scholarly journals A kinetic study of the soluble 5′-nucleotidase of rat liver

1977 ◽  
Vol 162 (3) ◽  
pp. 611-616 ◽  
Author(s):  
G van den Berghe ◽  
C van Pottelsberghe ◽  
H G Hers
Keyword(s):  
Uric Acid ◽  
Rat Liver ◽  
Liver Cell ◽  
Adenine Nucleotides ◽  
Enzymic Activity ◽  
Kinetic Properties ◽  
Stimulatory Action ◽  
Physiological Conditions ◽  
Hydrolysis Of ◽  
Stimulation Of

1. The kinetic properties of the 5′-nucleotidase (EC 3.1.3.5) present in the cytosol of rat liver were investigated in relation to the conversion of adenine nucleotides into uric acid, with particular reference to the stimulation of this process by fructose. The enzyme was assayed by the release of Pi and by a new and more sensitive radiochemical procedure. 2. When IMP was used as substrate, the partially purified enzyme displayed almost hyperbolic kinetics (h = 1.1) with S0.5 = 1.2 mM. Similar kinetics were observed with GMP and other nucleoside 5′-monophosphates, except AMP. 3. Vmax. of the enzyme for AMP was about the same as for IMP, but the kinetics were sigmoidal (h = 1.6) with S 0.5 = 10 mM. 4. The hydrolysis of IMP was inhibited competitively by GMP. IMP, at concentrations up to 0.5 mM, had a paradoxical stimulatory action on the hydrolysis of 2-5 mM-AMP and was inhibitory at higher concentrations. 5. The activity of the enzyme towards AMP and IMP was stimulated by ATP and GTP, and inhibited by Pi. Activators and inhibitor approximately cancelled each others' effects. At pH 7.4, the enzymic activity with 0.2 mM-AMP was undetectable under physiological conditions. 6. It is concluded that, in the liver cell, AMP is not hydrolysed by the soluble 5′-nucleotidase, but that its degradation requires prior deamination to IMP.

Thyroid hormone induction of Na(+)-K(+)-ATPase and its mRNAs in a rat liver cell line

1990 ◽  
Vol 258 (3) ◽  
pp. C544-C551 ◽  
Author(s):  
G. G. Gick ◽  
F. Ismail-Beigi
Keyword(s):  
Atpase Activity ◽  
Cell Line ◽  
Rat Liver ◽  
Liver Cell ◽  
Northern Blot Analysis ◽  
Fold Increase ◽  
Beta Subunits ◽  
Inhibit Protein Synthesis ◽  
Liver Cell Line ◽  
Stimulation Of

The expression of mRNAs encoding the alpha- and beta-subunits of Na(+)-K(+)-ATPase (Na(+)-K+ pump) was examined in a rat liver cell line, Clone 9, in various thyroidal states. Northern blot analysis of total RNA isolated from cells incubated in hypothyroid serum-containing medium revealed the expression of mRNAs encoding Na(+)-K(+)-ATPase alpha 1-(mRNA alpha 1) and beta- (mRNA beta) subunits; mRNAs encoding the alpha 2- and alpha 3-subunits were undetectable. There was a discrepancy in the abundance of mRNA alpha 1 relative to mRNA beta such that mRNA alpha 1 exceeded the sum of the multiple mRNA beta bands by approximately 35-fold. 3,3',5-Triiodothyronine (T3) produced a coordinate augmentation of mRNA alpha 1 and mRNA beta contents that was demonstrable within 2 h and preceded the stimulation of Na(+)-K(+)-ATPase activity. After incubation of cells with T3 for 48 h, Na(+)-K(+)-ATPase activity was stimulated by 1.32-fold, whereas mRNA alpha 1 and mRNA beta abundances were increased 1.46- and 2.87-fold, respectively. Treatment of cells for 6 h with 10 micrograms/ml cycloheximide, a concentration sufficient to inhibit protein synthesis by 95%, elicited a 3.5- and 5.1-fold increase in mRNA alpha 1 and mRNA beta content, respectively. Cycloheximide abrogated the stimulatory effect of T3 on mRNA beta abundance, whereas the T3-induced increase in mRNA alpha 1 content was not prevented.(ABSTRACT TRUNCATED AT 250 WORDS)

