scholarly journals Involvement of a single thiol group in the conversion of the NAD+-dependent activity of rat liver xanthine oxidoreductase to the O2-dependent activity

1982 ◽  
Vol 207 (2) ◽  
pp. 341-346 ◽  
Author(s):  
Z W Kamiński ◽  
M M Jezewska
Keyword(s):  
Rat Liver ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Xanthine Oxidoreductase ◽  
Complete Protection ◽  
First Order ◽  
Nitrobenzoic Acid ◽  
Modification Process ◽  
Dependent Activity ◽  
Iodosobenzoic Acid

The effects of 2-iodosobenzoic acid, 4-chloromercuribenzoate, 5,5′-dithiobis-(2-nitrobenzoic acid) and tetraethylthioperoxydicarbonic diamide (disulphiram) on the NAD+-dependent activity of xanthine oxidoreductase from rat liver were investigated. Only disulphiram converted the NAD+-dependent activity into the O2-dependent activity quantitatively, without changing the xanthine hydroxylation rate. The modification process was a first-order reaction with respect to time (min) and disulphiram concentration (microM). The kinetic data showed that modification of single thiol group is sufficient for loss of the enzymic activity towards NAD+ as electron acceptor. The complete protection afforded by NAD+ against the action of disulphiram suggests that the essential thiol group may be involved in binding of NAD+ to the xanthine oxidoreductase molecule.

scholarly journals Effect of NADH on hypoxanthine hydroxylation by native NAD+-dependent xanthine oxidoreductase of rat liver, and the possible biological role of this effect

1981 ◽  
Vol 200 (3) ◽  
pp. 597-603 ◽  
Author(s):  
Z W Kamiński ◽  
M M Jezewska
Keyword(s):  
Uric Acid ◽  
Rat Liver ◽  
Time Course ◽  
De Novo ◽  
Xanthine Oxidoreductase ◽  
Reaction Sequence ◽  
Oxidoreductase Activity ◽  
Dependent Activity ◽  
Sepharose 4B

The course of the reaction sequence hypoxanthine leads to xanthine leads to uric acid, catalysed by the NAD+-dependent activity of xanthine oxidoreductase, was investigated under conditions either of immediate oxidation of the NADH formed or of NADH accumulation. The enzymic preparation was obtained from rat liver, and purified 75-fold (as compared with the 25000 g supernatant) on a 5′-AMP-Sepharose 4B column; in this preparation the NAD+-dependent activity accounted for 100% of total xanthine oxidoreductase activity. A spectrophotometric method was developed for continuous measurements of changes in the concentrations of the three purines involved. The time course as well as the effects of the concentrations of enzyme and of hypoxanthine were examined. NADH produced by the enzyme lowered its activity by 50%, resulting in xanthine accumulation and in decreases of uric acid formation and of hypoxanthine utilization. The inhibition of the Xanthine oxidoreductase NAD+-dependent activity by NADH is discussed as a possible factor in the regulation of IMP biosynthesis by the ‘de novo’ pathway or (from unchanged hypoxanthine) by ther salvage pathway.

scholarly journals Reversible sensitization and desensitization of carnitine palmitoyltransferase I to inhibition by malonyl-CoA in isolated rat liver mitochondria. Significance for the mechanism of malonyl-CoA-induced sensitization

1983 ◽  
Vol 214 (3) ◽  
pp. 1027-1030 ◽  
Author(s):  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Reduced Glutathione ◽  
Thiol Group ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Nitrobenzoic Acid ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I

Preincubation of rat liver mitochondria with 5,5′-dithiobis-(2-nitrobenzoic acid) (Nbs2) followed by removal of excess reagent by washing the mitochondria with 0.5 mM-reduced glutathione resulted in a desensitization of carnitine palmitoyltransferase (CPT) I activity to malonyl-CoA inhibition. The effect was not observed if mitochondria were washed with 0.5 mM-dithiothreitol. The desensitization effect of Nbs2 could be reversed by a second incubation in the presence of 8 microM-malonyl-CoA. In addition, malonyl-CoA, when present simultaneously with Nbs2, protected CPT I activity against the desensitization effect of the thiol-group reagent. These results suggest that malonyl-CoA exerts an effect on one or more thiol groups of the enzyme, and that this effect is related to the ability of the metabolite to sensitize CPT I to malonyl-CoA inhibition.

