scholarly journals Adenylate cyclase in bloodstream forms of Trypanosoma (Trypanozoon) brucei sp

1978 ◽  
Vol 175 (1) ◽  
pp. 207-212 ◽  
Author(s):  
B R Martin ◽  
H P Voorheis ◽  
E L Kennedy
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Trypanosoma Brucei ◽  
Product Inhibition ◽  
Low Concentrations ◽  
Mammalian Enzyme ◽  
High Concentrations ◽  
Opposing Effects ◽  
Substrate Binding Domain

1. The adenylate cyclase in Trypanosoma brucei is located in the plasma membrane. 2. A partial kinetic analysis of the properties of the enzyme revealed a Km for ATP of 1.75 mM and a Km for Mg2+ of 4mM. 3. At low concentrations, Mg2+ activated the enzyme directly in addition to its effect of lowering the concentration of inhibitory free ATP species. 4. At high concentrations, Mg2+ inhibited the enzyme. Furthermore, the enzyme was inhibited at any Mg2+ concentration if the concentration of ATP exceeded that of Mg2+. 5. The opposing effects of Mg2+ at low and high concentrations would be consistent with more than one binding site for Mg2+ on the enzyme. 6. A study of the patterns of product inhibition revealed little or no effect of 3′:5′-cyclic AMP, but a profound inhibition by pyrophosphate, which was competitive with respect to ATP (Ki 0.135 mM). This result suggests that the substrate-binding domain on T. brucei adenylate cyclase interacts mainly with the triphosphate portion of the ATP molecule. 7. The enzyme activity was unaffected by the usual mammalian enzyme effectors glucagon, adrenaline, adenosine, GTP and guanyl-5′-yl imidodiphosphate. 8. The enzyme was not activated by fluoride, instead a powerful inhibition was found. The enzyme was also inhibited by relatively high concentrations of Ca2+ (1 mM).

scholarly journals Insight into the Mechanistic Basis of the Hysteretic-Like Kinetic Behavior of Thioredoxin-Glutathione Reductase (TGR)

2018 ◽  
Vol 2018 ◽  
pp. 1-17 ◽  
Author(s):  
Juan L. Rendón ◽  
Mauricio Miranda-Leyva ◽  
Alberto Guevara-Flores ◽  
José de Jesús Martínez-González ◽  
Irene Patricia del Arenal ◽  
...  
Keyword(s):  
Glutathione Reductase ◽  
Reductase Activity ◽  
Active Form ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Hysteretic Behavior ◽  
Low Concentrations ◽  
Mechanistic Basis ◽  
High Concentrations ◽  
Thioredoxin Glutathione Reductase

A kinetic study of thioredoxin-glutathione reductase (TGR) from Taenia crassiceps metacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproduced in silico.

EGTA-sensitive and -insensitive forms of particulate adenylate cyclase in rat cerebral cortex: regulation by divalent cations and GTP

1985 ◽  
Vol 63 (8) ◽  
pp. 1007-1016 ◽  
Author(s):  
P. V. Sulakhe
Keyword(s):  
Enzyme Activity ◽  
Cerebral Cortex ◽  
Adenylate Cyclase ◽  
Gray Matter ◽  
Rank Order ◽  
Divalent Cations ◽  
Rat Cerebral Cortex ◽  
Chloride Salt ◽  
Low Concentrations ◽  
Stimulation Of

Interactions of several divalent cations (Mn2+, Ca2+, Co2+, Sr2+, and Zn2+) with EGTA-inhibitable adenylate cyclase were investigated in washed membranes (particles) isolated from the gray matter of rat cerebral cortex. The EGTA-inhibitable (called sensitive) enzyme activity was assayed in the presence of Triton X-100 since this detergent caused a marked increase (up to 20-fold) in the enzyme activity. The effects of various divalent metals (all added as chloride salt) indicated the presence of two distinct sites called site I and site II. At low concentrations (less than micromolar) Mn2+, Co2+, and Ca2+ increased (up to 10-fold) the enzyme activity to the same extent and appeared to act via binding to site I (high affinity site). The rank order of affinity was Mn2+ ≥ Co2+ > Ca2+. Zn2+ showed the highest affinity and Sr2+ the lowest towards binding to site I; both these metals increased the enzyme activity to lesser extents than Mn2+, Co2+, or Ca2+. GTP was not required for the stimulation of this enzyme by low concentrations of Ca2+. The interaction of Mn2+ with site II (low affinity site) caused further increase in the enzyme activity, whereas Co2+, Ca2+, and Sr2+ were inhibitory at concentrations >10 μM. Isolated fraction contained loosely and tightly associated pools of calmodulin. Myelin basic protein, but not calcineurin, inhibited the EGTA-sensitive adenylate cyclase activity. The EGTA-insensitive enzyme activity was increased by norepinephrine by mechanisms that depended on GTP and was inhibited by Ca2+. The stimulation of the EGTA-insensitive enzyme modulated the Mg2+ requirement such that Mg2+ binding to the low affinity site (site II) apparently occurred with higher affinity. The likely significance of these results is discussed with regard to (i) the presence of two classes of adenylate cyclase in rat cerebral cortex gray matter and (ii) the regulation of their activities by calmodulin-requiring and GTP-requiring mechanisms.