RESPIRATORY AND ENZYMIC CHANGES IN RESPONSE TO STEROID HORMONES

1970 ◽  
Vol 47 (2) ◽  
pp. 159-166 ◽  
Author(s):  
T. DALTON ◽  
R. S. SNART
Keyword(s):  
Rat Liver ◽  
Enzymic Activity ◽  
Toad Bladder ◽  
Target Tissue ◽  
Transaminase Activity ◽  
Bladder Tissue ◽  
Cellular Processes ◽  
Receptor Sites ◽  
Common Effect ◽  
Stimulation Of

SUMMARY Changes in respiration and enzymic activity were measured in toad bladder tissue and homogenates in response to aldosterone and in rat liver tissue and homogenates in response to corticosterone. The results showed that the stimulation of target tissue may be associated with the saturation of two types of receptor sites for these hormones. The mechanism of action of both hormones is discussed in terms of a common effect on the control of respiration of the target tissue, and a second effect on the control of cellular processes such as the entry of sodium into the epithelial cells of toad bladder or increased transaminase activity in rat liver.

Stimulation of Active Na+and K+Transport by Thyroid Hormone in a Rat Liver Cell Line: Role of Enhanced Na+Entry*

Endocrinology ◽  
1986 ◽  
Vol 119 (6) ◽  
pp. 2527-2536 ◽  
Author(s):  
FARAMARZ ISMAIL-BEIGI ◽  
RICHARD S. HABER ◽  
JOHN N. LOEB
Keyword(s):  
Thyroid Hormone ◽  
Cell Line ◽  
Rat Liver ◽  
Liver Cell ◽  
Liver Cell Line ◽  
K Transport ◽  
Stimulation Of

scholarly journals Guanine nucleotides stimulate production of inositol trisphosphate in rat cortical membranes

1985 ◽  
Vol 232 (3) ◽  
pp. 799-804 ◽  
Author(s):  
R A Gonzales ◽  
F T Crews
Keyword(s):  
Inositol Phosphates ◽  
Adenine Nucleotides ◽  
Inositol Trisphosphate ◽  
Guanine Nucleotides ◽  
Guanine Nucleotide ◽  
Inositol Phospholipids ◽  
Cortical Membranes ◽  
Gamma S ◽  
Hydrolysis Of ◽  
Stimulation Of

The guanine nucleotides guanosine 5′[beta, gamma-imido]triphosphate (Gpp[NH]p), guanosine 5′-[γ-thio]-triphosphate (GTP gamma S), GMP, GDP and GTP stimulated the hydrolysis of inositol phospholipids by a phosphodiesterase in rat cerebral cortical membranes. Addition of 100 microM-Gpp[NH]p to prelabelled membranes caused a rapid accumulation of [3H)inositol phosphates (less than 30 s) for up to 2 min. GTP gamma S and Gpp [NH]p caused a concentration-dependent stimulation of phosphoinositide phosphodiesterase with a maximal stimulation of 2.5-3-fold over control at concentrations of 100 microM. GMP was as effective as the nonhydrolysable analogues, but much less potent (EC50 380 microM). GTP and GDP caused a 50% stimulation of the phospholipase C at 100 microM and at higher concentrations were inhibitory. The adenine nucleotides App[NH]p and ATP also caused small stimulatory effects (64% and 29%). The guanine nucleotide stimulation of inositide hydrolysis in cortical membranes was selective for inositol phospholipids over choline-containing phospholipids. Gpp[NH]p stimulated the production of inositol trisphosphate and inositol bisphosphate as well as inositol monophosphate, indicating that phosphoinositides are substrates for the phosphodiesterase. EGTA (33 microM) did not prevent the guanine nucleotide stimulation of inositide hydrolysis. Calcium addition by itself caused inositide phosphodiesterase activation from 3 to 100 microM which was additive with the Gpp[NH]p stimulation. These data suggest that guanine nucleotides may play a regulatory role in the modulation of the activity of phosphoinositide phosphodiesterase in rat cortical membranes.

scholarly journals The control of isocitrate oxidation by rat liver mitochondria

1969 ◽  
Vol 114 (2) ◽  
pp. 215-225 ◽  
Author(s):  
D. G. Nicholls ◽  
P. B. Garland
Keyword(s):  
Respiratory Chain ◽  
Rat Liver ◽  
Isocitrate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Kinetic Studies ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Dependent Inhibition ◽  
Nad 4