scholarly journals Subcellular localization of a rat liver enzyme converting thyroxine into tri-iodothyronine and possible involvement of essential thiol groups

1976 ◽  
Vol 157 (2) ◽  
pp. 479-482 ◽  
Author(s):  
T J Visser ◽  
I Does-Tobé ◽  
R Docter ◽  
G Hennemann
Keyword(s):  
Rat Liver ◽  
Essential Role ◽  
Microsomal Fraction ◽  
Thiol Group ◽  
Subcellular Fractionation ◽  
Enzymic Activity ◽  
Marker Enzyme ◽  
Converting Enzyme ◽  
Thiol Groups ◽  
Liver Homogenates

Experiments with rat liver homogenates showed that on subcellular fractionation the ability to catalyse the conversion of thyroxine into tri-iodothyronine was lost. The activity could in part be restored by addition of the cytosol to the microsomal fraction. Both components were found to be heat labile. The necessity of the presence of cytosol could be circumvented by incorporation of thiol-group-containing compounds in the medium. Optimal enzymic activity was observed in the presence of dithiothreitol and EDTA in medium of low osmolarity. By comparing the distribution of the converting enzyme over the subcellular fractions with a microsomal marker enzyme, glucose 6-phosphatase, it was demonstrated that the former is indeed of microsomal origin. Finally, it was shown that thiol groups play an essential role in the conversion of thyroxine into tri-iodothyronine.

scholarly journals On the reactivity of metallothioneins with 5,5′-dithiobis-(2-nitrobenzoic acid)

1981 ◽  
Vol 193 (2) ◽  
pp. 441-446 ◽  
Author(s):  
T Y Li ◽  
D T Minkel ◽  
C F Shaw ◽  
D H Petering
Keyword(s):  
Metal Ions ◽  
Rat Liver ◽  
Guanidine Hydrochloride ◽  
First Order ◽  
Nitrobenzoic Acid ◽  
Model Complex ◽  
Pseudo First Order ◽  
Hydrochloride Solution ◽  
Metal Ratios ◽  
Good Agreement

Rat liver and horse kidney metallothioneins react with 5,5′-dithiobis-(2-nitrobenzoic acid) (Nbs2) to release 5-thio-2-nitrobenzoate and metal ions. The reactions are slow and exhibit biphasic kinetics with each process having an empirical rate law of the form: rate - k[RSM] X [Nbs2] + k'[RSM], where RSM represents mental-bound thiolate groups. The pseudo-first-order rates are insensitive to pH but are modified in guanidine hydrochloride solution. Rat liver metallothioneins of variable zinc, copper and cadmium composition react similarly and give observable thiol/total metal ratios in good agreement with stoichiometries of SH/(Cd + Zn) of 3 and SH/Cu of 1. A model complex cadmium-2,3-dimercaptopropanol, resembles the proteins in its reaction with Nbs2.

scholarly journals The reactivity of thiol groups and the subunit structure of aldolase

1970 ◽  
Vol 117 (2) ◽  
pp. 291-298 ◽  
Author(s):  
P. J. Anderson ◽  
R. N. Perham
Keyword(s):  
Group Analysis ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Native Enzyme ◽  
Cysteine Residues ◽  
Subunit Structure ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Prolonged Incubation ◽  
Nitrobenzoic Acid

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-14C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5′-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5′-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5′-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.

