Glutamate kinases from winter-wheat leaves and some properties of the proline-inhibitable glutamate kinase

1982 ◽  
Vol 47 (1) ◽  
pp. 349-359 ◽  
Author(s):  
Ludmila Vašáková ◽  
Miroslav Štefl
Keyword(s):  
Molecular Weight ◽  
Triticum Aestivum ◽  
Winter Wheat ◽  
Deae Cellulose ◽  
Salting Out ◽  
Molecular Weights ◽  
Steady State Kinetics ◽  
Optimum Ph ◽  
Wheat Leaves ◽  
Ph Range

Three glutamate kinases (GK 1, GK 2 and GK 3) have been found in the leaves of winter wheat (Triticum aestivum L). They were separated by salting out with ammonium sulphate and by chromatography on DEAE-cellulose in a concentration gradient of KCl and on Sephadex G-100. GK 1 belongs to the biosynthesis of L-proline, which inhibits it according to the principle of feedback, GK 2 to the biosynthesis of glutamine; the function of GK 3 has not been found. The partically purified oligomer GK 1 had a molecular weight of 254 000 and dissociated to subunits of molecular weights 84 000 and 42 000. In a K+-phosphate buffer, 50 mmol/l, it was active in a pH range of 6.3 to 8.0, with an optimum of 7.2. An amine buffer inhibited it completely. The reaction required Mg2+ ions as activators and L-glutamate and ATP as substrates. ATP in a concentration above 70 mmol/l inhibited it. The steady-state kinetics at the optimum pH have shown that GK 1 produces a complex co-operativity of the substrates and belongs to the slowly dissociating hysteretic enzymes of the type Np P.

scholarly journals α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

scholarly journals Characterization of proteins structurally related to human N-acetyl-β-d-glucosaminidase

1978 ◽  
Vol 173 (1) ◽  
pp. 191-196 ◽  
Author(s):  
M Carroll
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Enzyme Preparations ◽  
Molecular Weights ◽  
Functional Relationships ◽  
Hexosaminidase A ◽  
Cellulose Column

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000–200000) were present both in the unadsorbed fraction and in the 0.05–0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000–70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.

scholarly journals Polymorphism of high-molecular-weight glutenin loci in Ukrainian wheat landraces Triticum aestivum L.

2021 ◽  
Vol 29 ◽  
pp. 111-116
Author(s):  
T. O. Sobko ◽  
G.M. Lisova ◽  
O.M. Blagodarova
Keyword(s):  
Molecular Weight ◽  
Triticum Aestivum ◽  
Winter Wheat ◽  
High Molecular Weight ◽  
Gene Pool ◽  
Allelic Diversity ◽  
Triticum Aestivum L ◽  
Hmw Glutenin ◽  
Wheat Landraces ◽  
In The Beginning

Aim. The aim of the study was to investigate allelic variability of high-molecular-weight glutenin loci Glu-A1, Glu-B1, Glu-D1 in Ukrainian winter wheat landraces and obsolete cultivars Triticum aestivum L. Methods. Allelic diversity at the Glu-1 loci were analyzed in 54 collection accessions, including 41 landraces (Krymka, Banatka, Girka, Theyka and others), and 13 first breeding cultivars that were developed in the beginning of the last century by selection from local wheat. Method of SDS-PAG electrophoresis according to Laemmli was used for fractionation of HMW glutenin subunits. Results. A total 11 alleles at the Glu-1 loci were identified, including 3 alleles at the Glu-A1 (a, b, c) and Glu-D1 (a, b, d) loci, and 5 – at the Glu- B1 (c, u, an, aj and subunit 9). Differences in frequencies of glutenin alleles were revealed. Conclusions. In the gene pool of Ukrainian winter bread wheat landraces the most widespread alleles were Glu-A1a (43.3 %), Glu-A1b (40.5 %), Glu-B1c (58 %), Glu-B1u (23 %), Glu-D1d (48.6 %), Glu-D1a (47.2 %). All these alleles (except of the Glu-D1a) are also predominant in the gene pool of modern commercial Ukrainian cultivars. A distinctive feature of Ukrainian landraces are the rare allelic variants of the Glu-B1 locus, which encode the subunits 1By9 and 1By8 (allele Glu-B1aj). Keywords: Triticum aestivum L., winter wheat, landraces, high-molecular-weight glutenin, alleles.

scholarly journals Tuning the Properties of High Molecular Weight Chitosans to Develop Full Water Solubility Within a Wide pH Range

2020 ◽  
Author(s):  
Anderson Fiamingo ◽  
Sergio Paulo Campana Filho ◽  
Osvaldo Novais Oliveira Junior
Keyword(s):  
Molecular Weight ◽  
Biomedical Applications ◽  
Random Distribution ◽  
Water Solubility ◽  
Aqueous Media ◽  
Degree Of Polymerization ◽  
Molecular Weights ◽  
H Nmr ◽  
Wide Ph Range ◽  
Ph Range

