scholarly journals New kinetic parameters for rat liver arginase measured at near-physiological steady-state concentrations of arginine and Mn2+

1992 ◽  
Vol 283 (3) ◽  
pp. 653-660 ◽  
Author(s):  
S Maggini ◽  
F B Stoecklin-Tschan ◽  
S Mörikofer-Zwez ◽  
P Walter
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Rat Liver ◽  
Urea Cycle ◽  
Hill Coefficient ◽  
Regulatory Effect ◽  
Free System ◽  
State Concentration ◽  
Good Agreement

A cytosolic cell-free system from rat liver containing the last three enzymes of the urea cycle, a number of cofactors and the substrates aspartate and citrulline was shown to synthesize urea at near-physiological rates ranging between 0.40 and 1.25 mumol/min per g of liver. This system was used to determine the kinetic parameters for arginase. With saturating amounts of Mn2+ (30 microM), arginine remained at a steady-state concentration of 5-35 microM depending on the aspartate and citrulline supply. Vmax. at micromolar arginine concentrations was between 1.10 and 1.25 mumol/min per g of liver, the K0.5 (arginine) between 6.0 and 6.5 microM and positive co-operativity was observed (Hill coefficient 2). Omission of Mn2+ caused a significant accumulation of arginine during the incubation, suggesting a regulatory effect of arginase. Under these conditions, Vmax. was 1.10-1.65 mumol/min per g of liver and the Km (arginine) increased up to 14.4-21.1 microM. The apparent Ka for Mn2+ in the presence of physiological concentrations of ATP, Mg2+ and arginine was calculated to be maximally 8 microM. Initial-velocity experiments with millimolar arginine concentrations as the direct substrate gave the following results, which are in good agreement with literature data. In the absence of Mn2+, Vmax. was 71.3 mumol/min per g of liver and the Km (arginine) 1.58 mM. With 30 microM-Mn2+, Vmax. was 69.4 mumol/min per g of liver and the Km (arginine) decreased to 0.94 mM. On the basis of our results, we propose the presence of high-affinity and low-affinity sites for arginine on rat liver arginase and postulate that alterations in arginase activity arising from changes in the concentration of arginine and of the cofactor Mn2+ may contribute to the regulation of ureagenesis in vivo.

Noradrenaline-induced calorigenesis in warm- or cold-acclimated rats. In vivo estimation of adrenoceptor concentration of noradrenaline effecting half-maximal response

1980 ◽  
Vol 58 (9) ◽  
pp. 1072-1077 ◽  
Author(s):  
Florent Depocas ◽  
Gloria Zaror-Behrens ◽  
Suzanne Lacelle
Keyword(s):  
Adipose Tissue ◽  
Steady State ◽  
Brown Adipose Tissue ◽  
Maximal Response ◽  
Brown Adipose ◽  
Acclimation Group ◽  
Arterial Plasma ◽  
State Concentration ◽  
Na Uptake

Desmethylimipramine (DMI, 1 mg DMI∙HCl kg−1) and normetanephrine (NMN, 1 μg min−1 g−0.74) were used to inhibit, respectively, neuronal and extraneuronal uptakes of noradrenaline (NA) during calorigenesis induced in barbital-sedated warm-acclimated (WA) or cold-acclimated (CA) rats by infusion of NA, a procedure which mimics the effects of NA released within calorigenic tissues in response to cold exposure. The doses of the inhibitors were selected for maximal effectiveness in potentiating calorigenic response and for minimal side effects. For rats of either acclimation group treated with DMI and NMN, with DMI only, or with neither inhibitor the doses of NA required to evoke approximately half-maximal calorigenic responses were, respectively, 0.5, 1.0, and 3.5 ng min−1 g−0.74. The corresponding steady-state concentrations of NA in arterial plasma averaged 14.3, 21.7, and 43.2 nM in the three groups of WA rats and 10.0, 14.8, and 31.9 nM in the three groups of CA rats. Reduction by NA uptake inhibitors of the circulating levels of NA necessary to stimulate calorigenesis, half-maximally, presumably in brown adipose tissue, indicates a reduction in the steepness of the NA concentration gradient between capillary plasma and synaptic clefts in that tissue. The steady-state concentration of NA in blood plasma of rats treated with DMI and NMN and infused with NA at a dose of 0.5 ng min−1 g−0.74 (~1 × 10−8 M) is a good estimate of the NA concentration required at calorigenic adrenoceptors to effect half-maximal activation. Presumably, this concentration is also an estimate of that resulting from NA released at nerve endings during cold-induced activation of nonshivering thermogenesis at half-maximal rates in brown adipose tissue.