The effect of polymeric and model imidazolium halides on the rate of hydrolysis of 4-acetoxy-3-nitrobenzoic acid

1985 ◽  
Vol 50 (4) ◽  
pp. 845-853 ◽  
Author(s):  
Miloslav Šorm ◽  
Miloslav Procházka ◽  
Jaroslav Kálal
Keyword(s):  
Catalyst Concentration ◽  
Low Molecular Weight ◽  
Imidazolium Chloride ◽  
First Order ◽  
Nitrobenzoic Acid ◽  
Rate Of Hydrolysis ◽  
Acid Catalyzed ◽  
Hydrolysis Of ◽  
Ethanol In Water ◽  
Direct Attack

The course of hydrolysis of an ester, 4-acetoxy-3-nitrobenzoic acid catalyzed with poly(1-methyl-3-allylimidazolium bromide) (IIa), poly[l-methyl-3-(2-propinyl)imidazolium chloride] (IIb) and poly[l-methyl-3-(2-methacryloyloxyethyl)imidazolium bromide] (IIc) in a 28.5% aqueous ethanol was investigated as a function of pH and compared with low-molecular weight models, viz., l-methyl-3-alkylimidazolium bromides (the alkyl group being methyl, propyl, and hexyl, resp). Polymers IIb, IIc possessed a higher activity at pH above 9, while the models were more active at a lower pH with a maximum at pH 7.67. The catalytic activity at the higher pH is attributed to an attack by the OH- group, while at the lower pH it is assigned to a direct attack of water on the substrate. The rate of hydrolysis of 4-acetoxy-3-nitrobenzoic acid is proportional to the catalyst concentration [IIc] and proceeds as a first-order reaction. The hydrolysis depends on the composition of the solvent and was highest at 28.5% (vol.) of ethanol in water. The hydrolysis of a neutral ester, 4-nitrophenyl acetate, was not accelerated by IIc.

scholarly journals Effects of dl-2-bromopalmitoyl-CoA and bromoacetyl-CoA in rat liver and heart mitochondria. Inhibition of carnitine palmitoyltransferase and displacement of [14C]malonyl-CoA from mitochondrial binding sites

1985 ◽  
Vol 230 (1) ◽  
pp. 169-179 ◽  
Author(s):  
M R Edwards ◽  
M I Bird ◽  
E D Saggerson
Keyword(s):  
Binding Site ◽  
Rat Liver ◽  
Binding Sites ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Heart Mitochondria ◽  
Nitrobenzoic Acid ◽  
Malonyl Coa ◽  
Tissue Differences ◽  
The Relationship

The overt form of carnitine palmitoyltransferase (CPT1) in rat liver and heart mitochondria was inhibited by DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA. S-Methanesulphonyl-CoA inhibited liver CPT1. The inhibitory potency of DL-2-bromopalmitoyl-CoA was 17 times greater with liver than with heart CPT1. Inhibition of CPT1 by DL-2-bromopalmitoyl-CoA was unaffected by 5,5′-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. In experiments in which DL-2-bromopalmitoyl-CoA displaced [14C]malonyl-CoA bound to liver mitochondria, the KD (competing) was 25 times the IC50 for inhibition of CPT1 providing evidence that the malonyl-CoA-binding site is unlikely to be the same as the acyl-CoA substrate site. Bromoacetyl-CoA inhibition of CPT1 was more potent in heart than in liver mitochondria and was diminished by 5,5′-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. Bromoacetyl-CoA displaced bound [14C]malonyl-CoA from heart and liver mitochondria. In heart mitochondria this displacement was competitive with malonyl-CoA and was considerably facilitated by L-carnitine. In liver mitochondria this synergism between carnitine and bromoacetyl-CoA was not observed. It is suggested that bromoacetyl-CoA interacts with the malonyl-CoA-binding site of CPT1. L-Carnitine also facilitated the displacement by DL-2-bromopalmitoyl-CoA of [14C]malonyl-CoA from heart, but not from liver, mitochondria. DL-2-Bromopalmitoyl-CoA and bromoacetyl-CoA also inhibited overt carnitine octanoyl-transferase in liver and heart mitochondria. These findings are discussed in relation to inter-tissue differences in (a) the response of CPT1 activity to various inhibitors and (b) the relationship between high-affinity malonyl-CoA-binding sites and those sites for binding of L-carnitine and acyl-CoA substrates.

