Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves

We examined the purine alkaloid content and purine metabolism in cacao (Theobroma cacao L.) plant leaves at various ages: young small leaves (stage I), developing intermediate size leaves (stage II), fully developed leaves (stage III) from flush shoots, and aged leaves (stage IV) from 1-year-old shoots. The major purine alkaloid in stage I leaves was theobromine (4.5 μmol g^–1 fresh weight), followed by caffeine (0.75 μmol g^–1 fresh weight). More than 75% of purine alkaloids disappeared with subsequent leaf development (stages II–IV). In stage I leaves, ¹⁴C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO₂) and to purine alkaloids (3- and 7-methylxanthine, 7-methylxanthosine and theobromine). In contrast, ¹⁴C-labelled xanthine and xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8-¹⁴C]Xanthosine was converted in stage I leaves to purine alkaloids. These observations are consistent with the following biosynthetic pathways for theobromine: (a) AMP → IMP → 5′-xanthosine monophosphate → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (b) GMP → guanosine → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (c) xanthine → 3-methylxanthine → theobromine. Although no caffeine biosynthesis from ¹⁴C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8-¹⁴C]Theobromine was converted to caffeine in young leaves. Conversion of theobromine to caffeine may, therefore, be slow in cacao leaves. No purine alkaloid synthesis was observed in the subsequent growth stages (stages II–IV). Significant degradation of purine alkaloids was found in leaves of stages II and III, in which [8-¹⁴C]Theobromine was degraded to CO₂ via 3-methylxanthine, xanthine and allantoic acid. [8-¹⁴C]Caffeine was catabolised to CO₂ via theophylline (1,3-dimethylxanthine) or theobromine.     

Methylation of Xanthosine by Tea-leaf Extracts and Caffeine Biosynthesis

N-Methyltransferase catalyzing the transfer of methyl groups from [¹⁴CH₃]SAM to xanthosine, producing 7- methylxanthosine, was extracted in cell-free systems from tea leaves. The purine nucleotides, nucleosides, and bases, except for xanthosine, were all inactive substrates in the methylation at the N-7 position of their purine rings. This confirmed that the methylation of xanthosine to 7- methylxanthosine is the first step of methylation in the pathway for caffeine biosynthesis. The optimum pH for the methyltransferase is 7.5 to 8.0. PCMB (0.5 mM), Zn^{2 +} (1 mM) and Cu²⁺ (1 mM) strongly inhibited the enzyme activity. The Km values for xanthosine and SAM are 0.25 mM at 3.9µM of SAM and 3.3µM at 0.40 mM of xanthosine, respectively.     

Catabolism of caffeine and related purine alkaloids in leaves ofCoffea arabica L.

In a study of purine alkaloid catabolism pathways in coffee,¹⁴C-labelled theobromine, caffeine, theophylline and xanthine were incubated with leaves ofCoffea arabica. Incorporation of label into¹⁴CO₂ was determined and methanol-soluble metabolites were analysed by high-performance liquid chromatography-radiocounting. The data obtained demonstrate catabolism of caffeine theophylline 3-methylxanthine xanthine. Xanthine is degraded further by the conventional purine catabolism pathway to CO₂ and NH₃ via uric acid, allantoin and allantoic acid. The conversion of caffeine to theophylline is the rate-limiting step in purine alkaloid catabolism and provides a ready explanation for the high concentration of endogenous caffeine found inC. arabica leaves. Although theobromine is converted primarily to caffeine, a small portion of the theobromine pool appears to be degraded to xanthine by a caffeine-independent pathway. In addition to being broken down to CO₂, via the purine catabolism pathway, xanthine is metabolised to 7-methylxanthine. Metabolism of [2-¹⁴C]xanthine byC. arabica leaves in the presence of 5 mM allopurinol results in very large increases in incorporation of radioactivity into 7-methylxanthine as degradation of the substrate via the purine catabolism pathway is blocked. The identity of 7-methylxanthine in these studies was confirmed by gas chromatography-mass spectrometry analysis.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting This work was supported by the British Council which provided H.A. with Japan-UK travel grants. F.M.G. was supported by a Biotechnology and Biological Sciences Research Council grant to A.C.     

