Use of affinity elution chromatography for the microassay of adenosine 3':5'-monophosphate.

Using the principle of affinity elution chromatography, a microassay has been developed for the determination of picomolar and even smaller quantities of adenosine 3'.5' -monophosphate (cyclic AMP) in biological samples. In principle, cyclic AMP along with the other anions is adsorbed onto an anion exchange cellulose and subsequently, the cyclic nucleotide is specifically eluted with cyclic-AMP -dependent protein kinase. In the actual assay cyclic AMP is determined by competitive displacement of radiolabelled cyclic AMP by non-labelled cyclic nucleotide. The described procedure results in a linear dependence for the displacement of the radioactive nucleotide on cyclic AMP concentration and obviates the usual procedure of sample enrichment by lyophilization. The degree of interference of different nucleotides including cyclic GMP was found to be minimal and in this respect the use affinity elution chromatography was comparable to radioimmunoassay.     

Cyclic adenosine 3',5'-monophosphate phosphodiesterase from Mucor rouxii: regulation of enzyme activity by phosphorylation and dephosphorylation.

Crude preparations of cyclic adenosine 3′, 5′-monophosphate phosphodiesterase were activated 1.5 to 2 fold by incubation with ATP, Mg²⁺ and cyclic AMP in a reaction which was both, time and temperature dependent. Cyclic AMP phosphodiesterase remained in an activated state upon filtration of the enzymatic preparation through Sephadex G-25 and ion-exchange chromatography. Activation of the enzyme in the presence of [γ ³²P]ATP resulted in a significant amount of [³²P] protein-bound radioactivity. Reversible deactivation of cyclic AMP phosphodiesterase was enhanced by Mg²⁺ and was accompanied by the release of [³²P] protein bound radioactivity. The evidence is consistent with a mechanism for controlling cyclic AMP phosphodiesterase through phosphorylation-dephosphorylation sequence.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

Phospholipid metabolism and lysosomal enzyme secretion by leukocytes. Effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP.

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

Refractoriness of cation transport in turkey erythrocytes to stimulation by cyclic adenosine 3':5'-monophosphate.

In turkey erythrocytes bidirectional fluxes of sodium and potassium develop a time-dependent refractoriness to stimulation by endogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP). The refractoriness of potassium influx and potassium outflux (both of which require extracellular sodium and potassium for stimulation by cyclic AMP) depends on the extracellular concentrations of sodium and potassium. In contrast, the refractoriness developed by sodium outflux (which does not require extracellular sodium or potassium for stimulation by cyclic AMP) does not depend on the extracellular concentrations of sodium or potassium. The refractoriness of these fluxes to cellular cyclic AMP reflects a decrease in the amount by which they can be maximally stimulated and appears to be proportional to the extent to which the transport system is utilized during the course of the incubation. Ouabain significantly reduces the rate at which cation transport in turkey erythrocytes becomes refractory to endogenous cyclic AMP. This effect of the glycoside is independent of the extracellular concentrations of sodium or potassium and does not correlate with how it alters the initial response of the transport systems to cyclic AMP.     

Use of novel assays to indicate that O-esters and S-esters are produced by the same enzyme in brewing yeast

Ethanol- and methanethiol-dependent removal of acetyl-CoA by crude extracts of ale yeast has been monitored using a decrease in OD232. Activity has also been detected in these extracts after fractionation on polyacrylamide gels, in this case using a novel assay in which the coenzyme A produced in the reaction is linked via DCPIP reduction to color formation from nitroblue tetrazolium. Ethanol- and methanethiol-dependent activities migrate identically on such gels, and only one band of color formation was observed. Furthermore they displayed closely similar sensitivity to heating at 40 degrees C and 60 degrees C and pH optima, with activity maximal at pH 7.5. It is likely that a single enzyme is responsible for the formation of O-esters and S-esters in yeast. Initial kinetic studies indicate that methanethiol has higher affinity for the enzyme than has ethanol and a higher maximum velocity. However, the enzyme has a much lower Km for acetyl-CoA, suggesting that the alcohol or thiol substrate is the more likely substrate to be limiting.     

