INHIBITION AND ACTIVATION OF ADENYLOSUCCINIC SYNTHETASE BY 8-AZAGUANOSINE TRIPHOSPHATE

1963 ◽  
Vol 41 (1) ◽  
pp. 1495-1501 ◽  
Author(s):  
L. H. Cohen ◽  
R. E. Parks Jr.
Keyword(s):  
Reaction Rate ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
High Concentrations

8-Azaguanosine triphosphate can function in place of GTP in the reaction catalyzed by adenylosuccinic synthetase. The Michaelis constant and maximal velocity for the reaction obtained with the analogue are lower than those obtained with GTP. As a result, 8-azaGTP increases the reaction rate at low GTP concentrations and inhibits competitively at high GTP concentrations. It is suggested that this type of inhibition might be exploited in the development of chemo-therapeutic analogues specific for tissues containing high concentrations of a coenzyme or metabolite.

INHIBITION AND ACTIVATION OF ADENYLOSUCCINIC SYNTHETASE BY 8-AZAGUANOSINE TRIPHOSPHATE

1963 ◽  
Vol 41 (7) ◽  
pp. 1495-1501 ◽  
Author(s):  
L. H. Cohen ◽  
R. E. Parks Jr.
Keyword(s):  
Reaction Rate ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
High Concentrations

8-Azaguanosine triphosphate can function in place of GTP in the reaction catalyzed by adenylosuccinic synthetase. The Michaelis constant and maximal velocity for the reaction obtained with the analogue are lower than those obtained with GTP. As a result, 8-azaGTP increases the reaction rate at low GTP concentrations and inhibits competitively at high GTP concentrations. It is suggested that this type of inhibition might be exploited in the development of chemo-therapeutic analogues specific for tissues containing high concentrations of a coenzyme or metabolite.

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.

Carrier-mediated urea transport across the mitochondrial membrane of an elasmobranch (Raja erinacea) and a teleost (Oncorhynchus mykiss) fish

2008 ◽  
Vol 294 (6) ◽  
pp. R1947-R1957 ◽  
Author(s):  
T. M. Rodela ◽  
J. S. Ballantyne ◽  
P. A. Wright
Keyword(s):  
Oncorhynchus Mykiss ◽  
Mitochondrial Membrane ◽  
Liver Mitochondria ◽  
Reverse Direction ◽  
Maximal Velocity ◽  
Urea Transport ◽  
Submitochondrial Particles ◽  
Raja Erinacea ◽  
Urea Uptake ◽  
High Concentrations

In osmoregulating teleost fish, urea is a minor nitrogen excretory product, whereas in osmoconforming marine elasmobranchs it serves as the major tissue organic solute and is retained at relatively high concentrations (∼400 mmol/l). We tested the hypothesis that urea transport across liver mitochondria is carrier mediated in both teleost and elasmobranch fishes. Intact liver mitochondria in rainbow trout ( Oncorhynchus mykiss) demonstrated two components of urea uptake, a linear component at high concentrations and a phloretin-sensitive saturable component [Michaelis constant ( Km) = 0.58 mmol/l; maximal velocity ( Vmax) = 0.12 μmol·h−1·mg protein−1] at lower urea concentrations (<5 mmol/l). Similarly, analysis of urea uptake in mitochondria from the little skate ( Raja erinacea) revealed a phloretin-sensitive saturable transport ( Km= 0.34 mmol/l; Vmax= 0.054 μmol·h−1·mg protein−1) at low urea concentrations (<5 mmol/l). Surprisingly, urea transport in skate, but not trout, was sensitive to a variety of classic ionophores and respiration inhibitors, suggesting cation sensitivity. Hence, urea transport was measured in the reverse direction using submitochondrial particles in skate. Transport kinetics, inhibitor response, and pH sensitivity were very similar in skate submitochondrial particle submitochondrial particles ( Km= 0.65 mmol/l, Vmax= 0.058 μmol·h−1·mg protein−1) relative to intact mitochondria. We conclude that urea influx and efflux in skate mitochondria is dependent, in part, on a bidirectional proton-sensitive mechanism similar to bacterial urea transporters and reminiscent of their ancestral origins. Rapid equilibration of urea across the mitochondrial membrane may be vital for cell osmoregulation (elasmobranch) or nitrogen waste excretion (teleost).

