scholarly journals Bonds broken and formed during the mixed-linkage glucan : xyloglucan endotransglucosylase reaction catalysed by Equisetum hetero-trans-β-glucanase

2017 ◽  
Vol 474 (7) ◽  
pp. 1055-1070 ◽  
Author(s):  
Thomas J. Simmons ◽  
Stephen C. Fry
Keyword(s):  
Gel Permeation Chromatography ◽  
Land Plants ◽  
Reaction Products ◽  
Glucose Residue ◽  
Substrate Specificities ◽  
Enzymic Digestion ◽  
The Third ◽  
Gel Permeation ◽  
Layer Chromatography

Mixed-linkage glucan∶xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-β-glucanase (HTG), which among land plants is known only from Equisetum species. The biochemical details of the MXE reaction were incompletely understood — details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-β-d-glucan (MLG), with radioactive oligosaccharides of xyloglucan as the acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and gel-permeation chromatography (GPC). Equisetum HTG consistently cleaved the MLG at the third consecutive β-(1→4)-bond following (towards the reducing terminus) a β-(1→3)-bond. It then formed a β-(1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its xyloglucan endotransglucosylase/hydrolase subfamily membership. Using size-homogeneous barley MLG as the donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative to the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.

scholarly journals Characterization of bovine κ-casein fractions and the kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel-permeation chromatography

1986 ◽  
Vol 240 (1) ◽  
pp. 87-97 ◽  
Author(s):  
H J Vreeman ◽  
S Visser ◽  
C J Slangen ◽  
J A Van Riel
Keyword(s):  
High Performance ◽  
Substrate Inhibition ◽  
Deae Cellulose ◽  
Free Fraction ◽  
Gel Permeation Chromatography ◽  
Reaction Products ◽  
Milk Clotting ◽  
Milk Clotting Enzyme ◽  
Gel Permeation

Bovine kappa-casein was fractionated at pH 8.0 on DEAE-Sepharose with an NaCl gradient, followed by DEAE-cellulose chromatography using a decreasing pH gradient from pH 6.0 to 4.5. At least ten components could be identified, each differing in N-acetylneuraminic acid (NeuAc) and/or phosphorus content. Two components appeared to be multiply-phosphorylated, but did not contain NeuAc. The possible significance of this finding in relation to the mode of phosphorylation and glycosylation in vivo is discussed. A carbohydrate-free fraction as well as two NeuAc-containing fractions were compared in their substrate behaviour towards the action of the milk-clotting enzyme chymosin at pH 6.6 and 30 degrees C. To this end the trichloroacetic acid-soluble reaction products were analysed by high-performance gel-permeation chromatography. In order of increasing carbohydrate content the kcat. values found ranged from 40 to 25 s-1 and the Km values from 9 to 3 microM; the overall substrate properties of these components as reflected by the kinetic parameter kcat./Km ranged from 5 to 8 microM-1 X S-1. Irreversible polymerization of the carbohydrate-free fraction brought about a more-than-2-fold increase in Km, the kcat. value remaining virtually constant. The kcat./Km found for the cleavage of whole kappa-casein at pH 6.6 was of the same magnitude as the kcat./Km found for the polymerized carbohydrate-free fraction (i.e. about 3 microM-1 X S-1). No indication of substrate inhibition was found for the carbohydrate-free fraction.

Synthesis and chromatographic characterization of [Tc-99m]technetium-humic acid species

2000 ◽  
Vol 88 (2) ◽  
Author(s):  
D. Rößler ◽  
K. Franke ◽  
R. Süß ◽  
E. Becker ◽  
H. Kupsch
Keyword(s):  
Humic Acid ◽  
Stannous Chloride ◽  
Gel Permeation Chromatography ◽  
Paper Electrophoresis ◽  
Gel Permeation ◽  
Soil Humic Acid ◽  
Chromatographic Characterization ◽  
Ph Dependent ◽  
Layer Chromatography