Caffeine biosynthesis in Camellia sinensis

The metabolism of adenine and guanine, relating to the biosynthesis of caffeine, in excised shoot tips of tea was studied with micromolar amounts of adenine-[8-¹⁴C] or guanine-[8-¹⁴C]. Among the presumed precursors of caffeine biosynthesis, adenine was the most effective, whereas guanine was the least effective. After administration of a ‘pulse’ of adenine-[8-¹⁴C], almost all of the adenine-[¹⁴C] supplied disappeared by 30 hr, and ¹⁴C-labelled caffeine and RNA purine nucleotide (AMP and GMP) synthesis increased throughout the experimental period, whereas the radioactivities of free purine nucleotides, 7-methylxanthine and theobromine increased during the first 10 hr incubation period, followed by a steady decrease. By contrast, more than 45% of the guanine-[8-¹⁴C] supplied remained unchanged even after a 120 hr period. The main products of guanine-[8-¹⁴C] metabolism in tea shoot tips were guanine nucleotides, theobromine, caffeine and the GMP of RNA. The results support the hypothesis that the purine nucleotides are synthesized from adenine and guanine via the pathway of purine salvage. Adenylate is readily converted into other purine nucleotides, whereas the conversion rate of guanylate into other purine nucleotides is very low.The results also support the view that 7-methylxanthine and theobromine are precursors of caffeine. For the origin of the purine ring in caffeine, purine nucleotides in the nucleotide pool rather than in nucleic acids are suggested.     

Biosynthesis and metabolism of purine alkaloids in leaves of cocoa tea (Camellia ptilophylla)

The biosynthesis and metabolism of purine alkaloids in leaves ofCamellia ptilophylla (cocoa tea), a new tea resource in China, have been investigated. The major purine alkaloid was theobromine, with theophylline also being present as a minor component. Caffeine was not accumulated in detectable quantities. Theobromine was synthesized from [8-¹⁴C] adenine and the rate of its biosynthesis in the segments from young and mature leaves from flush shoots was approximately 10 times higher than that from aged leaves from 1-year old shoots. Neither cellfree extracts nor segments fromC. ptilophylla leaves could convert theobromine to caffeine. A large quantity of [2-¹⁴C] xanthine taken up by the leaf segments was degraded to¹⁴CO₂ via the conventional purine catabolic pathway that includes allantoin as an intermediate. However, small amounts of [2-¹⁴C] xanthine were also converted to theobromine. Considerable amounts of [8-¹⁴C] caffeine exogenously supplied to the leaf segments ofC. ptilophylla was changed to theobromine. These results indicate that leaves ofC. ptilophylla exhibit unusual purine alkaloid metabolism as i) they have the capacity to synthesize theobromine from adenine nucleotides, but they lack adequate methyltransferase activity to convert of theobromine to caffeine in detectable quantities, ii) the leaves have a capacity to convert xanthine to theobromine, probably via 3-methylxanthine.     

Proton magnetic resonance study of the binding of purine to polyuridylic acid

The interaction of unsubstituted purine with polyuridylic acid in D₂O solution at neutral pD has been studied by high resolution proton magnetic resonance spectroscopy. The poly U proton resonances were shifted to higher fields by the added purine, indicating that purine binds to the uracil bases of the polymer by base stacking. Severe broadening of the purine proton resonances was also observed, providing strong evidence for the intercalation of purine between adjacent uracil bases of the polymer. The line widths of the poly U proton resonances were not noticeably broadened in the presence of purine; thus, the binding of purine to poly U does not result in a more rigid or ordered structure for the polymer.     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [¹⁴C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2′-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5′-nucleotidase (EC 3.1.3.5), reflecting the markedly higher V_max/K_m ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher V_max/K_m ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

