Absence Of Adenosine Research Articles

Adenine-derived purines increase adenosine triphosphate (ATP) levels in the luteal cell: evidence that cell levels of ATP may limit the stimulation of adenosine 3',5'-monophosphate accumulation by luteinizing hormone.

To assess if amplification of LH-stimulated cAMP accumulation and steroidogenesis by adenine-derived purines may be due to an increase in cell ATP levels, dispersed and enriched rat luteal cells were incubated with the purines under short term culture conditions. Adenosine produced an increase of 2-fold or more in the cell content of ATP; basal levels were about 160 ± 25 pmol/105 cells. An increase in ATP was een as early as 5 min, and the effect was maximal within 15 min of incubation. Incubation alone had little effect on the steady state concentration of ATP, no detectable ATP was seen in the incubation media, and the cells degraded extracellular ATP at a rate of 1 nmol/min · 105 cells. Concentrations of LH up to 500 ng/ml and of prostaglandin F2α at concentrations up to 1 μM had no effect on cell ATP levels in the presence or absence of adenosine. Cell ATP levels were increased by ADP, AMP, adenosine, inosine, adenine, and hypoxanthine, and these effects were paralleled by an increase in cAMP accumula...

On the mechanism by which theophylline inhibits histamine release from rat mast cells.

Theophylline (2.5 mM) did not influence the spontaneous release of histamine but inhibited histamine release induced by antigen, compound 48/80 or phosphatidylserine. The effect on 48/80-induced histamine release could not be reversed by increasing extracellular Ca2+. Exogenous adenosine (10(-8) to 10(-4) M) did not influence spontaneous histamine release or 48/80-induced release but potentiated antigen-induced release. The adenosine potentiation was competitively inhibited by theophylline in concentrations (10(-5) to 10(-4) M) lower than those required to inhibit antigen-induced histamine release in the absence of adenosine. In order to see if endogenous adenosine levels are high enough to potentiate an anaphylactic histamine release in vivo, adenosine was determined in mast cell incubates and in plasma from 4 different strains of rat. The levels were 0.18 to 0.99 microM in plasma, which is sufficient to cause significant potentiation of histamine release, but only 3 x 10(-8) M in mast cell incubates. Theophylline (2.5 mM) increased cAMP levels about 100%, whereas adenosine (10(-5) M) had little effect on cAMP and cGMP levels. However, when incubated together, adenosine could inhibit the theophylline-induced increase in cAMP levels but not the inhibition of histamine release. It is concluded that the effect of low concentrations of theophylline could be due partly to antagonism of adenosine effects. In addition, in higher doses, theophylline appears to exert an inhibitory action that is unrelated to cyclic nucleotides, extracellular calcium and adenosine.

Rabbit corneal endothelial permeability in the presence and absence of adenosine and glutathione.

Rabbit corneal endothelial fluxes of sodium, bicarbonate, inulin, and dextran have been measured in ambient solutions containing either 0.5 mM adenosine alone (A), 0.3 mM glutathione alone (G), both adenosine and glutathione, or neither adenosine nor glutathione. Addition of A alone to a solution lacking both additives caused a decrease in both Jstrendo and Jendostr sodium fluxes coupled with an increase in Jstrendo net. Addition of G alone caused no effect, whereas addition of A and G together caused a decrease in both Jstrendo and Jendostr sodium fluxes and a large increase in Jstrendo net. Addition of A or G caused an increase in Jstrendo bicarbonate flux, but no change in Jendostr flux. Addition of A and G caused an increase in both Jstrendo and Jendostr bicarbonate flux and a decrease in Jstrendo net. None of the solution variations caused an alteration in either inulin or dextran permeability. Since both A and G influence Jstrendo net bicarbonate and sodium transport, this may provide an explanation of the beneficial effects of these compounds on the endothelium to both enhance and prolong corneal thickness and hydration regulation.

Studies on purine transport and on purine content in vacuoles isolated from Saccharomyces cerevisiae

The transport of purine derivatives into vacuoles isolated from Saccharomyces cerevisiae was studied. Vacuoles which conserved their ability to take up purine compounds were prepared by a modification of the method of polybase-induced lysis of spheroplasts. Guanosine > inosine = hypoxanthine > adenosine were taken up with decreasing initial velocities, respectively; adenine was not transported. Guanosine and adenosine transporting systems were saturable, with apparent K m values 0.63 mM and 0.15 mM respectively, while uptake rates of inosine and of hypoxanthine were linear functions of their concentrations. Adenosine transport in vacuoles appeared strongly dependent on the growth phase of the cell culture. The system transporting adenosine was further characterized by its pH dependency optimum of 7.1 and its sensitivity to inhibition by S- adenosyl- l-methionine . In the absence of adenosine in the external medium, [ 14C]adenosine did not flow out from preloaded vacuoles. However, in the presence of external adenosine, a very rapid efflux of radioactivity was observed, indicating an exchange mechanism for the observed adenosine transport in the vacuoles. In isolated vacuoles the only purine derivative accumulated was found to be S- adenosyl- l-homocysteine .

