Firefly luciferase reacts with p1,p5-di(adenosine-5′-)pentaphosphate and adenosine-5′-tetraphosphate

Purified luciferase from firefly tails produces light not only with ATP but also with adenosine-5′-tetraphosphate and P¹,P⁵-di(adenosine-5′-)pentaphosphate. The latter compound is a potent and specific inhibitor of adenylate kinase. P¹,P⁵-Di(adenosine-5′-)pentaphosphate produces light with an intensity of about 0.75 per cent relative to ATP and adenosine-5′-tetraphosphate produces light with an intensity of about 2.2 per cent relative to ATP, even if efforts were made to remove contaminating ATP.     

Characterization by deletion mutagenesis in vitro of the promoter region of ompF, a positively regulated gene of Escherichia coli

The ompF gene codes for a major outer membrane protein whose expression is positively regulated by the ompR and envZ genes. Two sets of promoter deletions, upstream deletions and downstream deletions, were generated in vitro, and the promoter function was studied by connecting them with the tet genes. One of the hybrid genes thus constructed had a functioning ompF-tet hybrid promoter. The 107 base-pair fragment was found to be functioning as the ompF promoter, 90 nucleotides upstream and 17 nucleotides downstream of the mRNA start site that was also determined in this study. The start site was preceded by a convenient Pribnow box. Although the sequence at the -35 region had a low degree of homology to the consensus sequence, analyses of the hybrid promoter suggested that this region is involved in the promoter function in relation to the Pribnow box. They also indicated that the domain responsible for regulation by the ompR gene is located within the -35 region and its upstream region.     

Variations du taux d'amp cyclique et des activités spécifiques de l'adénylate cyclase et de l'amp cyclique-phosphodiestérase pendant le cycle cellulaire d'un actinomycète

The variations in the concentrations of intra- and extracellular cyclic AMP and in the specific activities of adenylate cyclase (EC 4.6.1.1) and cyclic AMP phosphodiesterase (EC 3.1.4.17) have been monitored in synchronized culture of Nocardia restricta, a prokaryote belonging to the group of Actinomycetes. At the beginning of the cell cycel, during a first period of RNA and protein synthesis, there is an increasing synthesis of adenylate cyclase which can be suppressed in the presence of chloramphenicol or rifampicin. Simultaneously, the specific activity of cyclic AMP phosphodiesterase decreases and the concentrations of intra- and extracellular cyclic AMP rise. After the end of DNA replication, during a second period of RNA and protein synthesis, the specific activity of cyclic AMP phosphodiesterase increases; during the same time, the specific activity of adenylate cyclase and the level of intracellular cyclic AMP drop. It appears that the overall metabolism of cyclic AMP is coordinated so that the cyclic AMP level will be high at the beginning of DNA replication and will fall thereafter. The results are discussed in comparison with known data about the variations of cyclic AMP during the cell cycle of mammalian cells in cultures.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

Modulation by gibberellic acid and adenosine-3′,5′-cyclic monophosphate of starch hydrolysing activity of cowpea seedlings

In cowpea seedlings starch hydrolysing activity increases 35–50 fold on germination for 4 days. This increase in enzyme activity was inhibited by the in vivo addition of 1% glucose but this inhibition was completely overcome by the addition of gibberellic acid (GA₃) (10^?5 M) and adenosine-3′,5′-cyclic monophosphate (cAMP) (10^?5 M). At 5% glucose, GA₃ and cAMP were only partially effective. Structural analogues of cAMP failed to relieve the inhibitory effect of glucose. The inhibition by glucose is not direct but RNA and protein synthesis may be involved. Glucose appears to reduce the internal pool of cAMP which causes inhibition of RNA synthesis and decrease in starch hydrolysing activity. Exogenous application of cAMP may replenish the endogenous pool of cyclic nucleotide and thus overcome inhibition of RNA synthesis and enzyme activity.     

