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Biomedical subjects

G Vereb

Publications and source records attributed to G Vereb.

At least 37 records · Page 2Linked to original sources
PubMed1993

Biphasic effect of extracellular ATP on the membrane potential of mouse thymocytes.

Extracellular ATP induced changes in the membrane potential of thymocytes from BALB/c mice were analyzed. At concentrations below 0.1 mM, ATP hyperpolarizes the cell membrane on the time scale of development of the Ca(2+)-signal. After a longer time hyperpolarization turns to depolarization. ATP concentrations higher than 0.5 mM caused rapid depolarization without previous hyperpolarization. Verapamil, quinine or the absence of extracellular Ca2+ blocked the hyperpolarization by ATP. In Na(+)-free medium the magnitude of depolarization decreased. Our data suggest a contribution of Ca(2+)-activated K+ channels to the hyperpolarizing effect of ATP at lower concentrations. The direction of membrane potential changes is determined presumably by a sensitive balance of ATP-receptor mediated Ca(2+)- and Na(+)-influx and the Ca(2+)-activated K(+)-channel activity.

Adenosine Triphosphate↗
PubMed1992

Synthesis and enzymatic activity of some new purine ring system analogues of adenosine 3',5'-cyclic monophosphate.

A series of novel adenosine 3',5'-cyclic monophosphate (cAMP) analogues, as well as their 6-deamino and 6-nitro derivatives, were synthesized where the purine ring was replaced by indazole, benzotriazole, and benzimidazole. The 3',5'-cyclic monophosphates of indazole and benzotriazole ribofuranosides, where the sugar-phosphate moiety is attached to the N-2 nitrogen atoms of the heterocycles, were also prepared. The biological efficiency of the analogues was tested by their ability to activate purified cAMP-dependent protein kinase I (PK-I) from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart. Each cyclic nucleotide is capable of activating both PK isozymes in half-maximum concentrations (Ka) ranging from 2.0 x 10(-8) to 4.8 x 10(-6) M. The cyclic phosphate of N-1-beta-D-ribofuranosylindazole (13) proved to be a very poor activator for both PK-I and PK-II, but when indazole binds by N-2 to ribose or when the hydrogen atom at C-4 is substituted by a nitro or amino group, activities of the analogues increase considerably. The activating potencies of benzotriazole derivatives are similar to that of cAMP, irrespective of the C-4 substituents. The Ka' values of cyclic nucleotides containing benzimidazole were found to be higher for PK-II than for PK-I; e.g. the activity of 4-nitro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphate (32) is nearly 20 times as high for PK-II than for PK-I.

Animals↗
PubMed1992

Phosphorylation-induced conformational changes in the phosphorylase ab hybrid as revealed by resolution of pyridoxal 5'-phosphate with imidazole citrate and cysteine.

The accessibility of pyridoxal 5'-phosphates of the phosphorylase ab hybrid to resolution by imidazole citrate and cysteine was studied and compared with that of the b and a forms. Promotion of resolution of phosphorylated forms by raising the temperature or in the presence of glycogen indicates that the resistance of phosphorylase a and ab to resolution at 0 degrees C is due rather to their tetrameric state than their phosphorylation-related active conformation. The pattern of resolution of the ab hybrid was similar to that of the a and differed from that of the b forms in that it occurred at 30 degrees C and 37 degrees C but not at 0 degrees C, moreover, it did not show first-order kinetics. On the other hand, inhibition of resolution by ligands binding to the nucleotide site of phosphorylase reflected an intermediate sensitivity of the ab form between that of the b and a forms. We conclude that partial phosphorylation of phosphorylase b elicits conformational change(s) in both subunits which influence the monomer-monomer interactions and resolution of pyridoxal 5'-phosphates. Resistance of ab hybrid to monomerizing agents as imidazole citrate, comparable to that of other forms, argues for its stability, ruling out its reshuffling into mixtures of phosphorylase b and a.

Adenosine Monophosphate↗
PubMed1992

Effect of some new cAMP analogs on cAMP-dependent protein kinase isoenzymes.