1. The factors capable of affecting the rate of isocitrate oxidation in intact mitochondria include the rate of isocitrate penetration, the activity of the NAD-specific and NADP-specific isocitrate dehydrogenases, the activity of the transhydrogenase acting from NADPH to NAD+, the rate of NADPH oxidation by the reductive synthesis of glutamate and the activity of the respiratory chain. A quantitative assessment of these factors was made in intact mitochondria. 2. The kinetic properties of the NAD-specific and NADP-specific isocitrate dehydrogenases extracted from rat liver mitochondria were examined. 3. The rate of isocitrate oxidation through the respiratory chain in mitochondria with coupled phosphorylation is approximately equal to the maximal of the NAD-specific isocitrate dehydrogenase but at least ten times as great as the transhydrogenase activity from NADPH to NAD+. 4. It is concluded that the energy-dependent inhibition of isocitrate oxidation by palmitoylcarnitine oxidation is due to an inhibition of the NAD-specific isocitrate dehydrogenase. 5. Kinetic studies of NAD-specific isocitrate dehydrogenase demonstrated that its activity could be inhibited by one or more of the following: an increased reduction of mitochondrial NAD, an increased phosphorylation of mitochondrial adenine nucleotides or a fall in the mitochondrial isocitrate concentration. 6. Uncoupling agents stimulate isocitrate oxidation by an extent equal to the associated stimulation of transhydrogenation from NADPH to NAD+. 7. A technique is described for continuously measuring with a carbon dioxide electrode the synthesis of glutamate from isocitrate and ammonia.

scholarly journals Enzymic characteristics of ecto-adenosine triphosphatase in rat epididymal intact spermatozoa

1981 ◽  
Vol 195 (1) ◽  
pp. 103-110 ◽  
Author(s):  
G C Majumder
Keyword(s):  
Atpase Activity ◽  
Enzymic Activity ◽  
Testicular Spermatozoa ◽  
Calf Thymus ◽  
Km Value ◽  
Vanadate Inhibition ◽  
Bivalent Metal Ions ◽  
High Degree ◽  
Hydrolysis Of ◽  
Stimulation Of

Ecto-ATPase in rat cauda-epididymal intact spermatozoa has a high degree of substrate specificity for the hydrolysis of ATP and dATP rather than of ADP, AMP, GTP, dGTP, CTP, dCTP, TTP and UTP. The enzyme is activated by bivalent metal ions in the order Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+. The apparent Km values of the enzyme for Mg2+, Mn2+, Co2+ and Ca2+ are approx. 80, 100, 100 and 150 microM respectively. Addition of Ca2+ (0.1 or 1 mM) gives no further stimulation of the Mg2+-activated ecto-ATPase activity. The apparent Km value of the enzyme for ATP is 95 microM. Pi (16 mM) inhibits the enzymic activity (by 25%), whereas Na+ (50 mM) or K+ (10 mM) alone or in combination, polyamines (spermine and spermidine; 1--12.5mM) and nucleic acids (yeast RNA and calf thymus DNA; 0.12 or 0.62 mg/ml) had no significant effect on the activity of the enzyme. Orthovanadate at a relatively low concentration (20 microM) strongly inhibits (approx. 50%) the ecto-ATPase activity. Vanadate inhibition can be reversed by noradrenaline (2.5 mM). The vanadate-sensitivity of the enzyme increases markedly during spermatozoal maturation in the epididymis. However, the activity of the spermatozoal ecto-ATPase decreases progressively during the epididymal transit of the testicular spermatozoa.

scholarly journals Adenine derivatives as neurohumoral agents in the brain. The quantities liberated on excitation of superfused cerebral tissues

1972 ◽  
Vol 130 (4) ◽  
pp. 975-981 ◽  
Author(s):  
Ian Pull ◽  
Henry McIlwain
Keyword(s):  
Electrical Stimulation ◽  
Guinea Pig ◽  
Adenine Nucleotides ◽  
Specific Radioactivity ◽  
Acid Extraction ◽  
Free Medium ◽  
Hydrolysis Of ◽  
The Brain ◽  
Stimulation Of ◽  
Adenine Derivatives