A natural moor soil humic acid (HA) was labeled with Tc-99m via reduction of pertechnetate with stannous chloride. The humic acid species obtained were characterized by thin layer chromatography (TLC), gel permeation chromatography (GPC), sequential chromatographic analysis (SCA), paper electrophoresis and micropore filtration. Labeling was found to take place in all ranges of molecular weight. Due to the complex humic acid composition and the formation of hydroxo species the labeling yields strongly depend on the separation conditions, ranging from 42% to 80%. The pH-dependent distribution of mobile and immobile species was determined by SCA for HTcO

scholarly journals Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

2004 ◽  
Vol 186 (19) ◽  
pp. 6501-6507 ◽  
Author(s):  
A. de la Fuente ◽  
J. F. Martín ◽  
A. Rodríguez-García ◽  
P. Liras
Keyword(s):  
Clavulanic Acid ◽  
Streptomyces Clavuligerus ◽  
Culture Conditions ◽  
Transferase Activity ◽  
Reaction Products ◽  
Acid Biosynthesis ◽  
Substrate Specificities ◽  
Acetyltransferase Activity ◽  
Standard Culture

ABSTRACT The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations.

scholarly journals Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1024-1029
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
KR Tanaka
Keyword(s):  
Pyruvate Kinase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Gel Permeation Chromatography ◽  
Metabolic Abnormalities ◽  
Gel Permeation ◽  
Reduced Nicotinamide Adenine Dinucleotide

Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1- pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3- diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK-deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs.

scholarly journals Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1024-1029 ◽  
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
KR Tanaka
Keyword(s):  
Pyruvate Kinase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Gel Permeation Chromatography ◽  
Metabolic Abnormalities ◽  
Gel Permeation ◽  
Reduced Nicotinamide Adenine Dinucleotide

Abstract Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1- pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3- diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK-deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs.

scholarly journals In Vivo Stability of Polyurethane-Based Electrospun Vascular Grafts in Terms of Chemistry and Mechanics

Polymers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 845
Author(s):  
Alexander A. Gostev ◽  
Inna K. Shundrina ◽  
Vitaliy I. Pastukhov ◽  
Alexey V. Shutov ◽  
Vera S. Chernonosova ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Molecular Weight ◽  
Tensile Strength ◽  
Wistar Rats ◽  
Vascular Grafts ◽  
Gel Permeation Chromatography ◽  
Initial Value ◽  
Gel Permeation ◽  
The Stability

The biostability of the polyurethanes Tecoflex EG-80A and Pellethane 2363-80A, used as basic polymers of the vascular grafts (VGs) produced by electrospinning, as well as the tensile strength of Tecoflex VGs, are studied. Solutions of Tecoflex or Pellethane with gelatin and bivalirudin in 1,1,1,3,3,3-hexafluoroisopropanol are used for VG production. After 1, 12, and 24 weeks of VG implantation in the infrarenal position of the abdominal aorta of Wistar rats, VGs are explanted, fixed in formalin, freed from outer tissues, dialyzed, and dried. The polyurethanes are extracted from VGs by dispersion/extraction in tetrahydrofuran (THF) and freed from the excess of THF-insoluble biopolymers. The stability of polyurethanes is assessed by IR spectroscopy and gel permeation chromatography. Pellethane has emerged to be stable at all experimental points. Tecoflex loses approximately 10% of its molecular weight (both Mn and Mw) after 3 months and restored its initial value within 6 months of its functioning as a graft. Mechanical testing demonstrates a 30% reduction in the tensile strength after 3 months in VG and a 10% increase after 6 months. The stability and mechanical properties of polyurethane-based VGs demonstrate their utility for the reconstitution of damaged arteries.

Development of Rubber and Asphalt Binders by Depolymerization and Devulcanization of Scrap Tires in Asphalt

1996 ◽  
Vol 1530 (1) ◽  
pp. 51-58 ◽  
Author(s):  
Ludo Zanzotto ◽  
Gerhard J. Kennepohl
Keyword(s):  
Gel Permeation Chromatography ◽  
Crumb Rubber ◽  
Asphalt Binders ◽  
Temperature Sensitive ◽  
High Shear ◽  
Reaction Products ◽  
Scrap Tires ◽  
Creep And Recovery ◽  
Conventional Tests ◽  
Gel Permeation

Scrap tires in three forms—crumb rubber 60 minus mesh, crumb rubber 20 minus mesh, and buffed rubber—were subjected to depolymerization and devulcanization by applying heat, high shear, and a combination of both. Different reaction temperatures and times of reaction were applied. The reaction products were submitted to the testing of rheological properties by measurement of creep and recovery, viscosity, and some conventional tests. The amount of unreacted rubber and tendency to separation during the hot storage were also tested. Selected materials were evaluated according to SHRP specification and by the gel permeation chromatography. The results from the SHRP testing indicate that these materials are less temperature sensitive than conventional asphalts prepared from the same crude oil as the base asphalt. These materials were prepared in larger quantities and are being tested for their performance in paving mixes.

