scholarly journals The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme

1972 ◽  
Vol 126 (3) ◽  
pp. 739-745 ◽  
Author(s):  
E D Corte ◽  
F. Stirpe
Keyword(s):  
Enzyme Activity ◽  
Xanthine Oxidase ◽  
Rat Liver ◽  
Copper Sulphate ◽  
Liver Homogenate ◽  
Nitrobenzoic Acid ◽  
Complete Inactivation ◽  
Type D ◽  
Pigeon Liver ◽  
Lung And Kidney

1. The ‘xanthine oxidase’ activity of rat liver supernatant, most of which behaves as an NAD+-dependent dehydrogenase (type D) can be rapidly converted into an oxidase (type O) by thiol reagents such as tetraethylthiuram disulphide, copper sulphate, 5,5′-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercuribenzoate. Treatment with copper sulphate, if prolonged, leads to almost complete inactivation of the enzyme. The effect of these reagents is prevented by dithioerythritol, and in all cases but that of N-ethylmaleimide is reversed by the same thiol. 2. Dithioerythritol prevents and reverses the conversion of xanthine oxidase from type D into type O brought about by storage of rat liver supernatant at -20°C, preincubation under anaerobic conditions, treatment with carbon or with diethyl ether, and reverses, but does not prevent, the conversion obtained by preincubation of the whole liver homogenate. 3. Conversion of the enzyme from type D into type O is effected by preincubation of rat liver supernatant with the sedimentable fraction from rat liver but not from chick or pigeon liver. The xanthine dehydrogenase activity of chick liver supernatant is not changed into an oxidase by preincubation with the sedimentable fraction from rat liver. 4. The enzyme activity of rat liver supernatant is converted from type D into type O during purification of the enzyme: the purified enzyme can be reconverted into type D by dithioerythritol. 5. The enzyme appears as an oxidase in the supernatant of rat heart, intestine, spleen, pancreas, lung and kidney. The enzyme of all organs but intestine can be converted into a dehydrogenase by dithioerythritol.

scholarly journals Inhibition of the thrombin–fibrinogen reaction by a macromolecular factor from rat liver

1972 ◽  
Vol 129 (1) ◽  
pp. 83-89 ◽  
Author(s):  
Ragnar Flengsrud ◽  
Bjarne Østerud ◽  
Hans Prydz
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Competitive Inhibition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Kinetic Studies ◽  
Disc Electrophoresis ◽  
Nitrobenzoic Acid

1. The supernatant obtained by centrifugation of a rat liver homogenate at 100000g for 1h contained a heat-labile macromolecular inhibitor of the thrombin–fibrinogen reaction. 2. The inhibitor was purified to electrophoretic homogeneity by repeated preparative polyacrylamide disc electrophoresis. Inhibition was observed with purified inhibitor equivalent to about 1μg of protein/ml. 3. The inhibitor had a pI of 3.50–3.75, a molecular weight (from sodium dodecyl sulphate–polyacrylamide-gel electrophoresis) of 72000±3000 and was inactivated by p-hydroxymercuribenzoate or 5,5′-dithiobis-(2-nitrobenzoic acid). 4. Kinetic studies revealed a non-competitive inhibition, with the inhibitor probably acting on the thrombin–fibrinogen complex.

scholarly journals The function of subcellular fractions in the oxidation of glutathione in rat liver homogenate

1970 ◽  
Vol 117 (5) ◽  
pp. 951-956 ◽  
Author(s):  
P. C. Jocelyn
Keyword(s):  
Uric Acid ◽  
Xanthine Oxidase ◽  
Rat Liver ◽  
Liver Homogenate ◽  
No Oxidation ◽  
Urate Oxidase ◽  
Supernatant Fraction ◽  
Endogenous Substrate ◽  
Additional Protein ◽  
Microsome Fraction