Adenine nucleotides of guinea-pig neocortical tissues were labelled by incubation with [14C]adenine and excess of adenine was then removed by superfusion with precursor-free medium. Adenine derivatives released from the tissue during continued superfusion, including a period of electrical stimulation of the tissue, were collected by adsorption and examined after elution and concentration. The stimulation greatly increased the 14C output, and material collected during and just after stimulation had a u.v. spectrum which indicated adenosine to be a major component. The additional presence of inosine and hypoxanthine was shown by chromatography and adenosine was identified also by using adenosine deaminase. Total adenine derivatives released from the tissue during a 10min period of stimulation were obtained as hypoxanthine, after deamination and hydrolysis of adenosine and inosine, and amounted to 159nmol/g of tissue. This corresponded to the release of approx. 7pmol/g of tissue per applied stimulus. The hypoxanthine sample derived from superfusate hypoxanthine, inosine and adenosine was of similar specific radioactivity to the sample of inosine separated chromatographically, and each was of higher specific radioactivity than the adenine nucleotides obtained by cold-acid extraction of the tissue.

Titration of Rat Liver with Digitonin: a Well Defined Short Term Damage of Cellular Metabolism

1981 ◽  
Vol 36 (9-10) ◽  
pp. 880-883 ◽  
Author(s):  
Stefan Postius ◽  
Dieter Platt
Keyword(s):  
Lactate Dehydrogenase ◽  
Rat Liver ◽  
Liver Cell ◽  
Plasma Membranes ◽  
Short Term ◽  
Isolated Rat Liver ◽  
Physiological Conditions ◽  
Cell Plasma ◽  
Molecular Compounds ◽  
Isolated Rat

Abstract Carefully performed pulse titration of the isolated rat liver in the course of continuous erythro­ cyte free perfusion with small amounts of digitonin causes a short term perm eability of liver cell plasma membranes with concomitant short lived release of intracellular low or high molecular compounds such as ATP or lactate dehydrogenase. Gluconeogenesis from lactate being completely inhibited during this period restores w ithin about one m inute up to a level that depends on the am ount of perfused digitonin. The described experimental m odel is suggested to be useful for the measurement of cytoplasmic m etabolites under physiological conditions. It moreover offers the possibility to im port foreign substances into liver cells th at normally do not penetrate liver cell plasma membranes.

Biosynthesis of Mitochondrial Phospholipids Using Endogenously Generated Diglycerides

1975 ◽  
Vol 53 (7) ◽  
pp. 784-795 ◽  
Author(s):  
W. C. McMurray
Keyword(s):  
Endoplasmic Reticulum ◽  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Rat Liver ◽  
De Novo ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Isolated Mitochondria ◽  
Hydrolysis Of ◽  
Stimulation Of

When isolated mitochondria or microsomes from rat liver were treated with phospholipase C, the incorporation of radioactive phospholipid precursors was markedly enhanced, presumably as a result of production of diglycerides by hydrolysis of endogenous phospholipids. Incorporation of CDP[14C]choline into lecithin in rat liver or BHK-21 mitochondria could be attributed to residual contamination from elements of the endoplasmic reticulum, with added diglycerides or with endogenous diglycerides produced by the phospholipase C treatment. A similar stimulation of [γ32P]ATP incorporation into phospholipids was observed with exogenous or endogenous diglycerides, but the mitochondrial diglyceride kinase in either case was also related to the degree of microsomal contaminants. It was concluded that previous studies showing negligible capacity of mitochondria for lecithin biosynthesis de novo were not explainable on the basis of limited accessibility of added diglycerides, and that formation of phosphatidic acid by diglyceride kinase was not of significance in rat liver mitochondria.

scholarly journals INORGANIC PYROPHOSPHATASE OF RAT LIVER MITOCHONDRIA

1969 ◽  
Vol 42 (1) ◽  
pp. 235-240 ◽  
Author(s):  
Lloyd Schick ◽  
Larry G. Butler
Keyword(s):  
Rat Liver ◽  
Catalytic Properties ◽  
Liver Mitochondria ◽  
Inorganic Pyrophosphatase ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Membranes ◽  
Physiological Conditions ◽  
Enzyme Stimulation ◽  
Pyrophosphatase Activity ◽  
Stimulation Of

Stimulation of Mg2+-dependent inorganic pyrophosphatase activity several fold by disruption of mitochondrial membranes does not appreciably alter the catalytic properties of the enzyme. Stimulation is due to increased accessibility of substrate to the enzyme, which is not solublized on activation. The enzyme is attached to the inside of the inner membrane, and under physiological conditions probably hydrolyzes only intramitochondrially-produced PPi.

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