scholarly journals Metabolism of inorganic sulphate in the isolated perfused rat liver. Effect of sulphate concentration on the rate of sulphation by phenol sulphotransferase

1978 ◽  
Vol 176 (3) ◽  
pp. 959-965 ◽  
Author(s):  
Gerard J. Mulder ◽  
Katja Keulemans
Keyword(s):  
Steady State ◽  
Plasma Concentration ◽  
Rat Liver ◽  
Isolated Perfused Rat Liver ◽  
Perfused Rat Liver ◽  
Perfusion Medium ◽  
Physiological Concentration ◽  
Sulphate Concentration ◽  
Inorganic Sulphate

1. The metabolism of inorganic [35S]sulphate (Na235SO4) was studied in the isolated perfused rat liver at three initial concentrations of inorganic sulphate in the perfusion medium (0, 0.65 and 1.30mm), in relation to sulphation and glucuronidation of a phenolic drug, harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole). 2. [35S]Sulphate rapidly equilibrated with endogenous sulphate in the liver. It was excreted in bile and reached, at the lowest concentration in the perfusion medium, concentrations in bile that were much higher than those in the perfusion medium; at the higher sulphate concentrations, these concentrations were equal. The physiological concentration of inorganic sulphate in the liver, available for sulphation of drugs, is similar to the plasma concentration. 3. At zero initial inorganic sulphate in the perfusion medium, the rate of sulphation was very low and harmol was mainly glucuronidated. At 0.65mm-sulphate glucuronidation was much decreased and considerable sulphation took place, indicating efficient competition of conjugation by sulphation. At 1.30mm-sulphate the sulphation increased still further. 4. The results suggest that an important factor in sulphation is the relatively high Km of synthesis of adenosine 3′-phosphate 5′-sulphatophosphate (the co-substrate of sulphation) for inorganic sulphate, which is of the order of the plasma concentration of inorganic sulphate. The steady-state adenosine 3′-phosphate 5′-sulphatophosphate concentration may determine the rate of sulphate conjugation of drugs in the rat in vivo.

Divergent effects of intravenous GSH and cysteine on renal and hepatic GSH

1992 ◽  
Vol 263 (2) ◽  
pp. R348-R352 ◽  
Author(s):  
S. Aebi ◽  
B. H. Lauterburg
Keyword(s):  
Steady State ◽  
De Novo ◽  
Feedback Inhibition ◽  
Therapeutic Use ◽  
Steady State Concentration ◽  
De Novo Synthesis ◽  
State Concentration ◽  
Cellular Thiol

There is a growing interest in the therapeutic use of sulfhydryls. To assess the effect of glutathione (GSH) and cysteine on the cellular thiol status, thiols were administered intravenously to rats in doses ranging from 1.67 to 8.35 mmol/kg with and without pretreatment with 4 mmol/kg buthionine-[S,R]-sulfoximine (BSO), an inhibitor of GSH synthesis. One hour after administration of 1.67 mmol/kg GSH, the concentration of GSH rose from 5.2 +/- 1.0 to 8.4 +/- 0.9 mumol/g and from 2.5 +/- 0.5 to 3.7 +/- 0.7 mumol/g in liver and kidneys, respectively. After 8.35 mmol/kg, hepatic GSH did not increase further, but renal GSH rose to 6.7 +/- 1.8 mumol/g. Infusion of cysteine increased hepatic GSH to the same extent as intravenous GSH, but renal GSH did not increase after 1.67 mmol/kg and even significantly decreased to 0.6 +/- 0.2 mumol/g after 8.35 mmol/kg. In the presence of BSO, GSH resulted in a significant increase in renal but not hepatic GSH, suggesting that the kidneys take up intact GSH and indicating that the increment in hepatic GSH was due to de novo synthesis. The present data show that hepatic GSH can be markedly increased in vivo by increasing the supply of cysteine. Measurements of hepatic cysteine indicate that up to a concentration of approximately 0.5 mumol/g cysteine is a key determinant of hepatic GSH, such that the physiological steady-state concentration of GSH in the liver appears to be mainly determined by the availability of cysteine. At higher concentrations GSH does not increase further, possibly due to feedback inhibition of GSH synthesis or increased efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of oxidative stress during imbibition of soybean embryonic axes

