scholarly journals Purification and some properties of glyoxylate reductase (NADP+) and its functional location in mitochondria in Euglena gracilis z

1985 ◽  
Vol 227 (1) ◽  
pp. 211-216 ◽  
Author(s):  
A Yokota ◽  
S Haga ◽  
S Kitaoka
Keyword(s):  
Gel Electrophoresis ◽  
Glyoxylate Cycle ◽  
Polyacrylamide Gel Electrophoresis ◽  
Intermembrane Space ◽  
Difference Spectra ◽  
Inner Membrane ◽  
Optimum Ph ◽  
Glycollate Dehydrogenase ◽  
Euglena Gracilis Z ◽  
Glyoxylate Reductase

Euglena mitochondria contain both glyoxylate reductase (NADP+) and glycollate dehydrogenase to constitute the glycollate-glyoxylate cycle [Yokota & Kitaoka (1979) Biochem. J. 184, 189-192]. Euglena glyoxylate reductase (NADP+) was purified and its submitochondrial location was determined in order to elucidate the cycle. The purified glyoxylate reductase was homogeneous on polyacrylamide-gel electrophoresis. Difference spectra of the purified enzyme revealed that the enzyme was a flavin enzyme. The Mr of the enzyme was 82 000. The enzyme was specific for NADPH, with an apparent Km of 3.9 microM, and for glyoxylate, with an apparent Km of 45 microM. It was 30% as active with oxaloacetate as with glyoxylate. NADH and hydroxypyruvate did not support the activity at all. The optimum pH was 6.45. Submitochondrial fractionation of purified mitochondria showed that the enzyme was located in the intermembrane space and loosely bound to the outer surface of the inner membrane. These properties and the submitochondrial localization of NADPH-glyoxylate reductase facilitate the operation of the glycollate-glyoxylate cycle in combination with glycollate dehydrogenase, which is tightly bound to the inner membrane of Euglena mitochondria.

β-D-Xylanases of Bacillus circulans WL-12

1982 ◽  
Vol 28 (7) ◽  
pp. 733-739 ◽  
Author(s):  
R. Esteban ◽  
J. R. Villanueva ◽  
T. G. Villa
Keyword(s):  
Gel Electrophoresis ◽  
Ph Value ◽  
Polyacrylamide Gel Electrophoresis ◽  
Negative Electrode ◽  
Polyacrylamide Gel ◽  
Bacillus Circulans ◽  
Michaelis Constant ◽  
Molecular Weights ◽  
Hydrolysis Products ◽  
Optimum Ph

Bacillus circulans WL-12 secretes two endo-β-D-xylanases (A and B, respectively) (EC 3.2.1.8.) and one β-D-xylosidase (EC 3.2.1.37) when cultured in liquid media with xylan as the sole carbon source. Xylanases A and B have been partially characterized with respect to their main physicochemical parameters and β-D-xylosidase to a lesser extent on account of its low stability. Both endo-β-D-xylanase A and β-D-xylosidase were adsorbed on DEAE-Biogel A, had similar molecular weights (approximately 85 000), and had optimum pH values of 5.5–7, but exhibited different isoelectric points (4.5 for β-D-xylanase A and 4.7 for β-D-xylosidase) and different mobilities in polyacrylamide gel electrophoresis. The apparent Michaelis constant for β-D-xylanase A was 8 mg∙mL−1 and the hydrolysis products produced were xylose, xylobiose, xylotriose, and xylotetraose.The second endo-β-D-xylanase (β-D-xylanase B) bound to CM-Biogel A and exhibited a molecular weight of approximately 15 000 and an optimum pH value in the range of 5.5–7. The isoelectric point was 9.1 and the apparent Michaelis constant was 4 mg∙mL−1. The hydrolysis products produced by this enzyme were xylobiose, xylotriose, and xylotetraose, but never xylose. In polyacrylamide gel electrophoresis at pH 8 the enzyme moved towards the negative electrode.

scholarly journals Arginase from human full-term placenta

1976 ◽  
Vol 159 (3) ◽  
pp. 579-583 ◽  
Author(s):  
R Porta ◽  
C Esposito ◽  
A Martin ◽  
G D Pietra
Keyword(s):  
Gel Electrophoresis ◽  
Competitive Inhibition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Full Term ◽  
Term Placenta ◽  
A Value ◽  
Optimum Ph

Arginase was purified about 1800-fold from extracts of human full-term placenta; the enzyme appeared to be homogenous by disc electrophoresis and molecular-sieve chromatography. The mol. wt. determination by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis yielded a value of 70000 for the most pure and the partially purified enzyme. The human placenta arginase is a metalloenzyme with an optimum pH of 9.1. The Km for L-arginine is 27 mM. L-Ornithine and L-lysine show competitive inhibition with Ki values of 6.3 and 14 mM respectively.

