A cAMP receptor from mouse liver cytosol whose binding capacity is enhanced by Mg++-ATP.

A receptor with a dissociation constant of 2·10^?6M for cyclic 3′,5′-AMP (cAMP) has been found in mouse liver cytosol. This cAMP binding activity can be differentiated from the cAMP-dependent protein kinase holoenzymes and the free regulatory subunits also found in the cytosol. Mg⁺⁺-ATP increases the number of binding sites for cAMP several fold. This increased capacity for cAMP binding persists after Sephadex G-25 filtration, and incubation for 14 hours in the presence of 5 mM EDTA. Among several adenosine- and guanosine-derivatives tested, only AMP, ADP and ATP compete efficiently with [³H] cAMP for the cAMP binding site.     

Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa: Purification by affinity chromatography and physicochemical properties

Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa was purified 150-fold by affinity chromatography on immobilized 2′-AMP. The binding of the enzyme is pH dependent. Elution was achieved with 2′-AMP, NADP⁺, or NADPH but not with 5′-AMP, NAD⁺, or NADH. The enzyme preparations appeared to be homogeneous in gel chromatography and ultracentrifugation, but only if these procedures were carried out in the presence of 2′-AMP or NADP⁺. With 2′-AMP a sedimentation coefficient of 34 S, a molecular weight of 1.6–1.7 million, and a Stokes' radius of 11.7 nm were determined. In the presence of NADP⁺ the sedimentation coefficient was 42 S and the molecular weight was 6.4 million. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed one kind of subunit with a molecular weight of 54,000. This was consistent with results from amino acid analyses and paper chromatography of peptides. Eight molar urea inactivated the enzyme but did not dissociate it into subunits. Full activity was restored after dialysis against urea-free buffer by mercaptoethanol and flavin-adenine dinucleotide.     

Identification and characterization of the adenosine 3′,5′-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3′,5′-[³²P]monophosphate (8-N₃-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5′-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.     

Cyclic AMP phosphodiesterase from dormant tubers of Jerusalem artichoke

An enzyme, which hydrolyzes 3′,5′-cyclic AMP to 3′-AMP and 5′-AMP, has been isolated from dormant tubers of Jerusalem artichoke and purified 850 × with a recovery of 15% of total activity. The partially purified enzyme differs greatly from both animal and bacterial phosphodiesterases in terms of pH optimum, substrate specificity, cation dependence and sensitivity to methylxanthines. The plant hormones are without effect, whereas ATP, 5′-AMP, 3′-AMP, inorganic phosphate and pyrophophosphate are inhibitors. The enzyme seems to be greatly inhibited in vivo by inorganic phosphate during dormancy.     

3′, 5′-Cyclic Adenosine Monophosphate Dependent Xylose Lethal Phenomenon in Escherichia coli

The growth of Escherichia coli W2252 was found to be inhibited when xylose and cAMP coexisted in the medium such as peptone or nutrient broth. Among other sugars, only arabinose imposed weaker effect. cAMP could not be replaced by adenine, adenosine, 5′-AMP, 3′-AMP and other 3′,5′-cyclic nucleoside monophosphates. Dose response was observed with reference to either xylose or cAMP. In the presence of both 1% xylose and 10 mm cAMP in peptone broth, 90% of logarithmic phase cells of E. coli W2252 were killed within 6 hr at 37°C. We call this phenomenon as cAMP dependent xylose lethal. This phenomenon was also observed with many substrains of E. coli K–12, E. coli C, Aerobacter aerogenes and Salmonella typhimurium, but not with their xylose negative mutants.     

Allosteric regulation of phosphodiesterase from Portulaca callus by cGMP and papaverin

Three fractions of phosphodiesterase activity capable of hydrolysing cyclic 3′,5′-AMP and cyclic 3′,5′-GMP were purified from Portulaca callus. Hydrolysing bis-(p-nitrophenyl)-phosphate, two fractions showed linear Lineweaver-Burk plots. One fraction showed positive cooperativity. This fraction can be activated competitively by blue dextran, indicating a possible allosteric regulation by nucleotides, demonstrated by changing from being positively cooperative, to following Michaelis-Menten kinetics by cGMP and papaverin. cGMP triggers an enzyme highly active against 3′,5′cAMP and 3′5′cGMP, and papaverin triggers high activity against 2′,3′cAMP, demonstrated by two separate enzyme fractions.     