1. Ten new cAMP analogs were synthesized by replacing the purine ring with with indazole, benzimidazole or benztriazole and/or their nitro and amino derivatives. 2. Each analog proved effective in activating cAMP-dependent protein kinase I (PK-I) purified from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart and chasing 8-[3H]cAMP bound to regulatory subunits in the half-maximal effective concentrations of 2 x 10(-8)-8 x 10(-6) M. 3. The N-1-beta-D-ribofuranosyl-indazole-3'5'-cyclophosphate(I) proved a very poor chaser and activator of both isoenzymes, but when indazole was attached at its N-2 to ribose (IV) or when its H at C-4 (equivalent to the position of amino-group in adenine) was substituted by an amino-(III) or especially nitro-group (II) its efficiency was dramatically increased. 4. Analogs containing benztriazole ring proved as powerful as cAMP irrespective of the presence of substituents (VII-X). 5. Benzimidazole derivatives with amino-(VI) or nitro-group (V) activated PK-II 3 and 20 times better than PK-I. 6. Attaching of ribose to N-2 of indazole or benztriazole increased the affinity to PK-II 10 and 4 times, respectively. 7. Chasing efficiency of cAMP analogs at half-saturating [3H]cAMP tended to correlate with activating potency only for PK-I but at saturating [3H]cAMP concentration for both isoenzymes. 8. On the basis of synergistic activation with 8-Br-cAMP a site 2-selective binding of nitro-benzimidazole (V) and unsubstituted benztriazole (VII) derivatives to PK-II is suggested.

Cyclic AMP↗
PubMed1991

Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent.

Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.

Calcium↗
PubMed1990

Effect of cyclosporin A on the membrane potential and Ca2+ level of human lymphoid cell lines and mouse thymocytes.

The effect of the immunosuppressive cyclosporin A (CsA) on the cytosolic free Ca2+ concentration ([Ca2+]i) and membrane potential of human B and T lymphoblastoid cells and mouse thymocytes was studied in order to reveal some features of the early stage of drug-cell interaction. Cytosolic free Ca2+ concentration of the cells was measured by spectrofluorimetry using indo-1 and quin2 fluorescent calcium indicators. Membrane potential was monitored in a flow cytometer with oxonol dye. CsA applied at 2-20 micrograms/ml final concentrations caused a dose-dependent, rapid, transient rise of [Ca2+]i in all cell types. This effect could be blocked by chelating the extracellular Ca2+ with EGTA but was not sensitive to Ca2+ channel blockers verapamil and nifedipine or K+ channel blocker 4-aminopyridine. A possible explanation for the calcium mobilizing effect of CsA is an ionophore-like mode of action at the cell membrane level. Besides directly interfering with mitogenic signals, the elevation of [Ca2+]i could be responsible for an initial hyperpolarization observed in CsA-treated T lymphocytes. This hyperpolarization, however, was not detectable in B lymphoblastoid cells. A further difference between B and T cells was the diverse pattern of depolarization following CsA treatment. This variance in the behaviour of T and B lymphocytes and the diversity of membrane transport systems in its background could account for the different final outcome of the drug-cell interaction.

Animals↗
PubMed1989

The role of autophosphorylation of cAMP-dependent protein kinase II in the inhibition of protein phosphatase-1.

1. The inhibition of the catalytic subunit of protein phosphatase-1 (PP-1c) by the regulatory subunit of cAMP-dependent protein kinase II (RII) was studied. 2. Phosphorylation or thiophosphorylation of RII increased its inhibitory potency up to 4- and 6-fold and rendered it competitive with respect to the substrate of PP-1c, phosphorylase a. The Ki values for thiophospho-RII and phospho-RII were 200 and 500 nM, respectively. 3. Though PP-1c was able to release phosphate from phospho-RII, its activity once incubated with phospho-RII, remained inhibited even 80% of the phosphate was released from phospho-RII. 4. The catalytic subunit of cAMP-dependent protein kinase was effective in suspending the inhibition employed either before or after the addition of phospho-RII to PP-1c. 5. No exclusive bindings of thiophospho-RII and heat-stable protein inhibitors to the PP-1c could be proved by double inhibition studies, however some synergism was observed in their effect.

Binding Sites↗
PubMed1988

The sequence around the phosphorylation site of the porcine heart type phosphorylase isoenzyme.

1. A tetradecapeptide containing the phosphorylation site was obtained from 32P-labelled pig heart phosphorylase a isoenzyme by alpha-chymotryptic digestion. 2. The peptide was purified by Mono S cation-exchange chromatography and reversed-phase HPLC. 3. The phosphorylated residue was identified as Ser and the sequence was determined: T D G E R R K Q I S V R G L. 4. The sequence was compared to the known sequences of muscle and liver type isophosphorylases and the structural consequences of the amino acid residue exchanges were predicted.

Amino Acid Sequence↗
PubMed1988

Activation/dephosphorylation of rabbit muscle glycogen synthase by the catalytic subunits of protein phosphatase-1 and 2A.