Heat shock increases cytosolic free Ca2+ concentration via Na(+)-Ca2+ exchange in human epidermoid A 431 cells

1992 ◽  
Vol 263 (1) ◽  
pp. C30-C38 ◽  
Author(s):  
J. G. Kiang ◽  
M. L. Koenig ◽  
R. C. Smallridge
Keyword(s):  
Heat Treatment ◽  
Heat Shock ◽  
Time Dependent ◽  
Maximal Velocity ◽  
Dependent Manner ◽  
Michaelis Constant ◽  
Exchange System ◽  
Maximal Increase ◽  
Normal Cells ◽  
Dimethyl Amiloride

This study characterized cytosolic free Ca2+ concentration ([Ca2+]i) in normal and thermally injured human epidermoid A 431 cells. The resting [Ca2+]i in normal cells at 37 degrees C was 87 +/- 5 nM (n = 105). When cells were subjected to hyperthermia (40-50 degrees C), [Ca2+]i increased in a temperature- and time-dependent manner. The maximal increase in cells exposed to 45 degrees C was observed at 20 min; [Ca2+]i returned to normal within 1 h. The heat-induced [Ca2+]i increase depended on the presence of external Ca2+. La3+ and Cd2+ but not Co2+, verapamil, or nifedipine attenuated the heat-induced [Ca2+]i increase. TMB-8 partially blocked the increase in [Ca2+]i but pertussis toxin and cholera toxin pretreatment did not. The magnitude of the heat-induced [Ca2+]i increase or 45Ca2+ uptake depended on the presence of extracellular Na+. Heat treatment reduced the apparent Michaelis constant for external Ca2+ from 490 +/- 91 to 210 +/- 60 microM, whereas the maximal velocity remained the same. The intracellular Na+ concentration decreased 62.5% after heating. The heat-induced [Ca2+]i increase was completely blocked by amiloride (5 microM) and 5'-(N,N-dimethyl)-amiloride (1 microM). These results suggest heat activates the Na(+)-Ca2+ exchange system so as to increase [Ca2+]i and reduce [Na+]i.

Kinetik und Mechanismus der enzymatischen Dextransynthese und Wirkung der Akzeptoren auf diese Reaktion

1968 ◽  
Vol 23 (6) ◽  
pp. 788-797 ◽  
Author(s):  
K. H. Ebert ◽  
G. Schenk
Keyword(s):  
Reaction Rate ◽  
Polymer Chains ◽  
Low Molecular Weight ◽  
Michaelis Constant ◽  
Acceptor Reaction ◽  
General Applicability ◽  
Polymer Molecules ◽  
Molecular Weights ◽  
Reaction Constants ◽  
Good Agreement

According to the proposed mechanism, the enzymic formation of dextran from sucrose consists of two reaction steps: the propagation reaction forming polymer molecules by an insertion type growth, and the acceptor reaction leading to the termination of the polymer chains. This mechanism is of a more general applicability; it explains hydrolysis, transfer and poly reactions.A complete kinetic analysis is given for the dextran formation and values for the reaction constants Vm, Km and Ka (the Michaelis constant of the acceptor reaction) have been evaluated. Very good agreement between the rate data calculated from the mechanism and the experimental data has been obtained.From experiments with a series of radioactively labelled acceptors it was established that the acceptor reaction, in fact, proceeds as proposed by the mechanism. Further, it was found that strong acceptors, leading to the formation of low molecular weight dextrans, do not inhibit the reaction rate, and that weak acceptors, which inhibit the reaction rate, have only a small effect on the molecular weights. This correlation has also been demonstrated in terms of the proposed reaction mechanism.