Detection of a 3′, 5′-cyclic-AMP phosphodiesterase activity in the lichenized fungus Evernia prunastri

Abstract

A 3′, 5′-cyclic-AMP phosphodiesterase (PDE) was detected and measured in the lichen Evernia prunastri. The percentage of hydrolysis of tritiated 3′, 5′-cyclic-adenosine monophosphate ([³H]-cAMP) and 3′, 5′-cyclic-guanosine monophosphate ([³H]-cGMP) by the PDE enzyme into tritiated 5′-adenosine-monophospahte ([³H]-AMP) and tritiated 5′-guanosine-monophospahte ([³H]-GMP) was measured by treating the PDE products with a 5′-nucleotidase enzyme present in snake venom. The lysate fraction (L) (plasma membranes and cell walls) and the supernatant (S) (soluble fraction of the cells) were tested. In both fractions, competition of unlabelled cAMP, but not unlabelled cGMP, was revealed. Specific competitive PDE inhibitors such as IBMX inhibited enzymatic activity. Although it is thought that in this species cAMP is regulated by red/far red light through PDE activity, this is the first report that seems to suggest the presence of a PDE activity specific for cAMP in lichenized fungi. However, this work is at a preliminary stage and despite the high levels of enzymatic activity with cAMP found in both fractions, data are still insufficient to state the absolute specificity for this nucleotide.     

Interaction of pyridoxal 5′-phosphate with the liver glucocorticoid receptor-DNA complex

The rat liver glucocorticoid receptor has been eluted from DNA-cellulose with pyridoxal 5′-phosphate at low ionic strength. This elution is concentration dependent with 80–90% of the receptor eluted in 30 rain at 0 °C when the concentration of pyridoxal 5′-phosphate is 10 mm. This elution is specific for the 4′-aldehyde group of pyridoxal 5′-phosphate since vitamin B₆ analogs lacking this group are inactive in eluting the steroid-receptor complex from DNA-cellulose. Receptor has also been eluted from rat liver nuclei with similar results. The receptor eluted with pyridoxal 5′-phosphate has been compared with the receptor eluted with 0.45 m NaCl. Both methods of elution yield a steroid-receptor complex which sediments at about 3.7 S. The pyridoxal 5′-phosphate-eluted receptor however, is less prone to aggregation at low ionic strength and more stable with respect to steroid binding than the 0.45 m NaCl-eluted steroid-receptor complex. The complement of proteins eluted from DNA-cellulose with pyridoxal 5′-phosphate is very similar to that eluted with NaCl as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Properties of a 5'-AMP specific nucleotidase which accumulates in one cell type during development of Dictyostelium discoideum

5′-AMP nucleotidase activity accumulates during the culmination stage of development in a thin layer of cells at the prestalk-prespore interface of Dictyostelium discoideum. In this report we characterize a highly purified preparation of this enzyme in an attempt to determine the physiological significance of the accumulation and localization of the activity during cellular differentiation. A pH optimum of 9.5 was determined using nine different buffer systems tested over a range of pH from 3 to 13.5. The Michaelis constants for p-nitrophenylphosphate (NPP) and 5′-AMP were 1.8 and 1.2 mm, respectively. Substrate concentrations of 5′-AMP in excess of 2.5 mm were found to inhibit the activity. Little or no effect on the activity of the enzyme was observed in the presence of EDTA, Mg²⁺, Mn²⁺, Ca²⁺, Fe²⁺, or Zn²⁺ ions. However, the enzyme appears to be a zinc metalloprotein as evidenced by its inhibition with 1,10-phenanthroline and recovery of activity in the presence of zinc. Other inhibitors of enzymatic activity include dithiothreitol and imidazole. The enzyme was bound by calcium phosphate, but could not be immobilized on matricies containing other substrate or product analogs, including 5′-AMP, cyclic AMP, ATP, phenylalanine, blue dextran, and Procion Red HE3B. The hydrophobicity of 5′-AMP nucleotidase was demonstrated by its strong affinity for immobilized alkyl and ω-amino alkyl ligands, as well as phenyl Sepharose. Isoelectric focusing of the enzyme in granulated gel required both the presence of detergent to prevent aggregate formation and precipitation of the enzyme, and the addition of zinc after focusing to reverse Ampholine inhibition. Apparently, Ampholine chelates zinc away from the enzyme much like 1,10-phenanthroline. Using this method, the isoelectric point of 5′-AMP nucleotidase was found to be 4.5–4.9, with a 30% recovery of the applied activity.     

Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.     