<p>The preparation of chitosans soluble in physiological conditions has been sought for years, but so far solubility in non-acidic aqueous media has only been achieved at the expense of lowering chitosan molecular weight. In this work, we applied the multistep ultrasound-assisted deacetylation process (USAD process) to β-chitin and obtained extensively deacetylated chitosans with high molecular weights (Mw ≥ 1,000,000 g mol<sup>-1</sup>). The homogeneous <i>N</i>-acetylation of a chitosan sample resulting from three consecutive USAD procedures allowed us to produce chitosans with a high weight average degree of polymerization (DPw ≈ 6,000) and tunable degrees of acetylation (DA from 5 to 80%). <i>N</i>-acetylation was carried out under mild conditions to minimize depolymerization, while preserving a predominantly random distribution of 2-amino-2-deoxy-D-glucopyanose (<i>GlcN</i>) and 2-acetamido-2-deoxy-D-glucopyanose (<i>GlcNAc</i>) units. This close to random distribution, inferred with deconvolution of nuclear magnetic resonance (<sup>1</sup>H NMR) spectra, is considered as responsible for the solubility within a wide pH range. Two of the highly <i>N</i>-acetylated chitosans (DA ≈ 60 % and ≈ 70 %) exhibited full water solubility even at neutral pH, which can expand the biomedical applications of chitosans. </p>

scholarly journals Production and Characterization of Crude Protease from RS1 Isolate from silage of Floating Bladderwort (Utricularia gibba)

2020 ◽  
Vol 10 (3) ◽  
pp. 289-293
Author(s):  
Ace Baehaki ◽  
Arif Hidayat ◽  
Nuni Gofar ◽  
Rodiana Nopianti
Keyword(s):  
Molecular Weight ◽  
Production Time ◽  
Molecular Weights ◽  
Sds Page ◽  
Optimum Ph ◽  
Utricularia Gibba ◽  
Optimum Production ◽  
Optimum Ph And Temperature

The purpose of this research was to produce and characterizing crude protease from RS1 isolate of swamp plant silage. The optimum production time of RS1 isolate was 40 h. The optimum pH and temperature of protease from RS1 isolate were 10 and 45℃, respectively.  Ion Mg3+ increased RS1 protease whereas ion of Na+, K+, Fe2+, and Zn2+ inhibited protease from RS1 isolate. Study on the effect of metals ion indicated that protease from RS1 isolate was metaloenzyme. Based analysis on SDS-PAGE, the molecular weight of RS1 protease had 12 bands with molecular weights ranging from 34.75 kDa to 263.53 kDa.

Proteolytic Inactivation of Glutamine Synthetase in Extracts From Wheat Leaves: Effects of pH, Inorganic Ions and Metabolites

1994 ◽  
Vol 21 (3) ◽  
pp. 303 ◽  
Author(s):  
V Frohlich ◽  
A Fischer ◽  
G Ochs ◽  
A Wild ◽  
U Feller
Keyword(s):  
Triticum Aestivum ◽  
Glutamine Synthetase ◽  
Inorganic Ions ◽  
Triticum Aestivum L ◽  
Major Effect ◽  
Substrate Protein ◽  
Effects Of Ph ◽  
Proteolytic Activities ◽  
Wheat Leaves ◽  
Ph Range

Glutamine synthetase in extracts from young wheat (Triticum aestivum L.) leaves was quite stable at pH 7.0-9.0, whereas it was remarkably more labile below and above this range. Added extract from senescing wheat leaves accelerated the inactivation over the whole investigated pH range (6.0-10.5) and was most effective around pH 8.5-9.0. At pH 7.5, glutamine synthetase inactivation by endogenous or other supplied endopeptidases was delayed in the presence of magnesium sulfate, magnesium chloride and L-lysine, while potassium chloride stabilised only slightly. No major effect was caused by the addition of sucrose, L-alanine, L-serine or glycine. These results, and the fact that the stabilities of various enzymes are affected differently by the same solutes, suggest stabilising interactions with the substrate protein (glutamine synthetase) rather than effects on the inactivating endopeptidases. From immunoblots, it became evident that the inactivation of glutamine synthetase was paralleled by the degradation of the intact subunit. A smaller fragment was detected on immunoblots during the catabolism of this enzyme. Stabilising solutes retarded the loss of the intact subunit and the formation of the fragment. Solute concentrations must be considered as factors regulating the catabolism of a particular protein by given proteolytic activities.

scholarly journals Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain

1973 ◽  
Vol 135 (2) ◽  
pp. 367-373 ◽  
Author(s):  
C.-C. Liu ◽  
C.-H. Chung ◽  
M.-L. Lee
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Mammalian Brain ◽  
Acid Activation ◽  
High Concentration ◽  
Molecular Weights ◽  
Fractionation Column ◽  
Quaternary Structures

l-Tryptophan-activating enzyme [l-tryptophan–tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different Km and Vmax. values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg2+, ATP, in any combination.