Aggregated, Conformationally Changed Fibrinogen Exposes the Stimulating Sites for t-PA-Catalysed Plasminogen Activation

1996 ◽  
Vol 75 (02) ◽  
pp. 326-331 ◽  
Author(s):  
Unni Haddeland ◽  
Knut Sletten ◽  
Anne Bennick ◽  
Willem Nieuwenhuizen ◽  
Frank Brosstad
Keyword(s):  
Conformational Changes ◽  
Gel Permeation Chromatography ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Chromogenic Substrate ◽  
Type Plasminogen Activator ◽  
Gel Permeation ◽  
Self Association ◽  
Fibrin Monomers ◽  
Stimulation Of

SummaryThe present paper shows that conformationally changed fibrinogen can expose the sites Aα-(148-160) and γ-(312-324) involved in stimulation of the tissue-type plasminogen activator (t-PA)-catalysed plasminogen activation. The exposure of the stimulating sites was determined by ELISA using mABs directed to these sites, and was shown to coincide with stimulation of t-PA-catalysed plasminogen activation as assessed in an assay using a chromogenic substrate for plasmin. Gel permeation chromatography of fibrinogen conformationally changed by heat (46.5° C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, had exposed the epitopes Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation. Fibrinogen subjected to heat in the presence of 3 mM of the tetrapeptide GPRP neither aggregates nor exposes the rate-enhancing sites. Thus, aggregation and exposure of t-PA-stimulating sites in fibrinogen seem to be related phenomena, and it is tempting to believe that the exposure of stimulating sites is a consequence of the conformational changes that occur during aggregation, or self-association. Fibrin monomers kept in a monomeric state by a final GPRP concentration of 3 mM do not expose the epitopes Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is no longer anti-polymerizing, results in exposure of these sites. Consequently, the exposure of t-PA-stimulating sites in fibrin as well is due to the conformational changes that occur during selfassociation.

The Spectrum of von Willebrand’s Disease

1967 ◽  
Vol 18 (01/02) ◽  
pp. 040-056 ◽  
Author(s):  
E. J Walter Bowie ◽  
P Didisheim ◽  
J. H Thompson ◽  
C. A Owen
Keyword(s):  
Factor Viii ◽  
Bleeding Time ◽  
Severe Bleeding ◽  
Platelet Adhesiveness ◽  
Platelet Activity ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
The Third ◽  
Generation Test

SummaryPatients (from 5 kindreds) with variants of von Willebrand’s disease are described. In one kindred the depression of factor VIII was moderate (20 to 40% of normal) and transfusion of 500 ml of normal plasma led to an increase higher than anticipated and to an almost normal level of factor VIII 17 to 24 hrs later. This represents the usual type of von Willebrand’s disease.In the second kindred the concentration of factor VIII was less than 2 % of normal in the son and daughter, who had severe bleeding and hemarthroses.The third kindred was characterized by reduction of factor VIII and a long bleeding time as well as by a serum defect in the thromboplastin-generation test comparable to that seen in patients with hemophilia B, yet with normal levels of factors IX, X, and VII. The severity of the serum defect, the positive result with the Rumpel-Leede test, and the reduced platelet activity in the thromboplastin-generation test are all compatible with the diagnosis of thrombopathy or ‘‘thrombopathic hemophilia.” In two other kindreds, one patient had a long bleeding time and normal levels of factor VIII and another had a normal bleeding time and decrease of factor VIII. The last patient had the type of response to transfusion usually seen in von Willebrand’s disease.In four kindreds, platelet adhesiveness in vivo was found to be strikingly abnormal (virtually absent).It would appear, therefore, that von Willebrand’s disease forms a spectrum, and whether the kindreds reported simply reflect variations of a single genetic disease state or represent separate entities will be answered only by clarification of the underlying etiology of that disease.

Sign in / Sign up

Export Citation Format

Share Document