Opposing actions of the enantiomers of BAY-K-8644 on calcium currents and ACTH secretion in clonal pituitary corticotrophs

1987 ◽  
Vol 65 (12) ◽  
pp. 2409-2414 ◽  
Author(s):  
J. F. MacDonald ◽  
Z. Miljkovic ◽  
S. Heisler
Keyword(s):  
Bay K 8644 ◽  
Calcium Currents ◽  
Similar Relationship ◽  
Acth Secretion ◽  
Calcium Dependent ◽  
Low Concentrations ◽  
Voltage Dependent ◽  
High Concentrations ◽  
Opposing Effects ◽  
Stimulation Of

BAY-K-8644 in low concentrations is known to stimulate, and in higher concentrations, to depress calcium-dependent ACTH secretion from mouse clonal (tumor) pituitary corticotrophs, AtT-20/D16-16 (AtT-20). In the present study, voltage-dependent inward calcium currents in these cells were potentiated by low concentrations of this compound and depressed by higher concentrations consistent with its actions on ACTH secretion. A similar relationship was demonstrated for a different but related compound, CGP 28 392. Each of BAY-K-8644's enantiomers, BAY-R(−)5417 and BAY-R(+)4407, had opposing effects upon these inward calcium currents and ACTH secretion. The (+)isomer antagonized both inward calcium currents and ACTH secretion. In contrast, the (−)enantiomer was responsible for the stimulatory effects of BAY-K-8644. Nevertheless, some antagonistic properties were noted with high concentrations of this latter enantiomer. The stimulation of ACTH secretion in AtT-20 cells by low concentrations of BAY-K-8644 can be attributed to a potentiation of voltage-activated calcium currents by one of its enantiomers, BAY-R-(−)5417. In contrast, the depression of secretion that occurs at higher concentrations is likely to be the result of the reduction of these currents by the other enantiomer (BAY-R(+)4407).

scholarly journals Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow-trout fructose diphosphatase

1969 ◽  
Vol 111 (3) ◽  
pp. 287-295 ◽  
Author(s):  
H. W. Behrisch ◽  
P. W. Hochachka
Keyword(s):  
Enzyme Activity ◽  
Rainbow Trout ◽  
Temperature Adaptation ◽  
Temperature Dependent ◽  
Ph Optimum ◽  
Physiological Temperature ◽  
Low Concentrations ◽  
High Concentrations ◽  
Regulation Of Enzyme Activity ◽  
Hill Plots

1. The properties of fructose diphosphatase from the liver of rainbow trout (Salmo gairdnerii) were examined over the physiological temperature range of the organism. 2. Saturation curves for substrate (fructose 1,6-diphosphate) and a cofactor (Mg2+) are sigmoidal, and Hill plots of the results suggest a minimum of two interacting fructose 1,6-diphosphate sites and two interacting Mg2+ sites per molecule of enzyme. 3. Mn2+-saturation curves are hyperbolic, and the Ka for Mn2+, which inhibits the enzyme at high concentrations, is 50–100-fold lower than the Ka for Mg2+. 4. Fructose diphosphatase is inhibited by low concentrations of AMP; this inhibition appears to be decreased and reversed by increasing the concentrations of Mg2+ and Mn2+. Higher concentrations of AMP are required to inhibit the trout fructose diphosphatase in the presence of Mn2+. 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn2+ appear to be temperature-independent, whereas the affinities for Mg2+ and AMP are highly temperature-dependent. 6. The pH optimum of the enzyme depends on the concentrations of Mg2+ and Mn2+. In addition, pH determines the Ka for Mg2+; at high pH, Ka for Mg2+ is lowered. 7. The enzyme is inhibited by Ca2+ and Zn2+, and the inhibition is competitive with respect to both cations. 8. The possible roles of these ions and AMP in the modulation of fructose diphosphatase and gluconeogenic activity are discussed in relation to temperature adaptation.