Distribution of endogenously phosphorylated proteins in subcellular fractions of rat cerebral cortex

The cerebral cortex from adult rats was separated into several subcellular fractions by using established methods of differential and sucrose density gradient centrifugation. Aliquots from each fraction were incubated with γ-(32)P-ATP, in the presence and absence of adenosine 3',5'-monophosphate (cyclic AMP), and its protein constituents were separated by means of SDS-slab gel electrophoresis. Fractions containing nuclei, synaptosomes, myelin, microsomes, and soluble proteins each showed a characteristic pattern of protein staining and of endogenously phosphorylated proteins detected by autoradiography of the gels. Cyclic AMP-stimulated phosphorylation of proteins with MW 78K and 84K can serve as markers for membranes of synaptic origin, while cyclic AMP-independent phosphorylation of low-molecular-weight proteins (15K-20K) is characteristic of myelin. The finding of different phosphoproteins in various subcellular fractions may be related to the diversity of cellular functions known to be regulated by phosphorylative activity.

Growth in vitro and metabolism of Plasmodium vinckei chabaudi.

An in vitro system, based on the rocker dilution technic, has been developed that supports intraerythrocytic growth of a rat-adapted strain of Plasmodium vinckei chabaudi from ring to schizont stages; some reinvasion was obtained, although invariably, this was associated with a decrease in parasite numbers. Pertinent features were the very high buffer content of the medium and the low oxygen tension of the gaseous phase. Lactate production, glucose utilization, and 3H-leucine and 3H-adenosine incorporations were investigated for their suitability to monitor parasite growth. Throughout an 18-hr incubation there was a continuous and increasing production of lactate and utilization of glucose, which correlated well with the development of the parasites from ring to schizont stages. During the same period, there was a low but continuous and increasing incorporation of 3H-leucine into parasite protein. However, 3H-adenosine was incorporated only for the 1st hr of incubation, after which time no net incorporation occurred. Parasites grew normally from ring to schizont stages even in the absence of adenosine from the dilution medium.

The kinetics of serum-induced initiation of DNA synthesis in BHK 21/C13 cells, and the influence of exogenous adenosine

After the re-addition of serum in the presence of adenosine (25 muM), the entry of quiescent, serum starved BHK 21 cells into DNA synthesis follows first order kinetics after a well defined lag period of eight hours, and with a rate constant dependent on serum concentration. Initiation of DNA synthesis under these conditions can therefore be considered to be a random event occurring with a "Transition Probability" determined by the serum concentration. In the presence of adenosine, the change of Transition Probability following the addition of serum occurs abruptly. In the absence of exogenous adenosine, however, the change of Transition Probability after serum addition appears to be both gradual and bi-phasic. The initial changes in the absence of adenosine, though smaller in magnitude, display a similar dependence on serum concentration to the changes occurring in the presence of the nucleoside. In contrast, the secondary gradual increase of Transition Probability in the absence of added purines exhibits a higher serum requirement. It is suggested that the regulation of Transition Probability by serum involves some purine-dependent process, and that in the absence of an exogenous supply this becomes limited by endogenous synthesis which in turn may be dependent on serum concentration.

Demonstration of phosphorylase in urines by polyacrylamide gel disc electrophoresis

Using polyacrylamide gel disc electrophoresis, a sensitive detection method for glycogen phosphorylase has been developed. The method was highly sensitive even at the level where the usual assay method failed. With this method the presence of phosphorylase in the urines of various diseases was demonstrated. Of 315 cases tested phosphorylase was found in 56 cases, of which 51 cases were of urogenital diseases. In none of the cases tested was phosphorylase activity demonstrated in serum. In contrast to tissue phosphorylases, which showed a marked decrease of activity in the absence of adenosine 5'-monophosphate, urinary phosphorylase was not affected by this. From the retardation of mobility as a function of glycogen concentration, the dissociation constant of urinary phosphorylase for glycogen was calculated. It was estimated to be 2.1–2.7 mM for urinary phosphorylase of uterine carcinoma and 1.4 mM for that of vesical carcinoma.

Formula omitted] regulation of hepatic protein kinase by adenosine 3′,5′-monophosphate mediated glucagon stimulation

In vivo administration of glucagon caused an increase in the dissociation of protein kinase subunits which was accompanied by elevated adenosine 3′,5′-monophosphate concentrations in the rat liver. Concomitantly, there was a decrease in non saturated adenosine 3′,5′-monophosphate binding sites. A reduction in protein kinase activity measured in the presence of the cyclic nucleotide was apparent at 5 minutes of glucagon administration while enzyme activity assayed in the absence of adenosine 3′,5′-monophosphate was already increased after one minute. Glucose, given through an intragastric tube, caused no changes in the effect of glucagon on hepatic protein kinase.

