scholarly journals Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases

1970 ◽  
Vol 120 (4) ◽  
pp. 809-814 ◽  
Author(s):  
F. J. Ballard
Keyword(s):  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Mitochondrial Enzymes ◽  
High Concentration ◽  
Michaelis Constant ◽  
Bivalent Cation ◽  
Sheep Liver ◽  
Michaelis Constants

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg2+ as bivalent cation, the Michaelis constant was approx. 2.5×10−5m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn2+ replaced Mg2+ in the reaction a lower Michaelis constant of 9×10−6m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn2+ was the added cation. With Mg2+ the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.

scholarly journals Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase

1979 ◽  
Vol 177 (3) ◽  
pp. 833-846 ◽  
Author(s):  
M C Scrutton ◽  
I Beis
Keyword(s):  
Rat Liver ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Detectable Effect ◽  
Formyltetrahydrofolate Dehydrogenase ◽  
Hydrolysis Of

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7–0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.

scholarly journals The mechanism of the reaction between glutathione and 1-menaphthyl sulphate catalysed by a glutathione S-transferase from rat liver

1973 ◽  
Vol 135 (4) ◽  
pp. 797-804 ◽  
Author(s):  
Brian Gillham
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Glutathione S Transferase ◽  
Michaelis Constant ◽  
Dead End ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Inhibition Pattern ◽  
Mechanism Of The Reaction

1. The glutathione S-transferase that catalyses the reaction of 1-menaphthyl (naphth-1-ylmethyl) sulphate with GSH was purified 76-fold from rat liver. 2. The properties of the purified enzyme were studied by gel filtration and isoelectric focusing. 3. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with an Ordered Bi Bi mechanism in which the GSH adds to the enzyme before 1-menaphthyl sulphate and the products are released in the order SO42−followed by S-(1-menaphthyl)glutathione. 4. Dead-end-inhibition studies with p-aminobenzoic acid, which has been shown to be competitive with GSH and non-competitive with 1-menaphthyl sulphate, support the suggestion that an Ordered Bi Bi mechanism is operative. 5. Values were determined for some of the dissociation and Michaelis constants for the reaction of the substrates and products with the enzyme. 6. It appears that S-(1-menaphthyl)glutathione activates the enzyme when the concentration of GSH is saturating and that of 1-menaphthyl sulphate is low (of the order of its Michaelis constant).

scholarly journals Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase

1978 ◽  
Vol 171 (3) ◽  
pp. 533-538 ◽  
Author(s):  
A K H MacGibbon ◽  
S J Haylock ◽  
P D Buckley ◽  
L F Blackwell
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Sheep Liver ◽  
Rate Limiting Step ◽  
Dehydrogenase Reaction ◽  
Steady State Rate ◽  
Michaelis Constants ◽  
State Rate ◽  
Liver Aldehyde Dehydrogenase

The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.

scholarly journals Properties of Lubrol-extracted uridine diphosphate glucuronyltransferase

1971 ◽  
Vol 125 (4) ◽  
pp. 991-997 ◽  
Author(s):  
R. D. Howland ◽  
A. Burkhalter ◽  
A. J. Trevor ◽  
S. Hegeman ◽  
D. Y. Shirachi
Keyword(s):  
Rat Liver ◽  
Uridine Diphosphate ◽  
Soluble Enzyme ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Ionic Detergent ◽  
Michaelis Constants

1. A partially purified UDP-glucuronyltransferase was obtained by extracting rat liver microsomal preparations with Lubrol, a non-ionic detergent. 2. The soluble enzyme catalysed conjugation of both o-aminophenol and p-nitrophenol and was extremely stable when compared with untreated microsomal preparations. 3. The characteristics of the conjugation of the two phenols were found to differ with respect to pH optimum, bivalent cation requirement and Michaelis constants, suggesting that more than one enzyme is involved in the conjugation reaction.

scholarly journals Kinetic studies of nitrogenase from soya-bean root-nodule bacteroids