Effects of thyrotropin,prostaglandin E1 and iodide on cyclic 3′,5′-AMP concentration in dog thyroid slices

The kinetics and concentration effect on the relationship of thyrotropin (TSH) action on cyclic 3′,5′-AMP concentration has been studied in dog thyroid slices in vitro. TSH markedly increased cyclic 3′,5′-AMP level after 5 min, the effect reached a plateau after 10–60 min and slowly declined afterwards. TSH enhanced in parallel the cyclic 3′,5′-AMP level and the binding of iodide to proteins. For this latter effect of TSH, the four criteria of the validity of the Sutherland model for a hormonal action are therefore fulfilled. The effect of TSH on cyclic 3′,5′-AMP concentration in thyroid did not require the presence of a methylxanthine inhibitor of cyclic 3′,5′-AMP phosphodiesterase in the medium. Prostaglandin E1 increased cyclic 3′,5′-AMP levels in control and stimulated slices. The omission of Ca²⁺ in the incubation medium decreased the action of TSH but partial replacement of Na⁺ by K⁺ had little effect. Iodide, 1 μM to 100 μM, inhibited the action of TSH. This inhibitory effect was relieved by NaClO₄, methimazole and propylthiouracil (1 mM). The possible role of this inhibitory effect in an intracellular regulatory mechanism is discussed.     

Microinjection of cyclic nucleotides provides evidence for a diffusional mechanism of intraneuronal control

Cyclic 3′,5′-AMP and cyclic 3′,5′-GMP injected into large neurons of the snail Helix lucorum altered neuron activity. The effect of cAMP is usually depolarizing and that of cGMP hyperpolarizing. The results are specific for 3′,5′-cyclic nucleotides. The experiments support the hypothesis that reaction-diffusion processes involving cyclic nucleotides from the basis of an intraneuronal system of information processing.     

Cyclic nucleotide-dependent protein kinase activity in malignant and cyclic AMP-induced "differentiated" neuroblastoma cells in culture.

The activity of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase was demonstrated in the supernatant (S) fraction (100,000 × g) of mouse and human neuroblastoma (NB) cells. In cAMP-induced “differentiated” mouse NB cells, the cAMP-dependent protein kinase (cAMP-PK) activity did not significantly change. Cyclic GMP did not stimulate the PK activity in S-fraction. The cAMP-PK or cGMP-PK activity was not detected in the membrane (M) fraction. The present results in combination with previous data support the concept that the major portion of binding proteins is distinct from the regulatory subunits of cAMP-PK. For example, the level of binding proteins markedly increases in S-fraction of “differentiated” NB cell, but cAMP-PK activity does not change. cAMP binding proteins are present in the M-fraction, but cAMP-PK activity is not demonstrable. Cyclic GMP binds with the soluble proteins with about 10-fold less binding affinity than cAMP; however, cGMP does not stimulate PK activity.     

Cyclic AMP binding protein from Jerusalem artichoke rhizome tissues

A cyclic AMP binding protein has been purified to electrophoretic homogeneity from Jerusalem artichoke rhizome tissues. Its MW is ca. 240 000 and the apparent constant of cyclic AMP binding to the protein is 2.3 × 10^?7 M. When tested using Millipore filter assay, cyclic AMP binding activity was enhanced by protamine and histone, but not by casein and phosvitin. Of several purine derivatives tested, only 5′-AMP and adenosine inhibited significantly the binding of cyclic AMP by the protein. The protein also binds adenosine and this binding is not affected by cyclic AMP or by other purine derivatives. The apparent binding constant for adenosine is 1.0 × 10^?6 M. The binding protein did not show protein kinase activity. In addition, it did not affect the chromatin-bound DNA dependent RNA polymerase of homologous origin, either in the presence or absence of cyclic AMP. The binding protein is devoid of the following activities: cyclic AMP phosphodiesterase, 5′-nucleotidase, adenosine deaminase and ATPase.     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.     

Schistosoma mansoni: Purification and characterization of malate dehydrogenases

Isoelectric focusing of a homogenate of Schistosoma mansoni, followed by malate dehydrogenase-specific staining, showed the presence of two major and five minor malate dehydrogenase isoenzymes (EC 1.1.1.37), with isoelectric points ranging from 7.3 to 9.5. The malate dehydrogenase isoenzymes were purified by gel filtration, followed by ion-exchange chromatography on DEAE- and CM-cellulose. The isoenzymes could be differentiated by their susceptibility to substrate inhibition. No differences in the Michaelis-Menten constants for substrate were found. One of the isoenzymes is inhibited by 5′-AMP. Further purification of this particular isoenzyme was achieved by affinity chromatography on 5′-AMP-Sepharose 4B. Analysis after subcellular fractionation indicated a mitochondrial origin for this isoenzyme. The mitochondrial isoenzyme (at a recovery of 80%) was purified 218-fold compared to the crude soluble extract, and contained about 40% of the total malate dehydrogenase activity. The enzyme has a molecular weight of 65,500 and showed absolute specificity for l-malic acid, NAD, and NADH. The final preparation has a specific activity of 451 U/mg protein. Physicochemical studies, including binding constants, substrate inhibition, thermostability, and pH optima, demonstrated differences between the mitochondrial and cytoplasmic enzymes. A role for malate dehydrogenase in Schistosoma mansoni metabolism is discussed.     