Adenosine and its analogue ( − )-N6-R-phenyl-isopropyladenosine modulate anterior pituitary adenylate cyclase activity and prolactin secretion in the rat

1990 ◽  
Vol 5 (1) ◽  
pp. 69-76 ◽  
Author(s):  
G. Schettini ◽  
E. Landolfi ◽  
O. Meucci ◽  
T. Florio ◽  
M. Grimaldi ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Anterior Pituitary ◽  
Primary Cultures ◽  
Prolactin Secretion ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Pituitary Cells ◽  
Prolactin Release ◽  
Low Concentrations ◽  
High Concentrations

ABSTRACT The effect of adenosine and its analogue ( − )-N6-R-phenylisopropyladenosine (PIA) on both anterior pituitary adenylate cyclase activity and prolactin secretion was examined in the rat. Adenosine inhibited basal adenylate cyclase activity in a dose-dependent manner and also reduced the stimulation of the enzyme by vasoactive intestinal peptide (VIP). Likewise, in primary cultures of anterior pituitary cells, adenosine decreased prolactin secretion in both basal and VIP-stimulated conditions. In perifusion experiments, adenosine also inhibited prolactin release in both basal and TRH-stimulated conditions. PIA produced a biphasic pattern of response of basal adenylate cyclase activity, being inhibitory at low and stimulatory at high concentrations. In VIP-stimulated conditions, low concentrations of PIA inhibited both adenylate cyclase activity and prolactin release from primary cultures of pituitary cells, while no additive stimulatory effect was seen at high concentrations. Similarly, low concentrations of PIA reduced both basal and TRH-stimulated prolactin release from perifused pituitaries, while increasing PIA concentrations restored prolactin release. These data show that adenosine affects basal and stimulated prolactin secretion from anterior pituitary cells. Adenosine receptors seem to be coupled to the adenylate cyclase system in the anterior pituitary gland, suggesting a possible relationship between the effect of adenosine on adenylate cyclase activity and prolactin secretion.

scholarly journals Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of non-nuclear adenylate cyclase

1977 ◽  
Vol 162 (3) ◽  
pp. 473-482 ◽  
Author(s):  
D E Snider ◽  
C W Parker
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Prostaglandin E1 ◽  
Plasma Membranes ◽  
Subcellular Fractions ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Marker Enzyme ◽  
Discontinuous Sucrose Gradient

Human peripheral lymphocytes were broken in a Dounce homogenizer and subcellular fractions enriched in plasma membranes or microsomal particles and mitochondria were isolated by centrifugation through a discontinuous sucrose gradient. Various agents that promote cyclic AMP accumulation in intact lymphocytes were compared in their ability to stimulate adenylate cyclase activity in the individual fractions. Plasma-membrane-rich fractions that were essentially free of other subcellular particles as judged by electron microscopy and marker enzyme measurements responded to fluoride, but weakly or not at all to prostaglandin E1 and other prostaglandins. Microsomal and mitochondrial-rich fractions responded markedly to both prostaglandin E1 and fluoride. In some, but not all, experiments phytohaemagglutinin produced a modest increase in enzyme activity in plasma-membrane-rich fractions. Catecholamines, histamine, parathyrin, glucagon and corticotropin produced little or no response. In the absence of theophylline, adenosine (1-10 micronM) stimulated basal enzyme activity, although at higher concentrations the responses to prostaglandin E1 and fluoride were inhibited. GTP (1-100 micronM) and GMP(5-1000 micronM) respectively inhibited or stimulated the response to fluoride, whereas the converse was true with prostaglandin E1.

scholarly journals Modulation of the response of bovine adrenocortical adenylate cyclase to corticotropin

1977 ◽  
Vol 168 (2) ◽  
pp. 277-282 ◽  
Author(s):  
P Glynn ◽  
D M F Cooper ◽  
D Schulster
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Guanine Nucleotide ◽  
Half Maximum ◽  
Basic Parameters ◽  
High Concentrations ◽  
Plasma Membrane Preparation ◽  
Stimulation Of