1994 ◽  
Vol 102 ◽  
pp. 279-286 ◽  
Author(s):  
Susana Puntarulo
Keyword(s):  
Oxidative Stress ◽  
Steady State ◽  
External Medium ◽  
Tocopherol Content ◽  
Active Species ◽  
Oxygen Species ◽  
Steady State Concentration ◽  
Embryonic Axes ◽  
State Concentration

SynopsisBoth respiration and generation by soybean embryonic axes showed a sharp increase upon germination, leading to a significant increase in the steady-state concentration of and H2O2 after 6 h of imbibition. An assay was developed to assess in vivo generation of reactive oxygen species, based upon DCFH-DA oxidation. Fluorescence of the external medium was dependent on reaction time and axes number and was inhibited by catalase.α-Tocopherol content declined significantly after 24 h of incubation, as compared to the content at the onset of germination. Incubation in the presence of redox cycling agent paraquat (4 mM) for 24 h increased α-tocopherol content to 1.9±0.2 nmol per axis from 1.0 ± 0.1 nmol per axis in the absence of paraquat. Supplementation of the incubation medium with 500 μM Fe-EDTA increased α-tocopherol content to 1.8±0.1 nmol/axis and DCFH-DA oxidation by two-fold.The data presented here showed that active metabolism at the onset of germination increased steady-state concentration of oxygen active species and suggest that cellular content of α-tocopherol is physiologically adjusted as a response to conditions of oxidative stress.

scholarly journals Reconstitution of an endosome-lysosome interaction in a cell-free system.

1989 ◽  
Vol 108 (6) ◽  
pp. 2093-2099 ◽  
Author(s):  
B M Mullock ◽  
W J Branch ◽  
M van Schaik ◽  
L K Gilbert ◽  
J P Luzio
Keyword(s):  
Intravenous Injection ◽  
Rat Liver ◽  
Specific Interaction ◽  
Free System ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Free Model ◽  
A Cell ◽  
Isopycnic Centrifugation

A cell-free model for the transfer of endocytosed material to lysosomes is described. Rat liver late endosomes, loaded in vivo with radiolabeled ligand by intravenous injection shortly before killing the animal, showed a specific interaction with lysosomes when incubated at 37 degrees C in the presence of cytosol and an ATP regenerating system. The location of the ligand, generally asialofetuin, was analyzed by isopycnic centrifugation on Nycodenz gradients. Appearance of radiolabel in the lysosomal position on such gradients was maximal after approximately 30 min at 37 degrees C and required the provision of undamaged cytosol, lysosomes, and an ATP regenerating system. It could not be accounted for by nonspecific bulk aggregation of membranes. Transfer occurred only from late endosomes; radiolabel in early endosomes was unaffected. Digestion of the asialofetuin, as shown by the appearance of TCA-soluble radioactivity, occurred on incubation at 37 degrees C and was increased by the provision of an ATP regenerating system.

scholarly journals Cytochrome P -450 synthesis in vivo and in a cell-free system from rat liver

FEBS Letters ◽  
1978 ◽  
Vol 89 (2) ◽  
pp. 337-340 ◽  
Author(s):  
Kolari S. Bhat ◽  
G. Padmanaban
Keyword(s):  
Rat Liver ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Cytochrome P 450

scholarly journals The regulation of extramitochondrial free calcium ion concentration by rat liver mitochondria

1978 ◽  
Vol 176 (2) ◽  
pp. 463-474 ◽  
Author(s):  
David G. Nicholls
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Weak Acid ◽  
Ion Concentration ◽  
Rat Liver Mitochondria ◽  
Free Calcium ◽  
Electrochemical Gradient

