Enzymatic characteristics of the catalytic subunit of the protein kinase complex of human lymphocytes]

In earlier reports we have shown the existence in human lymphocytes homogenate, of a cyclic-AMP dependent protein-kinase activity. We demonstrate by affinity chromatography that two subunits display respectively cyclic-AMP binding and phosphorylating properties. Divalent cations such as Ca++, Mg++ or Mn++ are required for enzymatic activity. ATP which is an obligatory cosubstrate acts as an inhibitor when its concentration is higher than 10(-6)M.     

Purification of rat brain guanylate cyclase]

The purification of guanylate cyclase has been achieved. After electrophoresis on polyacrylamide gel only one protein band was observed. The low factor of purification must therefore be ascribed to the loss of an activator of guanylate cyclase. Stimulation of the activity by nitroprusside is also lost during purification. The purified enzyme follows Michaelis--Menten kinetics, it is activated by Mn++ ions and inhibited by triphospho nucleotides.     

Adenylate cyclase activation and its effects on intracellular cAMP in infected KB cells during trypsin-induced infectious Senda? virus production (author's transl)]

KB cells infected by Senda? virus can produce infectious virus if they are trypsinated twice over 24 h. Adenylate cyclase activity in infected KB cells is higher and more strongly activated by trypsin than that of control cells, but intracellular concentration of cAMP is the same, except during a short time after trypsinations, especially after the second trypsination which causes infectious virus production. During this short time, intracellular cAMP is slightly higher in infected cells. This miseffect of adenylate cyclase activation on intracellular cAMP concentrations might be related to an increased cell permeability caused by trypsin.     

Activation de l'adénylate cyclase et ses répercussions sur le taux intracellulaire d'AMP cyclique dans les cellules KB infectées produisant du virus Sendaï, infectieux sous l'influence de la trypsine

Summary KB cells infected by Sendaï virus can produce infectious virus if they are trypsinated twice over 24 h. Adenylate cyclase activity in infected KB cells is higher and more strongly activated by trypsin than that of control cells, but intracellular concentration of cAMP is the same, except during a short time after trypsinations, especially after the second trypsination which causes infectious virus production. During this short time, intracellular cAMP is slightly higher in infected cells. This miseffect of adenylate cyclase activation on intracellular cAMP concentrations might be related to an increased cell permeability caused by trypsin.     

Adenylate cyclase activation by trypsin in KB cell cultures

Summary Trypsin commonly used for cell dispersion increases adenylate cyclase activity of KB cells. It acts on catalytic receptors, since the apparent K_m for ATP is lowered, and it alters the dependence of adenylate cyclase on Mg⁺⁺ ions.     

Influence of ethanol on calcium, inositol phospholipids and intracellular signalling mechanisms

Studies have implicated Ca++ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca++ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca++/Mg++-ATPase, the high affinity membrane Ca++ pump. Current research suggests that a subtype of the voltage-dependent Ca++ channel, the dihydropyridine-sensitive Ca++ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases 45Ca++-influx and [3H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca++/Mg++-ATPase activity in neuronal membranes. Changes in Ca++ channel and Ca++/Mg++-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca++ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca++/Mg++-ATPase activity. The increased sensitivity of three Ca++-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Possible role of intestinal alkaline phosphatase activity in thiamine transport.

Thiamine deficiency caused a marked decrease of intestinal alkaline phosphatase (al-Pase) activity, but had no effect on the Ca++-ATPase activity and Ca++-absorption in rats. The al-Pase activity was significantly decreased 1 h after oral administration of ethanol at 0.5 and 2.5 g/kg. In contrast, Mg++-, Ca++-and (Na+ + K+)-ATPase activities did not change after the administration of ethanol. These findings show that the al-Pase activity, unlike the Ca++-ATPase activity, is not related to Ca++-absorption. A possible role of al-Pase activity in the active transport of thiamine in the intestine was discussed.     

Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists

Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of E _max values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase E _max values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.     

Adenyl cyclase activity of the cell membrane throughout the cell cycle: ultrastructural study of the cell line BHK 21/C 13]

Adenylate cyclase activity in the surface membrane of baby Hamster kidney (BHK 21/C13) cells is ultrastructurally studied as a function of the cell cycle. Cells are synchronized by a physical method suited to structural and functional investigations. The activity is highest through G1, decreases in S, markedly drops as cells traverse G2 and is very low or often seems not present at all in M.     

Specific phosphorylation of a calf serum protein by a kinase from the cell surface]

A protein of molecular weight approximately 200,000, which exists in trace amounts in calf serum, is specifically phosphorylated by an enzyme located at the surface of cultured cells. The emzymatic reaction utilizes ATP, is enhanced by Mg++ and Zn++ ions, and is not dependent on cyclic AMP. This kinase activity is associated with normal growing fibroblasts but disappears when they are contact-inhibited. It remains high, however, in transformed and malignant cells, whatever their growth state.     

Absence of dopamine sensitive adenylate cyclase in the A10 region, the origin of mesolimbic dopamine neurones.

Dopamine (DA) failed to stimulate the adenylate cyclase of the mesolimbic A10 DA nerve cell body area, in contrast to tis activating effect in the nigrostriatal A9 DA cell body area. The enzyme was stimulated by GMPPNP (a GTP analog) and NaF. This indicates the absence in the A 10 cell area of DA receptors with functional coupling on adenylate cyclase, in contrast to the A9 cell area where such DA receptors are believed to be located on afferent axon terminals.     

