygous F4 line by 2-D IEF/SDS-PAGE showed that recombination between the class I/II and class III subfamilies of genes had occurred, indicating that they are spatially separate within the Hor 2 locus. RFLP analysis using a B hordein-related cDNA clone confirmed that recombination had occurred, while similar analysis using a genomic clone related to γ-type hordein (encoded by the Hor 5/HrdF locus) indicated the order of the two subfamilies of genes on the short arm of chromosome 5, the class I/II genes being closer to the centromere. The results are consistent with the origin of the B hordein gene family from initial duplication of a single ancestral gene to give two genes which diverged to become the ancestors of the class I/II and class III subfamilies. Subsequent cycles of duplication and divergence have resulted in the present high degree of polymorphism.

scholarly journals Isolation of palmitoyl-CoA hydrolases from human blood platelets

1981 ◽  
Vol 199 (3) ◽  
pp. 639-647 ◽  
Author(s):  
R K Berge ◽  
L E Hagen ◽  
M Farstad
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Cytosol Fraction ◽  
Molecular Weights ◽  
Apparent Homogeneity ◽  
Coa Hydrolase ◽  
Human Blood Platelets

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].

scholarly journals Isolation and partial purification of anticoagulant fractions from the venom of the Iranian snake Echis carinatus.

2012 ◽  
Vol 60 (1) ◽  
Author(s):  
Mahdi Babaie ◽  
Hossein Zolfagharian ◽  
Hossein Salmanizadeh ◽  
Abbas Zare Mirakabadi ◽  
Hafezeh Alizadeh
Keyword(s):  
Blood Coagulation ◽  
Gel Filtration ◽  
Partial Purification ◽  
Crude Venom ◽  
Molecular Weights ◽  
Time Value ◽  
Sds Page ◽  
Echis Carinatus ◽  
The Many ◽  
Coagulation Pathway

Many snake venoms comprise different factors, which can either promote or inhibit the blood coagulation pathway. Coagulation disorders and hemorrhage belong to the most prominent features of bites of the many vipers. A number of these factors interact with components of the human blood coagulation. This study is focused on the effect of Echis carinatus snake venom on blood coagulation pathway. Anticoagulant factors were purified from the Iranian Echis carinatus venom by two steps: gel filtration (Sephadex G-75) and ion-exchange (DEAE-Sephadex) chromatography, in order to study the anticoagulant effect of crude venom and their fractions. The prothrombin time was estimated on human plasma for each fraction. Our results showed that protrombin time value was increase from 13.4 s to 170 s for F2C and to 280 s for F2D. Our study showed that these fractions of the venom delay the prothrombine time and thus can be considered as anticoagulant factors. They were shown to exhibit proteolytic activity. The molecular weights of these anticoagulants (F2C, F2D) were estimated by SDS/PAGE electrophoresis. F2C comprises two protein bands with molecular weights of 50 and 79 kDa and F2D a single band with a molecular weight of 42 kDa.

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.

Human Leucocyte Urokinase Inhibitor - Purification, Characterization and Comparative Studies Against Different Plasminogen Activators

1985 ◽  
Vol 54 (04) ◽  
pp. 750-755 ◽  
Author(s):  
M Kopitar ◽  
B Rozman ◽  
J Babnik ◽  
V Turk ◽  
D E Mullins ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Inhibitory Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Plasminogen Activator Inhibitor ◽  
Exchange Chromatography ◽  
Sds Page ◽  
Peripheral Leukocytes ◽  
Ph Range ◽  
Activator Inhibitor

SummaryA plasminogen activator inhibitor (PA-I) which inhibits primarily plasminogen activator of the urokinase type (u-PA) was isolated from the cytosol of human peripheral leukocytes. The inhibitor was isolated using ion exchange chromatography, gel filtration and FPLC. This inhibitor has an apparent molecular weight of 45 kDa, determined by SDS-PAGE, and a pi of 5.5-5.7. The inhibitor is a fast reacting inhibitor, is thermally unstable and is inactivated outside the pH range 7-9. Treatment of cytosol to pH 9 for 30 min at 37° C resulted in a large increase in inhibitory activity. Antibodies against human placental UK-I completely quenched the inhibitory activity of human leucocyte UK-I.

Interference in a radioimmunoassay for human thyrotropin.

1977 ◽  
Vol 23 (3) ◽  
pp. 584-588 ◽  
Author(s):  
C A Spencer ◽  
G S Challand
Keyword(s):  
Clinical Evidence ◽  
Chemical Nature ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
High Plasma ◽  
Normal Serum ◽  
Freezing And Thawing ◽  
Separation Stage ◽  
Molecular Weights ◽  
Repeated Freezing

Abstract Abnormally high plasma thyrotropin values were found by radioimmunoassay in some patients when an antiserum to porcine thyrotropin was used, normal results being obtained with an antiserum raised to human thyrotropin. These discrepancies were found in some subjects with no biochemical or clinical evidence of hypothyroidism and occasionally in sera from patients with unequivocal hyperthyroidism. We found a substance in serum that cross reacts with the anti-porcine antiserum, is stable on repeated freezing and thawing, and is independent of the 125I-labeled tracer preparation. It is unlikely that this substance is a separation-stage artefact related to immunoglobulins. Its apparent molecular weight (gel filtration) is 114 000, as compared with apparent molecular weights for standard thyrotropin and endogenous thyrotropin (as found in idiopathic hypothyroidism) of 34 700 and 38 000, respectively. We believe the substance is a normal serum constituent that is present in large quantities in a minority of subjects. Apparently unrelated to TSH, its exact chemical nature remains unidentified.

Homologues Of Fibrin X Oligomers

1981 ◽  
Author(s):  
R Hafter ◽  
H Graeff ◽  
R v Hugo
Keyword(s):  
Gel Filtration ◽  
Advanced Ovarian Cancer ◽  
Simultaneous Action ◽  
Coagulation Disorder ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Sds Page ◽  
D Dimer

Crosslinked fibrin derivatives signalize intravascular coagulation. D-dimer, Y-D and X oligomers are observed in plasma from obstetric patients with severe coagulation disorder. They are also found in ascitic fluid from patients with advanced ovarian cancer and can be produced in vitro by simultaneous action of thrombin, plasmin and factor XIII with fibrinogen. The study was aimed to evaluate the subunit structure of separated molecular entities. The derivatives were separated by 4% SDS-PAGE preceded in case of the in vivo products by gel filtration and/or by immunoabsorption technique. The gels were sliced at the respective migration positions and derivatives therein reelectrophoresed on 7,5% gels after reduction. Subunit characterisation revealed that D-dimer is composed of the chain remnants γ1-γ1, β2, α2, while Y-D is composed of γ-γ1, β2, α3, α2, besides αE, βE and γE Crosslinked X oligomers are composed of γ-γ, γ-γ1, β, β1, γ2, α1 and α2 besides αE, βE and γE Three possible combinations of plasmin degraded and undegraded dimeric γ-chains were observed in vivo and in vitro: γ-γ γ-γ1 and γ1-γ1. The ratio of degraded (γ1) to undegraded γ-chains in dimeric γ-chain patterns indicates the mol. structure of the respective derivative. Two X oligomers could be demonstrated in which the ratio of γ-γ to γ-γ1 in terms of stain intensity was either 1:1 or 2:1. Their subunit compositions are in accordance with structures describ- able as D-X-Y and D-X-X-Y. Their molecular weights, calculated from the subunit compositions are 476,000 and 716,000 respectively. - It is proposed that crosslinked X oligomers exist as a homologous family with increasing X fragment content.

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