Improved automated solid-phase microsequencing of peptides using DABITC

The methylated purines O⁶-methyl- and 7-methylguanine were isolated from mouse liver DNA hydrolysates by means of a column cleanup employing a Sep Pak C-18 reverse-phase cartridge. The purine bases were eluted from the cartridge with methanol, evaporated to dryness, and then dissolved in mobile phase for liquid chromatographic analysis by normalphase chromatography. The system consisted of a LiChrosorb Si 60 column with a watersaturated mobile phase of 20% methanol in chloroform containing 0.001% H₃PO₄. The two methylated bases eluted before adenine or guanine. For extremely low-level (<300 pmol) quantitation, the peaks corresponding to O⁶-methyl- and 7-methylguanine were collected and then analyzed by reverse-phase chromotography with a LiChrosorb RP-18 column and a mobile phase of 5% methanol in pH 7 phosphate buffer (for 7-methylguanine) or 9.5% methanol/buffer (for O⁶-methylguanine). Comparisons were made with fluorescence detection and with scintillation counting (in animal studies where [¹⁴C]dimethylnitrosamine was used). Minimum detectable levels at 254 nm were about 3 ng (3:1 signal to noise ratio) for each of the title compounds. As low as 10 pmol/mg of each could be detected in DNA hydrolysates. Recoveries of O⁶-methyl- and 7-methylguanine from DNA spiked at 750 pmol/mg were greater than 80%.     

Metabolic Relations between Methylxanthines and Methyluric Acids in Coffea L

Metabolism of purine alkaloids in the leaves of Coffea dewevrei De Wild et Durand var excelsa Chev, Coffea liberica Bull ex Hiern and Coffea abeokutae Cramer was studied by analyzing leaf discs collected during vegetative development and by feeding the following radioactive tracers: [¹⁴C]theobromine, [¹⁴C]caffeine, and [¹⁴C]theacrine (1,3,7,9-tetramethyluric acid). Their principal metabolites were quantitatively and qualitatively determined. All three species convert the precursors to the same radioactive products, and proceed through the same four maturity stages characterized by the alkaloid accumulation pattern and by a particular transformation potency: (stage 1) young plant accumulating caffeine, transforms theobromine to caffeine; (stage 2) caffeine is gradually replaced by theacrine, theobromine and caffeine are converted to theacrine; (stage 3) theacrine disappears whereas liberine (O(2), 1,9-thrimethyluric acid) accumulates, theacrine is metabolized to liberine; (stage 4) branched-out plant containing liberine but no theacrine, caffeine is converted rapidly to liberine via theacrine. Methylliberine (O(2),1,7,9-tetramethyluric acid), presumably the direct precursor of liberine, is occasionally found in low concentrations at stage 3 and 4.

The collective term `liberio-excelsoid' introduced by geneticists for the numerous races or species of Pachycoffea is in accordance with the phytochemical equality found in this work.

     

Adenylate cyclase from guinea pig lung: further characterization and inhibitory effects of substrate analogs and cyclic nucleotides

A potent (K_i = 0.01 mM), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′O-palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N⁶,2′O-dibutyryl cyclic AMP at concentrations of up to 1 mm or more. The possibility that 2′O-palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 mm Mg²⁺ in the presence of 1.2 mm ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 mm isoproterenol is dependent on the Mg²⁺ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E₁, E₂, and F_2α, histamine, and glucagon.     