Steady-state kinetic studies with the polysulfonate U-9843, an HIV reverse transcriptase inhibitor

The tetramer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT^* and possesses excellent antiviral activity at nontoxic doses in HIV-1 infected lymphocytes grown in tissue culture. Kinetic studies of the HIV-1 RT-catalyzed RNA-directed DNA polymerase activity were carried out in order to determine if the inhibitor interacts with the template: primer or the deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using steady-state Briggs-Haldane kinetics assuming that the template:primer binds to the enzyme first, followed by the binding of the dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived which allows the calculation of all the specific forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The calculated rate constants are in agreement with this model and the results indicated that U-9843 acts as a noncompetitive inhibitor with respect to both the template:primer and dNTP binding sites. Hence, U-9843 exhibits the same binding affinity for the free enzyme as for the enzyme-substrate complexes and must inhibit the RT polymerase by interacting with a site distinct from the substrate binding sites. Thus, U-9843 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either an event leading up to the formation of the phosphoester bond, the formation of the ester bond itself or translocation of the enzyme relative to its template:primer following the formation of the ester bond.     

Adenyl cyclase activity at different environmental temperatures in the isolated rat anterior pituitary membranes

Summary The effect of different environmental temperatures on adenyl cyclase was studied. An increase in temperature appears to increase TRH-induced activity of adenyl cyclase, and possibly causes an increased sensitivity to the hormone. Cyclic AMP levels of the pituitaries showed change at different environmental temperatures.     

Adenyl cyclase activity at different environmental temperatures in the isolated rat anterior pituitary membranes.

The effect of different environmental temperatures on adenyl cyclase was studied. An increase in temperature appears to increase TRH-induced activity of adenyl cyclase, and possible causes an increases sensitivity to the hormone. Cyclic AMP levels of the pituitaries showed change at different environmental temperatures.     

Manganese action potentials in mammalian cardiac muscle.

The membrane potential in guinea-pig's papillary muscles from right ventricle was recorded by glass microelectrodes and stimulation was effected by current pulses applied through a sucrose-gap. Action potentials with overshoot were recorded in the solution lacking Na+ and Ca++ but containing 2-95 mM Mn++. The overshoot was increased with the increase of [Mn++]o by about 30 mV/decade. Similar Mn++ dependent action potentials were also obtained in Na-free solution containing 0.6 mM Ca++. The results indicate that Mn inward current is sufficient to generate action potentials in cardiac muscle.     

Characterization of the (Na+-K+)-ATPase from endometrial plasma membranes of immature mammals]

The (Na+-K+)-ATPase in plasma membrane from Mammiferous endometrium is characterized by the Mg/ATP ratio equal to one, and by a distinct affinity for Na+ (1.3 mM) and K+ (2 mM). The activity is maximum for pH 7.4-7.5 in presence of Mg++ 2mM and ATP 2 mM, Na+ 140 mM and K+ 10 mM.     

Ethanol and opioid receptor signalling

Ethanol may modulate endogenous opioid systems by disrupting opioid receptor signalling. Low concentrations of ethanol slightly potentiate mu-opioid receptor binding by increasing receptor Bmax, and, in some cases, chronic ethanol exposure decreases the density or affinity of the mu-opioid receptors. By contrast, high concentrations of ethanol acutely decrease delta-opioid receptor binding by decreasing receptor affinity, whereas chronic exposure of animals and neuronal cell lines to lower concentrations of ethanol leads to possibly adaptive increases in the density or affinity of the delta-opioid receptors. In the neuronal cell line NG108-15, ethanol does not up-regulate the delta-opioid receptor by blocking receptor degradation or endocytosis, but protein synthesis is required for this response. Up-regulation of the delta-opioid receptor renders ethanol-treated NG108-15 cells 3.5-fold more sensitive to opioid inhibition of adenylyl cyclase. Long-term treatment with ethanol also increases maximal opioid inhibition in NG108-15 cells, possibly by decreasing levels of G alpha s and its mRNA. Ethanol differentially modulates signal transduction proteins in three additional neuronal cell lines, N18TG2, N4TG1, and N1E-115. Ethanol-treated N18TG2 cells show the least up-regulation of the delta-opioid receptor, little heterologous desensitization of adenylyl cyclase, and no changes in G alpha s or G alpha i. By contrast, ethanol-treated N1E-115 cells show the largest up-regulation of the delta-opioid receptor, the most heterologous desensitization of adenylyl cyclase, and concentration-dependent decreases in G alpha s and increases in G alpha i. Further analysis of these related neuronal cell lines may help to identify the molecular elements that endow some, but not all, neuronal cells with the capacity to adapt to ethanol.     

Connection between 3H 5-HT and adenyl cyclase activation induced by 5-HT in preparations of cerebral glial membranes

Purified glial membrane preparations have been isolated from horse brain striatum. Tritiated 5-HT bound to these membranes with a high affinity (KD = 10 nM); the corresponding binding is reversible and appears specific of the serotoninergic structure. In parallel, 5-HT activates an adenylate cyclase with a low affinity (KD = 1 microM). The sites involved in this binding and in this adenylate cyclase activation appear different from the serotoninergic sites reported in the neuronal membrane preparations.     

Effect of aldosterone and methylprednisolone on cardiac NaK-ATPase.

Aldosterone (15 microgram BID) and methylprednisolone (8 mg QD) administration to female guinea-pigs augmented both the total and the specific activity of NaK-ATPase but not the activity of adenylate cyclase in the cardiac sarcolemma. The rise in NaK-ATPase was due to increase in the number of enzyme molecules; catalytic activity and ouabain-sensitivity of individual molecules did not change.     

Behaviour of the prolyl hydroxylase to collagen synthesis in KB cells]

KB cells, which synthetized collagen at a low rate, shown a prolyl hydroxylase activity at the same rate that fibroblast. The relationship between collagen synthesis and prolyl hydroxylase activity in these cells was discussed.