scholarly journals Ganglioside incorporation and release by the isolated perfused rat liver

1991 ◽  
Vol 274 (2) ◽  
pp. 581-585 ◽  
Author(s):  
S C Kivatinitz ◽  
A Miglio ◽  
R Ghidoni
Keyword(s):  
Rat Liver ◽  
The Other ◽  
Regulatory Role ◽  
Isolated Perfused Rat Liver ◽  
Order Kinetic ◽  
Perfused Rat Liver ◽  
Ganglioside Gm1 ◽  
First Order ◽  
Pseudo First Order

The fate of exogenous ganglioside GM1 labelled in the sphingosine moiety, [Sph-3H]GM1, administered as a pulse, in the isolated perfused rat liver was investigated. When a non-recirculating protocol was employed, the amount of radioactivity in the liver and perfusates was found to be dependent on the presence of BSA in the perfusion liquid and on the time elapsed after the administration of the ganglioside. When BSA was added to the perfusion liquid, less radioactivity was found in the liver and more in the perfusate at each time tested, for up to 1 h. The recovery of radioactivity in the perfusates followed a complex course which can be described by three pseudo-first-order kinetic constants. The constants, in order of decreasing velocity, are interpreted as: (a) the dilution of the labelled GM1 by the constant influx of perfusion liquid; (b) the washing off of GM1 loosely bound to the surface of liver cells; (c) the release of gangliosides from the liver. Process (b) was found to be faster in the presence of BSA, probably owing to the ability of BSA to bind gangliosides. The [Sph-3H]GM1 in the liver underwent metabolism, leading to the appearance of products of anabolic (GD1a, GD1b) and catabolic (GM2, GM3) origin; GD1a appeared before GM2 and GM3 but, at times longer than 10 min, GM2 and GM3 showed more radioactivity than GD1a. At a given time the distribution of the radioactivity in the perfusates was quite different from that of the liver. In fact, after 60 min GD1a was the only metabolite present in any amount, the other being GM3, the quantity of which was small. This indicates that the liver is able to release newly synthesized gangliosides quite specifically. When a recirculating protocol was used, there were more catabolites and less GD1a than with the non-recirculating protocol. A possible regulatory role of ganglioside re-internalization on their own metabolism in the liver is postulated.

scholarly journals Anomeric specificity of d-glucose phosphorylation by rat liver glucose-6-phosphatase

1989 ◽  
Vol 261 (2) ◽  
pp. 509-513
Author(s):  
R Ramirez ◽  
D Zähner ◽  
G Marynissen ◽  
A Sener ◽  
W J Malaisse
Keyword(s):  
Rat Liver ◽  
Glycogen Synthesis ◽  
Liver Microsomes ◽  
Diabetic Rats ◽  
Enzymic Activity ◽  
Rat Liver Microsomes ◽  
Maximal Velocity ◽  
Carbamoyl Phosphate ◽  
Glucose Phosphorylation ◽  
Anomeric Specificity

The anomeric specificity of D-glucose phosphorylation by hepatic glucose-6-phosphatase was examined in rat liver microsomes incubated in the presence of carbamoyl phosphate. At 10 degrees C, the Km for the equilibrated hexose and phosphate donor was close to 56 mM and 11 mM, respectively. The enzymic activity, which was increased in diabetic rats, was about 40% lower in untreated than in sonicated microsomes. No anomeric difference in affinity was found in sonicated microsomes. In untreated microsomes, however, the Km for beta-D-glucose was slightly lower than that for alpha-D-glucose. The maximal velocity was higher with beta- than alpha-D-glucose in both untreated and sonicated microsomes. These data indicate that the phosphotransferase activity of glucose-6-phosphatase cannot account for the higher rate of glycolysis and glycogen synthesis found in hepatocytes exposed to alpha- rather than beta-D-glucose.

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