Selectivity in enzyme catalysed reaction: Preferential hydrolysis of saturated methyl ester over the corresponding unsaturated ester by pig liver esterase

Summary Saturated methyl ester is preferentially hydrolysed in presence of corresponding ,-unsaturated ester by Pig Liver Esterasse. This selectivity is known for aliphatic, aromatic and heterocyclic esters.Chemo, regio and stereoselective transformations are an important goal towards synthetic efficiency (Bartman and Trost, 1984). Among the various methods that are employed to achieve such reactions, enzymatic transformations are in the forefront of current research activities (Jones, 1986) because of several advantages over the chemical methods. These include extremely high degree of selectivity, easier isolation of products, specially with immobilized enzymes and their reusability. In the past decade this class of biocatalysts has provided a plathora of valuable selective transformations (Boland et.al, 1991). Pig Liver Esterase (PLE) is one such enzyme which has been thoroughly studied and used for asymmetric (Zhu and Tedford, 1990) and regioselective (Adachi et.al, 1986) hydrolysis of esters. Several chiral building blocks for the synthesis of complex natural products (Imori et.al., 1983) have been synthesized through PLE. In this communication, we report the preferential hydrolysis of a saturated ester over its unsaturated counterpart. The preference is total and works well for aliphatic, aromatic as well as heterocyclic systems.     

Studies on nucleotidases in plants. Dimerization of the crystalline mung bean nucleotide pyrophosphatase by 5'-adenosine monophosphate and the properties of the dimerized enzyme.

The addition of AMP to the crystalline and homogeneous mung bean nucleotide pyrophosphatase [EC 3.6.1.9]altered its electrophoretic mobility. AMP was tightly bound to the enzyme and was not removed on passage through a column of Sephadex G-25 or on electrophoresis. The molecular weight of the native and AMP-modified enzymes were 65,000 and 136,000, respectively. The properties of the native enzyme such as the pH (9.4) and temperature (49 °C) optima, inhibition by EDTA, reversal of EDTA-inhibition by Zn²⁺ and Co²⁺, were not altered on dimerization by AMP. The AMP-modified enzyme had a linear time-course of reaction, unlike the native enzyme which exhibited a biphasic time-course of reaction. The AMP-modified enzyme was irreversibly denatured by urea. AMP concentrations larger than 100 μM inhibited linearly the activity of the AMP-modified enzyme. ADP and ATP inhibited the activity in a sigmoidal manner. K_m and V of the native and AMP-modified enzymes were, 0.25 mm and 0.58 mm; and 3.3 and 2.5, respectively.     

The relation of endogenous adenosine cyclic 3':5'-monophosphate to the antagonistic effects of adenosine and colchicine on cell shape.

Adenosine and colchicine have antagonistic effects on cell shape. When Chinese Hamster lung fibroblasts (CHE36-6) or SV40 transformed 3T3 (SV3T3) cells are incubated with colchicine (1 muM) for one hour at 37 degrees C, they round up into spheres with short spikes. Cells treated with adenosine (1 muM-minus 4 mM) for one hour become refractile and develop spindly processes. However, when the two compounds are added simultaneously, the characteristic responses to either drug are abolished and the cells appear normal. The counteraction is specific for adenine and its derivatives, adenosine being the most effective of the compounds we tested. Accumulation of colchicine or adenosine is not altered significantly by the presence of the other drug, ruling out decreases in uptake as the basis of the mutual antagonism. The morphological changes can be observed under conditions where there are no changes in intracellular cAMP levels (such as incubation with low concentrations of adenosine or cordycepin, an adenosine analog that cannot be directly converted to cAMP). Colchicine does not alter cAMP content of control or adenosine-treated cells. These data show that adenine compounds have potent effects on cell shape, and the antagonistic effects of adenosine and colchicine on cell shape are not mediated through changes in intracellular cAMP levels.     

Nitric oxide degradation by potato tuber mitochondria: evidence for the involvement of external NAD(P)H dehydrogenases

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O2(-)). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O2(-) generation, besides complex III, stimulated NO degradation. Larger amounts of O2(-) were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O2(-) production were stimulated by free Ca2+ concentration. Together, these results indicate that Ca2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O2(-) production that favors NO degradation in potato tuber mitochondria.     

Calcium ion effects on cyclic adenosine 3':5'-monophosphate bindings to the plasma membrane of Dictyostelium discoideum.

The study of cell surface cyclic adenosine 3':5'-monophosphate binding to Dictyostelium discoideum amoebae indicates that Ca2+ increases the number of binding sites without significantly affecting their affinity constant(s). The effects of the ion are observed immediately (within 4 s after addition) and appear to be readily reversible. Ca2+ effects are observed at various temperatures and pH values and are not blocked by the presence of various metabolic inhibitors. Increases, and decreases, in the apparent number of cyclic nucleotide binding sites could also be effected by concanavalin A treatments which respectively stimulate, and inhibit cell differentiation.     