Mass spectrometric identification of adenosine 3′:5′-cyclic monophosphate isolated from a higher plant tissue

Mass spectrometric evidence is presented confirming the identification of the adenosine nucleotide previously isolated from tissues of Phaseolus vulgaris as adenosine 3′: 5′-cyclic monophosphate.     

Guanosine 3′:5′-cyclic monophosphate changes during germination of Hordeum vulgare

Guanosine 3′: 5′-cyclic monophosphate (cGMP) isolated from barley seeds and seedlings was purified using neutral alumina and anion-exchange column chromatography, then descending paper chromatography, and finally estimated by means of radioimmunoassay. The putative compound was identified on cellulose chromatography in three solvent systems.During the early phase of the germination, the cGMP content decreased steadily from 30 fmol/g of dry seeds to undetectable amounts in seeds after 18 h of germination. The process of seedling growth was associated with the increase of cGMP concentration. Nine-day-old seedlings contained 147 and 200 fmol/g of fresh weight in the roots and in the coleoptiles plus leaves, respectively.     

Enzymatic synthesis of 2′-ara and 2′-deoxy analogues of c-di-GMP

The substrate specificity of recombinant full-length diguanylate cyclase (DGC) of Thermotoga maritima with mutant allosteric site was investigated. It has been originally shown that the enzyme could use GTP closest analogues – 2′-deoxyguanosine-5′-triphosphate (dGTP) and 9-β-D-arabinofuranosyl-guanine-5′-triphosphate (araGTP) as the substrates. The first demonstrations of an enzymatic synthesis of bis-(3′-5′)-cyclic dimeric deoxyguanosine monophosphate (c-di-dGMP) and the previously unknown bis-(3′-5′)-cyclic dimeric araguanosine monophosphate (c-di-araGMP) using DGC of T. maritima in the form of inclusion bodies have been provided.     

Adrenocorticotropin regulates angiotensin II receptors in bovine adrenal cells in vitro

The role of ACTH-(1–24) on angiotensin II receptors has been studied in bovine adrenal glomerulosa cells in primary culture. Angiotensin II receptors were measured in cells pretreated or not by ACTH-(1–24) on day 4 of culture. ACTH-(1–24) decreased angiotensin II binding sites in a time and a dose-dependent manner. After 24 hours of treatment the minimal effective dose of ACTH-(1–24) was 10^?11M and the maximal effect was obtained with 10^?8M. Moreover, ACTH-(1–24) 10^?8M decreased significantly angiotensin II receptors after 6 hours of treatment. Scatchard plot analysis showed that ACTH-(1–24) treatment did not modify the affinity of angiotensin II receptors (Ka = 0.42 and 0.44 × 10⁹M^?1 in control and treated cells respectively) but reduced by about half the number of angiotensin II sites per cell. Like ACTH-(1–24), 8-Bromo-cAMP, forskolin and cholera toxin decreased angiotensin II receptors. Factors such as prolactin, somatostatin, ACTH-(11–24) and dopamine which are bound to adrenal membranes without increasing cAMP production had no effect. In conclusion, these studies $\text{in vitro}$ demonstrate for the first time that ACTH decreases angiotensin II receptors by a direct mechanism acting on glomerulosa cells, and they also suggest that this effect could be mediated by cAMP.     

Synthesis and release of adenosine 3′: 5′-cyclic monophosphate by Chlamydomonas reinhardtii

One of the labeled compounds synthesized by Chlamydomonas reinhardtii when ³²Pi was supplied was isolated from both the cells and the medium in which the cells had grown. This compound copurified with authentic [8-³H]cAMP by TLC to a constant ratio of ³²P/³H. The compound was degraded by beef heart cyclic nucleotide phosphodiesterase to a product which cochromatographed with authentic 5′AMP, at the same rate as the hydrolysis of authentic cAMP-[³H] to 5′AMP-[³H]. In both cases, 1-Me-3-isoBu-xanthine, a specific inhibitor of the phosphodiesterase, totally blocked the reaction. It is concluded that the compound synthesized by C. reinhardtii was cAMP, 85% of which was released into the medium.     