Albumin absorption by canine bronchial epithelium

1989 ◽  
Vol 66 (6) ◽  
pp. 2772-2777 ◽  
Author(s):  
L. G. Johnson ◽  
P. W. Cheng ◽  
R. C. Boucher
Keyword(s):  
Gel Filtration ◽  
Fluid Phase ◽  
Bronchial Epithelium ◽  
Bathing Solution ◽  
Michaelis Constant ◽  
Airway Epithelia ◽  
Ussing Chambers ◽  
Bronchial Epithelia ◽  
Surface Liquid ◽  
High Concentrations

Albumin concentrations in airway surface liquid are low compared with plasma. To investigate the mechanisms that generate albumin gradients across airway epithelia, we have investigated whether active albumin absorption is a feature of bronchial epithelia. Freshly excised canine bronchi were mounted in Ussing chambers and short-circuited. Permeability coefficients of 14C-labeled canine albumin (Palb) were measured in the mucosal-to-submucosal (M----S) and submucosal-to-mucosal (S----M) directions in conductance-matched tissues. Mean steady-state values for Palb in the absorptive (M----S) direction (5.97 +/- 1.89 x 10(-7) cm/s) were significantly greater than rates in the S----M direction (1.09 +/- 0.41 x 10(-7) cm/s). Simultaneous measurements detected no asymmetry of transport of the fluid phase marker [3H]inulin. Gel filtration chromatography demonstrated that the majority of the radiolabel released into the submucosal bathing solution represented albumin fragments. Albumin fragments per se were not transported because no asymmetries in permeabilities of albumin fragments isolated from spontaneous degradation of tracer were detected. Decreasing the temperature of the bathing solution from 37 to 4 degrees C completely inhibited net albumin absorption. [14C]albumin transport was saturated by addition of high concentrations of unlabeled albumin (estimated Michaelis constant = 1.6 x 10(-3) M). These results demonstrate that albumin is absorbed by a low-affinity process that may contribute to the maintenance of low albumin concentrations in secretions.

Neurohormonal regulation of histamine synthesis in isolated rabbit fundic mucosal cells

1994 ◽  
Vol 266 (3) ◽  
pp. G395-G402
Author(s):  
F. Hollande ◽  
J. P. Bali ◽  
R. Magous
Keyword(s):  
Endocrine Cells ◽  
Inhibitory Concentration ◽  
Histidine Decarboxylase ◽  
Effective Concentration ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
High Potency ◽  
Cholecystokinin Octapeptide ◽  
Muscarinic Receptor Antagonists ◽  
Mucosal Cells

In a population of rabbit fundic mucosal cells enriched in mucous and endocrine cells, gastrin and cholecystokinin octapeptide (CCK-8) were shown to increase dose-dependently histidine decarboxylase (HDC) activity with the same efficacy and high potencies [50% effective concentration (EC50) 0.389 +/- 0.041 and 0.275 +/- 0.011 nM, respectively], whereas pentagastrin was less potent (EC50 2.90 +/- 0.13 nM). L-365,260 and PD-135,666 inhibited gastrin- and CCK-8-stimulated HDC activity with a high potency [50% inhibitory concentration (IC50) 1.00 +/- 0.08 and 4.2 +/- 0.7 nM for gastrin-stimulated and 1.95 +/- 0.21 and 1.78 +/- 0.12 nM for CCK-8-stimulated HDC activity, respectively], whereas L-364,718 was 50 to 100 times less potent (EC50 100 +/- 2.5 and 91.2 +/- 3.1 nM, respectively on gastrin- and CCK-8-stimulated HDC activity). Carbachol also dose-dependently increased HDC activity (EC50 7.08 +/- 0.32 nM), and its effect was reversed by selective muscarinic-receptor antagonists with the following order of potency: pirenzepine (IC50 15.1 +/- 1.2 nM) > para-fluoro-hexahydro-siladifenidol (IC50 0.316 +/- 0.02 microM) > 11-2[(2-[(diethyl-amino)-methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (IC50 28.5 +/- 1.1 microM). Moreover, gastrin and carbachol were able to modify slightly but significantly both the Michaelis constant (Km) and the maximal velocity (Vmax) of HDC in the same way (18-20% reduction of the Km and 25-30% increase of the Vmax).(ABSTRACT TRUNCATED AT 250 WORDS)

Exponential function for calculating saturable enzyme kinetics.