1. The aerobic loss of GSH added to the supernatant fraction from rat liver is much increased by including the microsome fraction, which both inhibits the concurrent reduction of the GSSG formed and also augments the net oxidation rate. 2. Oxidation occurs with a mixture of dialysed supernatant and a protein-free filtrate; the latter is replaceable by hypoxanthine and the former by xanthine oxidase, whereas fractions lacking this enzyme give no oxidation. 3. In all these instances augmentation occurs with microsomes, with fractions having urate oxidase activity and with the purified enzyme; uric acid and microsomes alone also support the oxidation. 4. Evidence implicating additional protein factors is discussed. 5. It is suggested that GSH oxidation by homogenate is linked through glutathione peroxidase to the reaction of endogenous substrate with supernatant xanthine oxidase and of the uric acid formed with peroxisomal urate oxidase.

Activity and stability of rat liver xanthine oxidase in the presence of pyridine

2011 ◽  
Vol 89 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Kaveh Amini ◽  
Mohammad-Hossein Sorouraddin ◽  
Mohammad-Reza Rashidi
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Xanthine Oxidase ◽  
Rat Liver ◽  
Oxidase Activity ◽  
Enzyme Inactivation ◽  
Native Enzyme ◽  
Buffer Solution ◽  
Stability Parameters ◽  
Xanthine Oxidase Activity

In the present study, rat liver xanthine oxidase activity and its thermostability in the presence of pyridine were investigated. The activity of the enzyme was determined by following the formation of uric acid spectrophotometrically. The thermal stability of the enzyme was studied in the presence of 0.0%–2.0% of pyridine in Sorenson’s buffer. Thermal stability parameters (half-life, inactivation constant, and activation energies for enzyme inactivation), thermodynamic constants (ΔH*, ΔS*, and ΔG*) and the kinetic parameters (Km and Vmax), were determined in pyridine-free and pyridine-containing buffer solution. A dramatic reduction was observed in xanthine oxidase activity in the presence of pyridine. However, the pyridine-treated enzyme showed a marked enhancement in thermal stability compared with the native enzyme. The ΔG values for the enzyme activity in the presence of pyridine were found to be about 1.5-fold larger than that calculated for the native enzyme, indicating that the enzyme becomes kinetically more stable in the presence of pyridine. The Km value for xanthine oxidase in the presence of 0.5% pyridine increased by 4.8-fold compared with the enzyme in the pyridine-free buffer solution; however, there was 1.8-fold reduction in the Vmax value in the hydro-organic solution compared with the enzyme activity in the buffer solution. As the stability of enzymes is one of the most difficult problems in protein chemistry, this thermostability property of xanthine oxidase could be of great value in developing novel strategies to improve and expand its application in various areas.

scholarly journals Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase). Purification, interconversion and some properties

1973 ◽  
Vol 131 (2) ◽  
pp. 191-198 ◽  
Author(s):  
M. G. Battelli ◽  
E. Lorenzoni ◽  
F. Stirpe
Keyword(s):  
Xanthine Oxidase ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Small Extent ◽  
Kinetic Constants ◽  
Cow’S Milk ◽  
Dihydrolipoic Acid ◽  
Cow's Milk ◽  
Type D ◽  
A Minor

1. The xanthine oxidase of cow's milk, crude or purified, appears as an oxidase (type O), and can be converted almost completely into a NAD+-dependent dehydrogenase (type D) by treatment with dithioerythritol or dihydrolipoic acid, but only to a small extent by other thiols. 2. The D form of the enzyme is inhibited by NADH, which competes with NAD+. 3. The kinetic constants of the two forms of the enzyme are similar to those of the corresponding forms of rat liver xanthine oxidase. 4. Milk xanthine oxidase is converted into an irreversible O form by pretreatment with chymotrypsin, papain or subtilisin, but only partially with trypsin. 5. The enzyme as purified shows a major faster band and a minor slower band on gel electrophoresis. The slower band is greatly reinforced after xanthine oxidase is converted into the irreversible O form by chymotrypsin.