1973 ◽  
Vol 131 (1) ◽  
pp. 61-75 ◽  
Author(s):  
F. J. Bergersen ◽  
G. L. Turner
Keyword(s):  
Reaction Mechanism ◽  
Protein Concentration ◽  
Root Nodule ◽  
Maximum Velocity ◽  
Kinetic Studies ◽  
Soya Bean ◽  
Michaelis Constant ◽  
Fe Protein ◽  
Catalytic Sites ◽  
Michaelis Constants

The apparent Michaelis constants [K′(N2) and K′(C2H2)] and the corresponding apparent maximum velocity values (V′) for soya-bean bacteroid nitrogenase increased concomitantly in response to increases in nitrogenase Fe–protein concentration and ATP concentration in cell-free assays and in response to O2 pressure in intact nodules and bacteroid suspensions. K′(C2H2) in cell-free assays was also affected by pH and by Na2S2O4 concentration. Nitrogenase Fe–protein behaved as a catalytic effector reacting at interacting sites on the nitrogenase Fe–Mo–protein. The results indicated that the Fe–Mo–protein probably bears the catalytic sites for N2 and C2H2 reduction. It is concluded that reduction of N2 or C2H2 by this nitrogenase involves a reaction mechanism with a sequence of unknown order. The sequence in which substrate, enzyme, effector, ATP and reductant react determines which of the various rate-constants are involved in the apparent Michaelis constant, whose true kinetic meaning was thus unresolved.

scholarly journals Synthesis of phosphoenolpyruvate from propionate in sheep liver

1971 ◽  
Vol 124 (5) ◽  
pp. 867-876 ◽  
Author(s):  
R. M. Smith ◽  
W. S. Osborne-White
Keyword(s):  
Rat Liver ◽  
Malic Enzyme ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Liver Cytosol ◽  
Sheep Liver ◽  
Mitochondrial Aconitase ◽  
Low Activity ◽  
Very High

1. Utilization of propionate by sheep liver mitochondria was stimulated equally by pyruvate or α-oxoglutarate, with formation predominantly of malate. Pyruvate increased conversion of propionate carbon into citrate, whereas α-oxoglutarate increased formation of phosphoenolpyruvate. The fraction of metabolized propionate converted into phosphoenolpyruvate was about 17% in the presence or absence of α-oxoglutarate and about 7% in the presence of pyruvate. Pyruvate consumption was inhibited by 80% by 5mm-propionate. 2. Compared with rat liver, sheep liver was characterized by very high activities of phosphoenolpyruvate carboxykinase and moderately high activities of aconitase in the mitochondria and by low activities of ‘malic’ enzyme, pyruvate kinase and lactate dehydrogenase in the cytosol. Activities of phosphoenolpyruvate carboxy-kinase were similar in liver cytosol from rats and sheep. Activities of malate dehydrogenase and NADP-linked isocitrate dehydrogenase in sheep liver were about half those in rat liver. 3. The phosphate–dicarboxylate antiport was active in sheep liver mitochondria, but compared with rat liver mitochondria the citrate–malate antiport showed only low activity and mitochondrial aconitase was relatively inaccessible to external citrate. The rate of swelling of mitochondria induced by phosphate in solutions of ammonium malate was inversely related to the concentration of malate. 4. The results are discussed in relation to gluconeogenesis from propionate in sheep liver. It is proposed that propionate is converted into malate by the mitochondria and the malate is converted into phosphoenolpyruvate by enzymes in the cytosol. In this way sufficient NADH would be generated in the cytosol to convert the phosphoenolpyruvate into glucose.