Oxidation Chemistry of Adenosine-3′, 5′-Cyclic Monophosphate at Pyrolytic Graphite Eletrode

The voltammetric oxidation of adenosine-3′,5′-cyclic monophosphate (3′,5′-CAMP) has been studied in the pH range 2.13–10.07 using pyrolytic graphite electrode (PGE). Voltammetric, coulometric, spectral studies, and product characterization indicate that the oxidation of 3′,5′-CAMP occurs in an EC reaction involving a 6H⁺, 6e process at pH 7.24. Electrooxidized products were seperated by semipreparative high performance liquid chromatography (HPLC) and were characterized by mp, ¹HNMR, FTIR, and GC-mass as allantoin cyclic ribose monophosphate and 3 dimers as the major products. A detailed interpretation of the redox mechanism of 3′,5′-CAMP also has been presented to account for the formation of various products.     

LDL and cAMP cooperate to regulate the functional expression of the LRP in rat ovarian granulosa cells

Rat ovarian granulosa rely heavily on lipoprotein-derived cholesterol for steroidogenesis, which is principally supplied by the LDL receptor- and scavenger receptor class B type I (SR-BI)-mediated pathways. In this study, we characterized the hormonal and cholesterol regulation of another member of the LDL receptor superfamily, low density lipoprotein receptor-related protein (LRP), and its role in granulosa cell steroidogenesis. Coincubation of cultured granulosa cells with LDL and N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (Bt2cAMP) greatly increased the mRNA/protein levels of LRP. Bt2cAMP and Bt2cAMP plus human hLDL also enhanced SR-BI mRNA levels. However, there was no change in the expression of receptor-associated protein, a chaperone for LRP, or another lipoprotein receptor, LRP8/apoER2, in response to Bt2cAMP plus hLDL, whereas the mRNA expression of LDL receptor was reduced significantly. The induced LRP was fully functional, mediating increased uptake of its ligand, alpha2-macroglobulin. The level of binding of another LRP ligand, chylomicron remnants, did not increase, although the extent of remnant degradation that could be attributed to the LRP doubled in cells with increased levels of LRP. The addition of lipoprotein-type LRP ligands such as chylomicron remnants and VLDL to the incubation medium significantly increased the progestin production under both basal and stimulated conditions. In summary, our studies demonstrate a role for LRP in lipoprotein-supported ovarian granulosa cell steroidogenesis.     

The effects of cyclic AMP on disaggregation-induced changes in activities of developmentally regulated enzymes in Dictyostelium discoideum.

The addition of cyclic AMP to the shaking medium of cells disaggregated from pseudoplasmodia of $\text{Dictyostelium discoideum}$ suppressed the accumulation of cell-bound phosphodiesterase which normally occurs (1) after disaggregation. The suppression was not secondarily brought about by its possible inhibitory effect of cyclic AMP on protein synthesis or by its stimulating effect on the release of the enzyme into the medium. The effect was reversible and specific to cyclic AMP. On the other hand, the inhibitory effect of cyclic AMP on the disaggregation-induced inactivation of UDP-galactose transferase was not apparent in the initial period, but thereafter it slowed down the decrease in the enzyme activity. These results indicate that exogenous cyclic AMP mimics at least in part the regulatory effects of cell-to-cell contact on certain enzymes.     

Occurrence of adenosine 3′:5′-cyclic monophosphate in plant tissues

A procedure is described which unequivocally demonstrates the presence of adenosine 3′:5′-cyclic monophosphate in Phaseolus vulgaris. Its concentration was determined spectrophotometrically at 2·6–9·2 nmol g^?1 of tissue (dry wt) for 6-day-old seedlings and about one-tenth of this in 13-day-old plants.