Adenosine 3′,5′-Monophosphate-dependent Protein Kinase from Bovine Anterior Pituitary Gland: I. PROPERTIES

Abstract Adenosine 3',5'-monophosphate-dependent protein kinase was purified from bovine anterior pituitary gland by pH and ammonium sulfate precipitations and chromatography on sulfoethyl-Sephadex. Maximal rates of histone phosphorylation were observed in the presence and absence of adenosine 3',5'-monophosphate (cyclic AMP) at pH 6.5 and 7.0, respectively. The apparent Km value for histone was 125 µg per ml in the presence and absence of cyclic AMP. The apparent Km values for ATP in the presence and absence of cyclic AMP were 1.7 and 1.3 x 10-5 m, respectively. Half-maximal stimulation by the cyclic nucleotide was observed at 2.5 x 10-8 m. Maximal activation of protein kinase activity was obtained at much higher concentrations of the other cyclic nucleotides. No significant phosphorylation of histones occurred when γ-labeled UTP, CTP, or GTP was used instead of ATP. The enzyme requires both Mg2+ and ATP-Mg2- for activity. Magnesium decreases the apparent Km of the enzyme for ATP and increases the Vmax of enzymatic activity. The two peaks of cyclic AMP-dependent protein kinase activity observed on sucrose gradients are converted to a single light peak in the presence of cyclic AMP.

Resistance to Catabolite Repression of Histidase and Proline Oxidase during Nitrogen-limited Growth of Klebsiella aerogenes

Abstract In Klebsiella aerogenes β-galactosidase and histidase are subject to strong catabolite repression by glucose in the presence of a good source of nitrogen. Under conditions of nitrogen limitation, the catabolite repression of histidase is relieved, while β-galactosidase remains strongly repressed. Exogenous adenosine 3',5'-monophosphate relieves the catabolite repression of both histidase and β-galactosidase. A mutant which requires exogenous adenosine 3',5'-monophosphate for the synthesis of many catabolite-sensitive enzymes is able to utilize histidine and synthesize histidase at a rapid rate under conditions of nitrogen limitation in the absence of adenosine 3',5'-monophosphate. Proline oxidase, like histidase, is subject to catabolite repression and is synthesized rapidly in the presence of glucose when the supply of nitrogen is limited. The adenosine 3',5'-monophosphate requiring mutant is able to utilize proline as a sole nitrogen source in the presence of glucose without exogenous adenosine 3',5'-monophosphate. In Salmonella typhimurium, histidase is strongly repressed by glucose, even in nitrogen-starved cells. When histidase genes from S. typhimurium were transferred on an episome to cells of K. aerogenes carrying a deletion of the histidase gene, catabolite repression of histidase was relieved by nitrogen limitation. It is concluded that in K. aerogenes certain catabolitesensitive enzymes which degrade nitrogenous compounds are synthesized rapidly in the presence of glucose if the supply of nitrogen is limited. This rapid synthesis is mediated by a cytoplasmic factor which acts independently of adenosine 3',5'-monophosphate.

Permeability to water of rabbit corneal membranes.

Permeability to water of rabbit corneal membranes.

Enzymes of glycogen metabolism in white blood cells. II. Activation and inactivation of glycogen phosphorylase of rat chloroma.

Summary 1. The properties of glycogen phosphorylase (α-1,4-glucan:orthophosphate glucosyl transferase, EC 2.4.1.1) of rat chloroma, a tumor composed entirely of immature granulocytes, and the interconversion of the active and inactive forms of the enzyme have been studied in crude chloroma extract. 2. Striking similarities in the properties of chloroma phosphorylase to liver phosphorylase have been noted. Thus, the following properties are shared by both enzymes: Two forms of enzyme can be identified, an active and an inactive form; the active form exhibits 65–80% of its activity in the absence of adenosine 5′-phosphate, the inactive enzyme is not significantly active even in the presence of the nucleotide; the activity of the enzyme is not enhanced by cysteine; the inactive enzyme shows considerable activity when assayed in the presence of high salt concentration; the enzyme is activated by glucagon. 3. A phosphorylase kinase (ATP:phosphorylase phosphotransferase, EC 2.7.1.38) capable of converting rabbit-muscle phosphorylase b to a and a phosphatase (phosphorylase phosphohydrolase, EC 3.1.3.17) catalyzing the reverse reaction both active at neutral pH have been demonstrated in chloroma tissue.