Peroxidases from Tulip Bulbs (Tulipa fosteriana, L.) Oxidize Xenobiotics NNitrosodimethylamine and N-Nitroso-N-methylaniline in vitro

1997 ◽  
Vol 62 (11) ◽  
pp. 1804-1814 ◽  
Author(s):  
Marie Stiborová ◽  
Hana Hansíková
Keyword(s):  
Cytochromes P450 ◽  
Kinetic Studies ◽  
The Other ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
Other Hand ◽  
Plant Peroxidases ◽  
Tulip Bulbs

Tulip bulbs (Tulipa fosteriana, L.) contain peroxidases catalyzing the oxidation of the xenobiotics N-nitrosodimethylamine (NDMA) and N-nitroso-N-methylaniline (NMA). Three anionic (A1, A2, A3) and four cationic (B, C, D, E) peroxidases were purified from this tissue, partially characterized and used for kinetic studies. Demethylation of NDMA and NMA producing formaldehyde is catalyzed by one anionic (A1) and three cationic (C, D, E) peroxidases. The oxidation of NDMA by tulip peroxidases exhibits the Michaelis-Menten kinetics. The apparent Michaelis constant and the maximal velocity values for this substrate were determined. On the other hand, non-Michaelian kinetics for the NMA oxidation were observed with tulip peroxidases. The most abundant cationic peroxidase (peroxidase C) was used for detailed enzymatic studies. In addition to formation of formaldehyde, methylaniline, aniline, 4-aminophenol and phenol were found to be metabolites formed from NMA. Phenol was formed presumably by N-demethylation via a benzenediazonium ion, while methylaniline, aniline and 4-aminophenol were products of denitrosation of the substrate. The efficiencies of plant peroxidases to oxidize NDMA and NMA in vitro are compared with those of cytochromes P450 and discussed.

scholarly journals Alterations in translatable messenger RNA coding for phosphoenolpyruvate carboxykinase (GTP) in rat liver cytosol during deinduction.

1978 ◽  
Vol 253 (12) ◽  
pp. 4327-4332
Author(s):  
D. Kioussis ◽  
L. Reshef ◽  
H. Cohen ◽  
S.M. Tilghman ◽  
P.B. Iynedjian ◽  
...  
Keyword(s):  
Rat Liver ◽  
Messenger Rna ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Liver Cytosol ◽  
Rat Liver Cytosol

Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

1971 ◽  
Vol 17 (3) ◽  
pp. 183-191 ◽  
Author(s):  
Philip J Garry
Keyword(s):  
Enzyme Activity ◽  
Sodium Fluoride ◽  
Phosphate Buffer ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Serum Cholinesterase ◽  
Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

scholarly journals Catalytic wet oxidation of high concentration pharmaceutical wastewater with Fe3+ as catalyst

2018 ◽  
Vol 2017 (3) ◽  
pp. 661-666
Author(s):  
Xu Zeng ◽  
Jun Liu ◽  
Jianfu Zhao
Keyword(s):  
Oxygen Pressure ◽  
Batch Reactor ◽  
Oxygen Demand ◽  
Kinetic Studies ◽  
Cod Removal ◽  
Wet Oxidation ◽  
Catalytic Wet Oxidation ◽  
Pharmaceutical Wastewater ◽  
High Concentration ◽  
Temperature Time

Abstract Catalytic wet oxidation of high concentration pharmaceutical wastewater with Fe3+ as catalyst was carried out in a batch reactor. Results showed that the degradation of pharmaceutical wastewater was enhanced significantly by Fe3+. The effects of reaction parameters, such as the catalyst dose, reaction temperature, time, and initial oxygen pressure, were discussed. The chemical oxygen demand (COD) removal increased with the increases of catalyst dose, temperature, time and oxygen supply. With the initial COD 34,000–35,000 mg/L, approximately 70% COD removal can be achieved under the conditions of catalyst 1.0 g and oxygen pressure 1.0 MPa at 250 °C after 60 min. The results of kinetic studies showed that two reaction steps existed in this oxidation process, which followed an apparent first-order rate law. This process provides an effective approach for the pretreatment of high concentration pharmaceutical wastewater.

Sign in / Sign up

Export Citation Format

Share Document