Observations on the binding of adenosine 3':5'-monophosphate to cell membrane fragments from ox cerebral cortex.

Microsomal or synaptosome membrane fragments from ox brain bind cyclic AMP with a pH optimum of 7.0. Scatchard analysis shows the presence of at least two binding sites. Cyclic GMP and cyclic IMP only inhibit binding at concentrations 5000 times that of cyclic AMP and even higher concentration ratios of ATP and AMP have no effect. Membrane fragments saturated with cyclic [3-H]AMP lost less than 7% of bound nucleotide on incubation at 0 degrees C for 45 min but lost 25 % in the same period in the presence of 10 muM non-radioactive cyclic AMP.     

Extreme resistance to potato virus V in clones of Solanum tuberosum that are also resistant to potato viruses Y and A: evidence for a locus conferring broad-spectrum potyvirus resistance

 Extreme resistance to the potato V potyvirus (PVV) was found in four potato cultivars that contain Ry genes from Solanum stoloniferum. When plants of these cultivars, were inoculated by grafting in shoot tips from PVV-infected tomato plants, necrotic symptoms developed in some cultivars, although a full hypersensitive reaction was not elicited, while other cultivars were symptomless. PVV replication was not detected in any of the inoculated plants by ELISA, an infectivity assay of leaf extracts by manual inoculation to Nicotiana benthamiana indicator plants, or by ‘return grafting’ of shoot tips taken from newly developed shoots of the potato plants to virus-free indicator plants of tomato. These methods readily detected PVV infection in inoculated plants of cv ‘Flourball’, which does not contain an Ry gene and is susceptible, and in cvs ‘Maris Piper’ and ‘Dr Macintosh’, which contain gene Nv conditioning a hypersensitive reaction to inoculation. One of the Ry-containing cultivars, ‘Barbara’, has been previously shown to contain two genes that control extreme resistance, defined as no viral replication in intact plants, to the potyviruses potato viruses Y and A (PVY and PVA). These genes are: Ry _sto , which conditions resistance to PVY and PVA, and gene Ra, which conditions resistance to PVA only. It was found that in genotypes from a progeny of the cross ‘Barbara’ (Ry _sto /Ra)בFlourball’ (ry/ra), extreme resistance to PVV segregated with gene Ry _sto . It is proposed that either gene Ry _sto conditions broad-spectrum extreme resistance to the distinct potyviruses PVY, PVA, and PVV or that Ry _sto represents a family of genetically closely linked genes each controlling resistance to a specific virus. Received: 27 December 1996 / Accepted: 9 June 1997     

Regulation of intracellular adenosine cyclic 3':5'-monophosphate levels in Escherichia coli and Salmonella typhimurium. Evidence for energy-dependent excretion of the cyclic nucleotide.

Sugars and other energy sources were found to lower intracellular concentrations of adenosine 3':5'-monophosphate (cyclic AMP) in strains of Escherichia coli and Salmonella typhimurium which were deficient for cyclic AMP phosphodiesterase. This effect required the presence of the specific transport system responsible for entry of that sugar into the cell and depended on the intracellular catabolic enzymes. Metabolizable sugars were more effective than nonmetabolizable sugars in reducing cellular cyclic AMP levels, and this reduction was blocked partially by uncouplers of oxidative phosphorylation. Electron donors such as lactate and ascorbate plus phenazine methosulfate reduced internal cyclic AMP levels in bacterial membrane vesicles which had been preloaded with the cyclic nucleotide. Uncouplers of oxidative phosphorylation, but not arsenate, blocked the energy-stimulated loss of intravesicular cyclic AMP. Employing intact cells, sugars were shown to have two primary effects on cyclic AMP metabolism: (a) they inhibited net synthesis of the cyclic nucleotide while promoting its degradation, and (b) they stimulated efflux of cyclic AMP into the extracellular fluid. While the former effect was elicited by metabolizable and nonmetabolizable sugars alike, stimulation of cyclic nucleotide excretion was only observed with metabolizable sugars. The results suggest that the extrusion of cyclic AMP from the bacterial cell is energy-dependent and is driven by an energized membrane state.