scholarly journals The enzymic oxidation of glutathione in rat liver homogenates

1970 ◽  
Vol 117 (5) ◽  
pp. 947-949 ◽  
Author(s):  
P. C. Jocelyn
Keyword(s):  
Xanthine Oxidase ◽  
Rat Liver ◽  
Aerobic Oxidation ◽  
Liver Homogenate ◽  
Enzymic Oxidation ◽  
Liver Homogenates

1. The aerobic oxidation of GSH and other thiols by rat liver homogenate is abolished either by previous dialysis or by removal of the proteins but is restored by a mixture of the protein-free filtrate and the dialysed homogenate. 2. The oxidation is prevented by previously heating the dialysed homogenate but not the protein-free filtrate and also by known inhibitors of xanthine oxidase. 3. A similar oxidation occurs with hypoxanthine in place of of protein-free filtrate.

scholarly journals The regulation of xanthine oxidase. Inhibition by reduced nicotinamide–adenine dinucleotide of rat liver xanthine oxidase type D and of chick liver xanthine dehydrogenase

1970 ◽  
Vol 117 (1) ◽  
pp. 97-100 ◽  
Author(s):  
E. Della Corte ◽  
F. Stirpe
Keyword(s):  
Xanthine Oxidase ◽  
Rat Liver ◽  
Nicotinamide Adenine Dinucleotide ◽  
Enzyme Activities ◽  
Xanthine Dehydrogenase ◽  
Nicotinamide Adenine ◽  
Type D ◽  
Oxidase Inhibition ◽  
Xanthine Oxidase Inhibition ◽  
Reduced Nicotinamide Adenine Dinucleotide

1. Rat liver xanthine oxidase type D (NAD+-dependent) and chick liver xanthine oxidase are inhibited by NADH, which competes with NAD+. 2. The addition of a NADH-reoxidizing system in the assay of these enzyme activities is proposed. 3. Rat liver xanthine oxidase type O (oxygen-dependent) is not affected by NADH.

scholarly journals The nature of inactivation of rat muscle 5'-adenylate aminohydrolase by fluorodinitrobenzene

1975 ◽  
Vol 145 (2) ◽  
pp. 145-151 ◽  
Author(s):  
A Raggi ◽  
C Bergamini ◽  
G Ronca
Keyword(s):  
Enzyme Activity ◽  
Kinetic Properties ◽  
Hydroxyl Groups ◽  
Amp Deaminase ◽  
Thiol Groups ◽  
Tyrosine Residues ◽  
Nitrobenzoic Acid ◽  
Complete Inactivation ◽  
Lysine Residues ◽  
Phenolic Hydroxyl Groups

1. The inactivation of rat skeletal muscle AMP deaminase by Dnp-F (1-fluoro-2,4-dinitrobenzene) is accompanied by the arylation of thiol, amino and phenolic hydroxyl groups. 2. The number of thiol groups that react with Dnp-F is about 12; this is the number that reacts with Nbs2 [5,5′-dithiobis-(2-nitrobenzoic acid)] and N-ethylmaleimide without loss of enzyme activity, and it appears to be the same thiol groups that all three reagents attack. 3. Dinitrophenylation of these reactive SH groups is not the cause of inactivation, since active N-ethylmaleimide-substituted enzyme is also inactivated by Dnp-F.4. Complete inactivation of the N-ethylmaleimide-treated AMP deaminase occurs when about six tyrosine and two lysine residues are dinitrophenylated. 5. Since the treatment of Dnp-enzyme with 2-mercaptoethanol restores much of the enzyme activity, inactivation of AMP deaminase by Dnp-F is probably largely due to modification of tyrosine residues. 6. The kinetic properties of the Dnp-enzyme indicate that a marked decrease in V occurs only after extensive enzyme modification. The decreased activity after slight inactivation results from modification of Km.

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