scholarly journals Purification and properties of α-D-mannosidase from the germinated seeds of Medicago sativa (alfalfa)

1980 ◽  
Vol 185 (2) ◽  
pp. 455-462 ◽  
Author(s):  
A Curdel ◽  
F Petek
Keyword(s):  
Medicago Sativa ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Purification Method ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Citrate Phosphate

alpha-Mannosidase of Medicago sativa (alfalfa) was purified 1340-fold. The purification method included dialysis of the crude extract against a citrate/phosphate buffer, pH 3.9, (NH4)SO4 precipitation, hydroxyapatite chromatography, chromatography on Sephadex G-200 and finally a preparatory electrophoresis on polyacrylamide-gel gradient by Doly & Petek's [(1977) J. Chromatogr. 137. 69-81] method. Each step of purification was checked by polyacrylamide-gel disc electrophoresis. The purified enzyme showed a single band, corresponding to alpha-mannosidase activity. alpha-Mannosidase has a mol.wt. 230 000 as estimated by Hedrick & Smith's [(1968) Arch. Biochem. Biophys. 126, 155-164] method and also by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by Weber & Osborn [(1969) J. Biol. Chem. 244, 4406-4412]. The enzyme comprises four subunits of different molecular weight. Optimum pH and Km values were determined with p-nitrophenyl alpha-D-mannoside as substrate. When incubated at a temperature between 20 and 62 degrees C before assay, alpha-mannosidase initially shows an increase in activity. alpha-Mannosidase is stable when the pH is about neutrality. It can be inactivated by several metal ions, including Zn2+. At a pH below 5 the enzyme undergoes irreversible inactivation. The presence of EDTA at acid pH considerably enhances the inactivation of the enzyme. This inactivation due to EDTA can be specifically reversed by incubation with Zn2+.

scholarly journals Purification and properties of Cellvibrio gilvus cellobiose phosphorylase

1983 ◽  
Vol 209 (3) ◽  
pp. 803-807 ◽  
Author(s):  
T Sasaki ◽  
T Tanaka ◽  
S Nakagawa ◽  
K Kainuma
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Competitive Inhibitor ◽  
Polyacrylamide Gel ◽  
Protamine Sulphate ◽  
Purification And Properties ◽  
Cellobiose Phosphorylase ◽  
Optimum Ph

The cellobiose phosphorylase (EC 2.4.1.20) of Cellvibrio gilvus, which is an endocellular enzyme, has been purified 196-fold with a recovery of 11% and a specific activity of 27.4 mumol of glucose 1-phosphate formed/min per mg of protein. The purification procedure includes fractionation with protamine sulphate, and hydroxyapatite and DEAE-Sephadex A-50 chromatography. The enzyme appears homogeneous on polyacrylamide-gel electrophoresis, and a molecular weight of 280 000 was determined by molecular-sieve chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a single band and mol.wt. 72 000, indicating that cellobiose phosphorylase consists of four subunits. The enzyme had a specificity for cellobiose, requiring Pi and Mg2+ for phosphorylation, but not for cellodextrin, gentibiose, laminaribiose, lactose, maltose, kojibiose and sucrose. The enzyme showed low thermostability, an optimum pH of 7.6 and a high stability in the presence of 2-mercaptoethanol or dithiothreitol. The Km values for cellobiose and Pi were 1.25 mM and 0.77 mM respectively. Nojirimycin acted as a powerful pure competitive inhibitor (with respect to cellobiose) of the enzyme (Ki = 45 microM). Addition of thiol-blocking agents to the enzyme caused 56% inhibition at 500 microM-N-ethylmaleimide and 100% at 20 microM-p-chloromercuribenzoate.

Purification and physical properties of glucokinase from Thiobacillus versutus (A2)

1986 ◽  
Vol 32 (12) ◽  
pp. 937-941 ◽  
Author(s):  
David P. Klein ◽  
A. Michael Charles
Keyword(s):  
Physical Properties ◽  
Divalent Cation ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Optimum Temperature ◽  
Original Activity ◽  
Absolute Requirement ◽  
Thiobacillus Versutus ◽  
Optimum Ph ◽  
Temperature Ranges

Glucokinase was purified 749-fold from Thiobacillus versutus. Polyacrylamide gel electrophoresis revealed the presence of five protein bands in the purified preparation. The purified enzyme retained its original activity after 4 weeks of storage at −20 °C, but not at higher temperatures. Glucose provided some protection at 25 °C. The optimum temperature for activity was between 20 and 25 °C, and an energy of activation (Ea) of 1.305 kcal/mol was calculated. A Q10 value of about 1.080 was determined over two 10 °C temperature ranges between 5 and 20 °C. With both Hepes and Tricine buffers, the optimum pH was 7.8. The enzyme was specific for glucose, with a Km of 0.86 mM. The most efficient phosphoryl donor was ATP with a Km of 0.78 mM. About 30% of the activity observed with ATP was obtained with equimolar amounts of ITP, while TTP and UTP gave 7.0 and 12%, respectively. The enzyme displayed an absolute requirement for a divalent cation, with Mg2+ (Km, 0.27 mM) being the most effective.