An assessment was made of some of the basic parameters responsible for the modulation of adenylate cyclase activity in a bovine adrenocortical plasma-membrane preparation. When determined at 0.1 mM-ATP, basal adenylate cyclase activity increased with increasing MgCl2 concentrations, whereas in the presence of corticotropin activity was essentially maximal at 10mM-MgCl2; high concentrations (25mM) of MgCl2 inhibited adenylate cyclase activity determined in the presence of both corticotropin and GTP. At all MgCl2 concentrations, corticotropin and GTP activated the enzyme in a synergistic fashion. The magnitude of the stimulation of basal activity produced by corticotropin was a function of Mg2+ concentration, whereas that produced by GTP appeared largely independent of Mg2+ concentration. Adenylate cyclase activity in the bovine adrenal membrane was half-maximally stimulated by corticotropin concentrations in the range 0.3—1.0 nM. The concentration of corticotropin evoking half-maximum response was not significantly affected by raising the free Mg2+ concentration from 0.4 to 4.9 mM, nor by the presence of GTP. In the presence of GTP, high concentrations (over 1 micrometer) of corticotropin inhibited adenylate cyclase activity, although no inhibition was apparent in the absence of guanine nucleotide.

scholarly journals The effects of the tylosin and Cd on soil enzyme activity

2020 ◽  
Vol 213 ◽  
pp. 01032
Author(s):  
Zhaohong Meng ◽  
Shuman Wang ◽  
Jia Zhou
Keyword(s):  
Heavy Metals ◽  
Enzyme Activity ◽  
Soil Enzyme ◽  
Soil Enzyme Activity ◽  
Germination Index ◽  
Soil Microbial ◽  
Low Concentrations ◽  
Long Time ◽  
High Concentrations ◽  
Microbial Environment

Soil microbial environment have been affected by different concentration heavy metals Cd (HM) and tylosin (TYL) and combination of TYL and HM interactions. Degradation of TYL was caused certain inhibition due to the addition of HM. The germination index of seed had been inhibited owing to the toxic effects of HM and TYL, but we found that the low concentrations of HM (4 mg/kg), the germination index higher than the soil which unadded HM and TYL in it. The soil enzyme activity was significantly suppressed by the addition of HM and TYL. Actinomycete was inhibited by high concentrations of HM for a long time. The studies demonstrated that the pollution of the soil micro-environment has been serious than only add HM or TYL in the soil.

scholarly journals Kinetic mechanism of sheep liver NADPH-dependent aldehyde reductase

1987 ◽  
Vol 242 (1) ◽  
pp. 143-150 ◽  
Author(s):  
K S De Jongh ◽  
P J Schofield ◽  
M R Edwards
Keyword(s):  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Enzyme Mechanism ◽  
Free Enzyme ◽  
Aldehyde Reductase ◽  
Binding Studies ◽  
Sheep Liver ◽  
Low Concentrations ◽  
High Concentrations ◽  
Inhibition Studies

The kinetic mechanism of the major sheep liver aldehyde reductase (ALR1) was studied with three aldehyde substrates: p-nitrobenzaldehyde, pyridine-3-aldehyde and D-glucuronate. In each case the enzyme mechanism was sequential and product-inhibition studies were consistent with an ordered Bi Bi mechanism, with the coenzymes binding to the free enzyme. Binding studies were used to investigate the interactions of substrates, products and inhibitors with the free enzyme. These provided evidence for the binding of D-glucuronate, L-gulonate and valproate, as well as NADP+ and NADPH. The enzyme was inhibited by high concentrations of D-glucuronate in a non-competitive manner, indicating that this substrate was able to bind to the free enzyme and to the E X NADP+ complex at elevated concentrations. Although the enzyme was inhibited by high pyridine-3-aldehyde concentrations, there was no evidence for the binding of this substrate to the free enzyme. Sheep liver ALR1 was inhibited by the ionized forms of alrestatin, sorbinil, valproate, 2-ethylhexanoate and phenobarbitone, indicating the presence of an anion-binding site similar to that described for the pig liver enzyme, which interacts with inhibitors and substrates containing a carboxy group. Sorbinil, valproate and 2-ethylhexanoate inhibited the enzyme uncompetitively at low concentrations and non-competitively at high concentrations, whereas phenobarbitone and alrestatin were non-competitive and uncompetitive inhibitors respectively. The significance of these results with respect to inhibitor and substrate binding is discussed.

Sign in / Sign up

Export Citation Format

Share Document