Purification and characterization of exo-β-1,3-glucanase from a hatching supernatant of Strongylocentrotus intermedius

1983 ◽  
Vol 61 (1) ◽  
pp. 54-62 ◽  
Author(s):  
Kiyoshi Takeuchi
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Protein Band ◽  
Divalent Ions ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Single Protein

Exo-β-1,3-glucanase from the sea urchin embryos was purified 114-fold from the initial hatching supernatant by the following procedures: (a) gel filtration on Sephadex G-100; (b) hydrophobic chromatography on 4-phenylbutylamine-Sepharose (PBA-Sepharose); (c) two ion-exchange chromatographic steps on DEAE-cellulose; (d) gel filtration on Ultrogel AcA 34; (e) gel filtration on Sephadex G-100. The purified enzyme contained 2.2% carbohydrate and gave a single protein band corresponding to a molecular weight of 136 000 following electrophoresis on sodium dodecyl sulfate (SDS) – urea – polyacrylamide gel. Gel filtration on Ultrogel AcA 34 with a nondenaturing solvent gave a molecular weight of 130 000 ± 6000. The enzyme displayed an optimum pH at 5.0–5.5 and hydrolysed laminarin and PS(curdlan)-beads at the nonreducing ends, releasing glucose. Although activity of the purified enzyme was not affected by SDS, urea, some divalent ions, and 2-mercaptoethanol, both dithiothreitol and Hg2+ were markedly inhibitory.

Bovine hepatic carboxylesterases chromatographic fractionation, gel filtration, and molecular weight estimation

1969 ◽  
Vol 47 (8) ◽  
pp. 799-805 ◽  
Author(s):  
D. J. Ecobichon
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bovine Liver ◽  
Weight Estimation ◽  
Molecular Weights ◽  
Starch Gel ◽  
Two Peaks ◽  
Second Peak

The carboxylesterase activity of bovine liver was fractionated by DEAE-cellulose chromatography into two peaks of activity. Electrophoresis in starch gel showed that the peak eluted first was composed of a group of five electrophoretically slow bands while the second peak was a single, rapidly migrating band. Gel filtration of the crude extract on Sephadex G-100 and G-200 yielded a single peak of esterase activity containing both the electrophoretically slow and fast bands. The determination of molecular weights by gel filtration on Sephadex G-200 and G-100 yielded estimates of 52 000 and 55 000, respectively. The molecular weight estimates of the DEAE-cellulose fractionated electrophoretically slow and fast bands on Sephadex G-100 were identical, namely 55 000.

scholarly journals Tuning the Properties of High Molecular Weight Chitosans to Develop Full Water Solubility Within a Wide pH Range

2020 ◽  
Author(s):  
Anderson Fiamingo ◽  
Sergio Paulo Campana Filho ◽  
Osvaldo Novais Oliveira Junior
Keyword(s):  
Molecular Weight ◽  
Biomedical Applications ◽  
Random Distribution ◽  
Water Solubility ◽  
Aqueous Media ◽  
Degree Of Polymerization ◽  
Molecular Weights ◽  
H Nmr ◽  
Wide Ph Range ◽  
Ph Range

<p>The preparation of chitosans soluble in physiological conditions has been sought for years, but so far solubility in non-acidic aqueous media has only been achieved at the expense of lowering chitosan molecular weight. In this work, we applied the multistep ultrasound-assisted deacetylation process (USAD process) to β-chitin and obtained extensively deacetylated chitosans with high molecular weights (Mw ≥ 1,000,000 g mol<sup>-1</sup>). The homogeneous <i>N</i>-acetylation of a chitosan sample resulting from three consecutive USAD procedures allowed us to produce chitosans with a high weight average degree of polymerization (DPw ≈ 6,000) and tunable degrees of acetylation (DA from 5 to 80%). <i>N</i>-acetylation was carried out under mild conditions to minimize depolymerization, while preserving a predominantly random distribution of 2-amino-2-deoxy-D-glucopyanose (<i>GlcN</i>) and 2-acetamido-2-deoxy-D-glucopyanose (<i>GlcNAc</i>) units. This close to random distribution, inferred with deconvolution of nuclear magnetic resonance (<sup>1</sup>H NMR) spectra, is considered as responsible for the solubility within a wide pH range. Two of the highly <i>N</i>-acetylated chitosans (DA ≈ 60 % and ≈ 70 %) exhibited full water solubility even at neutral pH, which can expand the biomedical applications of chitosans. </p>

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