scholarly journals Occurrence and operation of the glycollate–glyoxylate shuttle in mitochondria of Euglena gracilis Z

1979 ◽  
Vol 184 (1) ◽  
pp. 189-192 ◽  
Author(s):  
A Yokota ◽  
S Kitaoka
Keyword(s):  
Euglena Gracilis ◽  
Higher Plants ◽  
Glycollate Dehydrogenase ◽  
Actual Operation ◽  
Nadph Oxidation ◽  
Euglena Gracilis Z ◽  
Glyoxylate Reductase

Both glyoxylate reductase (NADP+) and glycollate dehydrogenase were located exclusively in mitochondria in Euglena gracilis and constitute the glycollate–glyoxylate shuttle, whose existence in higher plants was thought doubtful, owing to different subcellular locations of the two enzymes. Disrupted Euglena mitochondria showed a glycollate-dependent NADPH oxidation, indicating actual operation of the shuttle in this protozoon.

scholarly journals Purification, partial characterization, and reactivity with aromatic compounds of two laccases fromMarasmius quercophilusstrain 17

2000 ◽  
Vol 46 (3) ◽  
pp. 189-194 ◽  
Author(s):  
A M Farnet ◽  
S Criquet ◽  
S Tagger ◽  
G Gil ◽  
J Le Petit
Keyword(s):  
Thermal Stability ◽  
Aromatic Compounds ◽  
Gel Electrophoresis ◽  
Liquid Culture ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phenol Degradation ◽  
Hydroxybenzoic Acid ◽  
Optimum Ph ◽  
Aromatic Cycle

Two isozymes of laccase were obtained from an induced liquid culture of Marasmius quercophilus with p-hydroxybenzoic acid as the inducer. Both the constitutive and the induced isozyme have a molecular mass of 60 kDa as determined by polyacrylamide gel electrophoresis. Using isoelectric focusing, we found three isozymes with the constitutive enzyme (pI 4, 4.2, 4.4) and four of the induced form (pI 4.75, 4.85, 4.95, 5.1). We observed certain differences between these two isozymes; the specific activity of the induced isozyme was twice as high, and two optimum pH levels (5 and 6) were observed with the induced isozyme (only one, pH 5, for the constitutive isozyme). However, both of these enzymes have the same thermal stability and the same temperature for their highest activity (80°C). Furthermore, the reactivity of both these enzymes with aromatic compounds was similar. The use of mediators extended the oxidized substrate range of the laccases studied. Various products of degradation were observed, depending on the mediator used. When laccase was used alone, the decrease of the signal corresponding to the aromatic cycle, without any formations of other peaks at different wavelengths, suggested polymerisation of aromatic compounds.Key words: laccase, Marasmius quercophilus, mediator, phenol degradation.

Ultrastructural localization of specific nucleoprotein fractions

1978 ◽  
Vol 36 (2) ◽  
pp. 204-205
Author(s):  
G. L. Brown
Keyword(s):  
Gel Electrophoresis ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ultrastructural Localization ◽  
Polyacrylamide Gel ◽  
Acid Extraction ◽  
Acid Solutions ◽  
Low Salt ◽  
Liver Nuclei ◽  
Phosphate Groups

Bismuth (Bi) stains nucleoproteins (NPs) by interacting with available amino and primary phosphate groups. These two staining mechanisms are distinguishable by glutaraldehyde crosslinking (Fig. 1,2).Isolated mouse liver nuclei, extracted with salt and acid solutions, fixed in either formaldehyde (form.) or gl utaraldehyde (glut.) and stained with Bi, were viewed to determine the effect of the extractions on Bi stainina. Solubilized NPs were analyzed by SDS-polyacrylamide gel electrophoresis.Extraction with 0.14 M salt does not change the Bi staining characteristics (Fig. 3). 0.34 M salt reduces nucleolar (Nu) staining but has no effect on interchromatinic (IC) staining (Fig. 4). Proteins responsible for Nu and glut.- insensitive IC staining are removed when nuclei are extracted with 0.6 M salt (Fig. 5, 6). Low salt and acid extraction prevents Bi-Nu staining but has no effect on IC staining (Fig. 7). When nuclei are extracted with 0.6 M salt followed by low salt and acid, all Bi-staining components are removed (Fig. 8).

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