Glycogen synthase a was purified from rabbit skeletal muscle by a procedure involving heparin-Sepharose chromatography. Glycogen synthase a was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to give synthase b1. Dephosphorylation and activation of synthase b1 was investigated using the catalytic subunits of protein phosphatase-1 and 2A. The dephosphorylation and activation of synthase b1 was biphasic with a larger rate constant for the initial phase. Analysis of tryptic phosphopeptides of glycogen synthase during the course of dephosphorylation revealed a faster initial phosphate release from site-2 by both phosphatases comparing to sites-1a and 1b. Ligand effects on synthase phosphatase reactions were also studied. Spermine was found to inhibit protein phosphatase-1 activity and to stimulate type-2A phosphatase using synthase b1 as substrate.

Animals↗
PubMed1987

Kinetic characterization of rabbit skeletal muscle phosphorylase ab hybrid.

Phosphorylase ab was prepared in vitro by partial phosphorylation of rabbit skeletal muscle phosphorylase b and was isolated by DEAE-Sephacel chromatography. Its phosphorylated and non-phosphorylated subunits could not be distinguished by different affinity to substrates, activators or inhibitors, indicating their coordinated function. In the absence of nucleotide activators, the Km values for Pi and glucose-1-P were 28 mM and 18 mM, respectively. Activity in the presence of 16 mM glucose-1-P was doubled by 10(-4) M AMP or 10(-3) M IMP, mainly by lowering the Km for glucose-1-P. Half-maximum activation was exerted by 2 microM AMP or 0.1 mM IMP. Activation by these nucleotides showed no cooperativity. Glucose exerted competitive inhibition with respect to glucose-1-P, while for the inhibition by glucose-6-P an allosteric mechanism is suggested; the appropriate Ki values were 4.5 mM and 1.5 mM, respectively. The Hill coefficient for glucose-1-P binding was about 1.0, even in the presence of glucose (up to 10 mM), but 10 mM glucose-6-P lowered it to 0.47, indicating a negative heterotropic cooperativity. Effective regulation of the activity of phosphorylase ab by physiological concentrations of Pi, AMP, IMP and glucose-6-P suggests its metabolic control under in vivo condition.

Adenosine Monophosphate↗
PubMed1986

Regulatory subunit of type II cAMP-dependent protein kinase as substrate and inhibitor of protein phosphatase-1 and -2A.

The dissociated regulatory subunit (RII) of autophosphorylated cAMP-dependent protein kinase II was dephosphorylated by the catalytic subunits of protein phosphatase-1 and -2A (phosphatase-1c and -2Ac) and by a high-Mr polycation-dependent form of phosphatase-2A (2Ao) with Km values of 5, 0.3 and 1 microM, respectively. Dissociation of protein kinase by cAMP preferentially increased the dephosphorylation of RII by phosphatase-1c, whereas polycations (histone Hl or polybrene) markedly stimulated phosphatase-2Ac and -2Ao even in the absence of cAMP. Thiophosphorylated RII inhibited the dephosphorylation of phosphorylase a by these phosphatases with half-maximum inhibitory concentrations of 0.1-0.36 microM.

Animals↗
PubMed1986

Catalytic unit-independent phosphorylation and dephosphorylation of type II regulatory subunit of cyclic AMP-dependent protein kinase in rat liver plasma membranes.

Rat liver plasma membranes contain a 55 kDa protein which proved to be identical with type II regulatory subunit (RII) of the cyclic AMP-dependent protein kinase (kinase A) by several criteria (gel electrophoretic behaviour, peptide map, position of the autophosphorylated site). Analysis of phosphopeptide maps revealed that the membrane-bound RII was phosphorylated by a kinase which is unrelated to the catalytic unit (C) of kinase A. Dephosphorylation of the membrane-bound RII by an endogenous phosphatase was stimulated by both cyclic AMP and fluoride. Addition of C did not stimulate dephosphorylation even in the presence of ADP; moreover, protein inhibitor of C did not modify the effects of cyclic AMP or fluoride. The effects of both cyclic AMP and fluoride were, however, inhibited by C. Results indicate that rat liver plasma membranes contain a phosphorylation-dephosphorylation system for which RII is a relatively specific substrate.

Animals↗
PubMed1986

Formation of partially phosphorylated phosphorylase in isoproterenol stimulated rat hearts.