The mechanism whereby rat liver mitochondria regulate the extramitochondrial concentration of free Ca2+ was investigated. At 30°C and pH7.0, mitochondria can maintain a steady-state pCa2+0 (the negative logarithm of the free extramitochondrial Ca2+ concentration) of 6.1 (0.8μm). This represents a true steady state, as slight displacements in pCa2+0 away from 6.1 result in net Ca2+ uptake or efflux in order to restore pCa2+0 to its original value. In the absence of added permeant weak acid, the steady-state pCa2+0 is virtually independent of the Ca2+ accumulated in the matrix until 60nmol of Ca2+/mg of protein has been taken up. The steady-state pCa2+0 is also independent of the membrane potential, as long as the latter parameter is above a critical value. When the membrane potential is below this value, pCa2+0 is variable and appears to be governed by thermodynamic equilibration of Ca2+ across a Ca2+ uniport. Permeant weak acids increase, and N-ethylmaleimide decreases, the capacity of mitochondria to buffer pCa2+0 in the region of 6 (1μm-free Ca2+) while accumulating Ca2+. Permeant acids delay the build-up of the transmembrane pH gradient as Ca2+ is accumulated, and consequently delay the fall in membrane potential to values insufficient to maintain a pCa2+0 of 6. The steady-state pCa2+0 is affected by temperature, incubation pH and Mg2+. The activity of the Ca2+ uniport, rather than that of the respiratory chain, is rate-limiting when pCa2+0 is greater than 5.3 (free Ca2+ less than 5μm). When the Ca2+ electrochemical gradient is in excess, the activity of the uniport decreases by 2-fold for every 0.12 increase in pCa2+0 (fall in free Ca2+). At pCa2+0 6.1, the activity of the Ca2+ uniport is kinetically limited to 5nmol of Ca2+/min per mg of protein, even when the Ca2+ electrochemical gradient is large. A steady-state cycling of Ca2+ through independent influx and efflux pathways provides a model which is kinetically and thermodynamically consistent with the present observations, and which predicts an extremely precise regulation of pCa2+0 by liver mitochondria in vivo.

Glucose transport and equilibrium across alveolar-airway barrier of rat

1996 ◽  
Vol 270 (2) ◽  
pp. L183-L190 ◽  
Author(s):  
G. Saumon ◽  
G. Martet ◽  
P. Loiseau
Keyword(s):  
Steady State ◽  
Glucose Transport ◽  
Glucose Concentration ◽  
Epithelial Lining Fluid ◽  
Uptake Sites ◽  
Maximum Uptake Rate ◽  
Isolated Lungs ◽  
Kinetics Of ◽  
State Concentration

The glucose concentration in the epithelial lining fluid (ELF) results from a balance between cellular uptake and paracellular leakage. The present study examines whether the ELF glucose concentration can be predicted from the kinetics of glucose transport obtained in fluid-filled lungs. Isolated rat lungs were filled via the trachea with instillate containing 0-10 mM glucose; the perfusate glucose concentration was 10 mM. The rate of glucose removal from airspaces depended on luminal glucose concentration and was saturable [maximum uptake rate = 101 +/- 8.6 mumol.h-1.g dry lung wt-1; apparent Michaelis constant K(m) = 1.5 +/- 0.43 mM; R2 = 0.79]. Glucose removal was inhibited by phloridzin but not by phloretin or by inhibiting glycolysis. The steady-state concentration in fluid-filled lungs was estimated to be 0.15 +/- 0.034 mM. It agreed with that (< 1/20 plasma) calculated using glucose transport kinetics and paracellular permeability. The ELF glucose concentration obtained by bronchoalveolar lavage was 0.39 +/- 0.012 plasma in vivo and 0.39 +/- 0.021 perfusate in air-filled isolated lungs. The equilibrium ELF/perfusate distribution ratio of alpha-methyl-glucose was similar to that of glucose. Thus there is a major difference between the alveolar steady-state glucose concentration in air- and fluid-filled lungs despite similar mechanisms of airspace glucose removal. This suggests that glucose kinetics or access to uptake sites differ in air- and fluid-filled lungs.

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