1988 ◽  
Vol 34 (12) ◽  
pp. 2486-2489 ◽  
Author(s):  
F Keller ◽  
C Emde ◽  
A Schwarz
Keyword(s):  
Steady State ◽  
Enzyme Kinetics ◽  
Exponential Function ◽  
Association Constant ◽  
Time Dependent ◽  
Mathematical Representation ◽  
Hyperbolic Function ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
General Meaning

Abstract Enzyme kinetics are usually described by the Michaelis-Menten equation, where the time-dependent decrease of substrate (-dS/dt) is a hyperbolic function of maximal velocity (Vmax), Michaelis constant (Km), and amount of substrate (S). Because the Michaelis-Menten function in its most general meaning requires an assumption of steady-state, it is less curvilinear than true enzyme kinetics. A saturation-type exponential function is more curvilinear than the hyperbolic function and more closely approximates enzyme kinetics: -dS/dt = Vmax [1 - exp(-S/Km)]. The mathematical representation of enzyme kinetics can be further improved by introducing a deceleration term (Vdec), to make the assumption of a steady state unnecessary. For the action of chymotrypsin on N-acetyltyrosylethylester, the Michaelis-Menten equation yields the following: Vmax = 3.74 mumol/min and Km = 833 mumol. According to decelerated enzyme kinetics, the values Vmax = 4.80 mumol/min, Vdec = 0.0118 mumol/min, and the association constant (Ka) = 0.00111/mumol are more nearly accurate for this reaction (where 1/Ka = 901 mumol approximately Km).

scholarly journals Syncatalytic inactivation of prolyl 4-hydroxylase by anthracyclines

1988 ◽  
Vol 251 (2) ◽  
pp. 365-372 ◽  
Author(s):  
V Günzler ◽  
H M Hanauske-Abel ◽  
R Myllylä ◽  
D D Kaska ◽  
A Hanauske ◽  
...  
Keyword(s):  
Reaction Rate ◽  
Total Radioactivity ◽  
Human Origin ◽  
Iron Ion ◽  
First Order ◽  
Irreversible Inhibitors ◽  
Semiquinone Radicals ◽  
Enzyme Subunits ◽  
High Concentrations ◽  
Reaction Cycles

The anthracyclines doxorubicin and daunorubicin were found to act as irreversible inhibitors of prolyl 4-hydroxylase. The reaction rate for enzyme from both chick and human origin was first order, the concentration of inhibitor giving 50% inhibition being 60 microM for both compounds after 1 h. The effect was dependent on the presence of iron ions in the reaction mixture. Inactivation could be prevented by addition of high concentrations of ascorbate, but not 2-oxoglutarate, before the inactivation period. The same results were obtained with competitive analogues of these cosubstrates. Lysyl hydroxylase from chick embryos was also susceptible to inactivation. Its activity was decreased by 50% after incubation for 1 h with a 150 microM concentration of the inhibitors. When chick-embryo prolyl 4-hydroxylase was incubated with [14-14C]doxorubicin, both enzyme subunits were radioactively labelled, about 70% of the total radioactivity being found in the alpha-subunit. Since the anthracyclines are known to undergo a redox reaction generating semiquinone radicals with Fe3+ only, the results suggest that the enzyme-bound iron ion is oxidized to a tervalent intermediate in uncoupled reaction cycles. The data also suggest that both enzyme subunits contribute to the catalytic site of prolyl 4-hydroxylase.

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