Phosphorylase ab hybrid was demonstrated in perfused rat hearts and during the in vitro conversion of purified rat heart phosphorylase b. Phosphorylase ab hybrid was determined in rat heart extracts by the activating effect of AMP in the presence of caffeine. These results were confirmed by the quantitative determination of incorporated 32P in vitro and through the characteristic inhibition of ab hybrid by glucose-6-phosphate. As shown by our results, in aerobically perfused control hearts only the ab hybrid represents the active form of phosphorylase, its activity reaching about 20% of the total. In response to isoproterenol (5-1000 ng), the amount of ab hybrid rose to about 30-40%, preceding the rise of the a form, which increased in a dose-dependent manner up to 45% of the total. The great sensitivity of the ab form to AMP activation and glucose-6-phosphate inhibition supports its physiological significance in heart under in vivo conditions as well. Our results strongly suggest that the activity ratio -AMP/ + AMP reflects rather the percentage ratio of phosphorylated subunits than that of the activated (partially or totally phosphorylated) phosphorylase molecules.

Adenosine Monophosphate↗
PubMed1984

Effect of immobilization on some glycolytic enzymes of skeletal muscle.

The effect of immobilization by plaster cast was studied on the activities of phosphoglucomutase (PGM), pyruvate kinase ( PyK ) and cAMP-dependent protein kinase in fast (extensor digitorum longus [EDL]), and slow (soleus) muscles of rats. In control untreated animals, PGM and PyK activities are 3 and 5 fold higher respectively in EDL than in soleus in correlation with the high glycolytic activity of fast muscles. During the four weeks period of immobilization a 20% decrease occurred in PGM activity, to which no limiting role in glycolysis is attributed, while PyK which has a regulatory function, showed a 35% decrease in EDL; at the same time in the soleus the activity of these enzymes did not change. The decrease of PGM and PyK activity in EDL diminished the difference between the slow and fast muscles, and it was evaluated as a tendency to dedifferentiation (transformation). The activity of cAMP-dependent protein kinase, in contrast to the glycolytic enzymes, was higher in the soleus than in the EDL and during immobilization it did not change significantly in either the muscles.

Animals↗
PubMed1983

Ligand effects on the dephosphorylation of heart and skeletal muscle specific phosphorylases.

Pseudo first order rate constants were determined for the dephosphorylation of heart and skeletal muscle specific phosphorylase a isoenzymes isolated from rabbit and pig using rabbit muscle phosphorylase phosphatase (mol. wt 34,000). The rate constants determined in the absence of ligands, were 4-5 fold lower for heart specific phosphorylases than for skeletal muscle specific ones. Glucose 6-phosphate (0.5-1 mM) enhances the rate of dephosphorylation of heart specific isophosphorylases 3-fold and suspends inhibition by 10(-5) M AMP, however, it has no significant effect on the dephosphorylation of skeletal muscle specific enzymes under the same conditions. Our data support characteristic functional differences between heart and skeletal muscle specific phosphorylases both in rabbit and pig.

Adenosine Monophosphate↗
PubMed1983

Effect of immobilization on the enzymes of glycogen metabolism.

Activity of the enzymes regulating glycogen metabolism was determined for glycogen phosphorylase (Ph), phosphorylase kinase (PhK) and glycogen synthetase (GS) in fast extensor digitorum longus (EDL) and slow (soleus) muscles of rats following immobilization by plastering. It was shown that the activity not only of Ph but also of PhK was about 8 times higher in the EDL than in the soleus muscle in correlation with the higher glycogenolytic activity of fast muscles. The activity of GS was approximately similar in the two muscles. PhK activity decreased significantly in the immobilized EDL muscle while in the soleus an early reduction of GS activity ensued. Reduction of the activity of the glycogenolytic enzymes caused dedifferentiation in the muscles with anaerobic metabolism and containing large amounts of glycogenolytic enzymes. The similar rate of Ph and PhK in fast and slow muscles, as well as the similar rate of their reduced activity, during immobilization indicated a close correlation between the two enzymes and raised the possibility of a combined regulation of their turnover.

Animals↗
PubMed1982

Ligand effects on the activity of the a form of various mammalian heart specific isophosphorylases.

1. The allosteric sensitivity of heart specific and skeletal muscle specific phosphorylase a-s from rabbit, pig, bovine and dog was compared. 2. 0.2-0.3 mM glucose 6-phosphate exerts 50% inhibition on the heart specific phosphorylase a-s without any inhibitory effect on the skeletal muscle specific ones. 3. AMP decreases the Km for substrates (glucose 1-phosphate, or Pi) of all heart specific phosphorylase a-s. It also enhances the Vmax of pig and bovine phosphorylase a-s 2-fold, but has no effect on the Vmax of rabbit and dog enzymes.

Adenosine Monophosphate↗