Evaluation of the binding of serotonin by isolated CNS acidic lipids

Binding of serotonin by rat lipids was examined in an organic solvent-aqueous partition system. Only phospholipids and sulfatide were found to have appreciable activity: this technique was unsuitable for gangliosides due to their poor extractibility. Binding by phospholipid was abolished and that by sulfatide was greatly inhibited by increasing ionic strength in the aqueous phase. At an ionic strength of 0.3 M the apparent affinity of sulfatide for serotonin was about 3×10³ M. Both tryptamine and 5-methoxytryptamine were much more effective than serotonin in inhibiting the binding of radioactive serotonin, suggesting that the observed binding is simply a charge neutralization with little specificity. Binding of serotonin by mixed brain gangliosides was examined in an equilibrium dialysis system. Without adequate precautions, the chemical lability of serotonin was found to produce spurious data when binding was assessed by the distribution of radiolabel. Binding of serotonin by ganglioside was also greatly inhibited by increasing ionic strength: at 0.3 M an apparent affinity of about 10³ M was found. While dopamine did not inhibit the binding of radioactive serotonin, tryptamine, 5-methoxytryptamine, and serotonin were equally effective inhibitors.     

Presence of one essential arginine that specifically binds the 2′-phosphate of NADPH on each of the ketoacyl reductase and enoyl reductase active sites of fatty acid synthetase

Two arginine modifying reagents, phenylglyoxal and 2,3-butanedione, inactivated fatty acid synthetase from goose uropygial gland. This inactivation could be partially prevented by NADP, 2′-AMP, and 2′,5′-ADP, whereas acetyl-CoA and/or malonyl-CoA provided very little protection. Ketoacyl reductase and enoyl reductase activities of fatty acid synthetase showed similar inactivation by phenylglyoxal and butanedione and protection by only NADP and its 2′-phosphate-containing analogs. Furthermore, 2′-AMP was found to be a competitive inhibitor of overall fatty acid synthetase, ketoacyl reductase, and enoyl reductase with apparent K_i values of 1.4, 0.2, and 14 mm, respectively. These results suggest that binding of NADPH to fatty acid synthetase involves specific interaction of the 2′-phosphate with the guanidino group of arginine residues at the active site of the two reductases. Quantitation of the number of arginine residues modified revealed that 4 out of 106 arginine residues per subunit of the synthetase showed high reactivity toward phenylglyoxal. Scatchard analysis showed that two rapidly reacting arginine residues had no effect on the catalytic activity, while modification of two additional arginine residues resulted in complete loss of enzyme activity. Under these conditions, of the seven partial reactions of fatty acid synthetase, only the ketoacyl reductase and enoyl reductase activities were inhibited by phenylglyoxal. The differential reversal of inhibition of the two reductases and the overall activity of fatty acid synthetase, resulting from dialysis of the modified enzyme, suggested that both ketoacyl reductase sites and enoyl reductase sites are required for the full expression of fatty acid synthetase activity. The results of the present chemical modification studies are consistent with the hypothesis that each subunit of fatty acid synthetase contains one ketoacyl reductase and one enoyl reductase and suggest that one essential arginine is present at each of these active sites.     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

S1 nuclease of Aspergillus oryzae: a glycoprotein with an associated nucleotidase activity

S₁ nuclease (EC 3.1.30.1) of Aspergillus oryzae has been purified 1600-fold by a procedure designed to remove traces of contaminating phosphatases. The nearly homogeneous enzyme was found to be a glycoprotein with a carbohydrate content of 18%. At pH 4.5 the enzyme preparation hydrolyzed single-stranded DNA, RNA, 3′-AMP, and 2′-AMP at relative rates of 100, 52, 13, and 0.05, respectively. The 3′-nucleotidase activity of this single-strand specific nuclease is inhibited by single-stranded DNA but not by double-stranded DNA. Three forms of the enzyme, with isoelectric points of 3.35, 3.53, and 3.67, were observed on electrofocusing, and each form exhibited the same relative activity on single-stranded DNA and 3′-AMP. Enzymatic hydrolysis of nucleotides occurred over a broad range of pH, with maximal activity at pH 6–7. Ribonucleotides were hydrolyzed approximately 100-fold more rapidly than deoxyribonucleotides. A high degree of base specificity was not observed. The 3′-nucleotidase activity was stimulated by Zn²⁺, but not by other divalent cations tested.