Adsorption of Adenine, Cytosine, Thymine, and Uracil on Sulfide-Modified Montmorillonite: FT-IR, M?ssbauer and EPR Spectroscopy and X-Ray Diffractometry Studies

In the present work the interactions of nucleic acid bases with and adsorption on clays were studied at two pHs (2.00, 7.00) using different techniques. As shown by Mössbauer and EPR spectroscopies and X-ray diffractometry, the most important finding of this work is that nucleic acid bases penetrate into the interlayer of the clays and oxidize Fe²⁺ to Fe³⁺, thus, this interaction cannot be regarded as a simple physical adsorption. For the two pHs the order of the adsorption of nucleic acid bases on the clays was: adenine????cytosine?>?thymine?>?uracil. The adsorption of adenine and cytosine on clays increased with decreasing of the pH. For unaltered montmorillonite this result could be explained by electrostatic forces between adenine/cytosine positively charged and clay negatively charged. However for montmorillonite modified with Na₂S, probably van der Waals forces also play an important role since both adenine/cytosine and clay were positively charged. FT-IR spectra showed that the interaction between nucleic acid bases and clays was through NH⁺ or NH ₂ ⁺ groups. X-ray diffractograms showed that nucleic acid bases adsorbed on clays were distributed into the interlayer surface, edge sites and external surface functional groups (aluminol, silanol) EPR spectra showed that the intensity of the line g????2 increased probably because the oxidation of Fe²⁺ to Fe³⁺ by nucleic acid bases and intensity of the line g?=?4.1 increased due to the interaction of Fe³⁺ with nucleic acid bases. Mössbauer spectra showed a large decreased on the Fe²⁺ doublet area of the clays due to the reaction of nucleic acid bases with Fe²⁺.     

Comparison of the π-stacking properties of purine versus pyrimidine residues. Some generalizations regarding selectivity

Aromatic-ring stacking is pronounced among the noncovalent interactions occurring in biosystems and therefore some pertinent features regarding nucleobase residues are summarized. Self-stacking decreases in the series adenine > guanine > hypoxanthine > cytosine ~ uracil. This contrasts with the stability of binary (phen)(N) adducts formed by 1,10-phenanthroline (phen) and a nucleobase residue (N), which is largely independent of the type of purine residue involved, including (N1)H-deprotonated guanine. Furthermore, the association constant for (phen)(A)^0/4? is rather independent of the type and charge of the adenine derivative (A) considered, be it adenosine or one of its nucleotides, including adenosine 5′-triphosphate (ATP^4?). The same holds for the corresponding adducts of 2,2′-bipyridine (bpy), although owing to the smaller size of the aromatic-ring system of bpy, the (bpy)(A)^0/4? adducts are less stable; the same applies correspondingly to the adducts formed with pyrimidines. In accord herewith, [M(bpy)](adenosine)²⁺ adducts (M²⁺ is Co²⁺, Ni²⁺, or Cu²⁺) show the same stability as the (bpy)(A)^0/4? ones. The formation of an ionic bridge between –NH₃ ⁺ and –PO₃ ^2?, as provided by tryptophan [H(Trp)^±] and adenosine 5′-monophosphate (AMP^2?), facilitates recognition and stabilizes the indole–purine stack in [H(Trp)](AMP)^2?. Such indole–purine stacks also occur in nature. Similarly, the formation of a metal ion bridge as occurs, e.g., between Cu²⁺ coordinated to phen and the phosphonate group of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA^2?) dramatically favors the intramolecular stack in Cu(phen)(PMEA). The consequences of such interactions for biosystems are discussed, especially emphasizing that the energies involved in such isomeric equilibria are small, allowing Nature to shift such equilibria easily.     

Origin of the intrinsic rigidity of DNA

The intrinsic rigidities of DNA and RNA helices are generally thought to arise from some combination of vertical base-stacking interactions and intra-helix phosphate–phosphate charge repulsion; however, the relative contributions of these two types of interaction to helix rigidity have not been quantified. To address this issue, we have measured the rotational decay times of a ‘gapped-duplex’ DNA molecule possessing a central, single-stranded region, dT₂₄, before and after addition of the free purine base, N⁶-methyladenine (^meA). Upon addition of ^meA, the bases pair with the T residues, forming a continuous stack within the gap region. Formation of the gapped duplex is accompanied by a nearly 2-fold increase in decay time, to values that are indistinguishable from the full duplex control for monovalent salt concentrations up to 90 mM. These results indicate that at least 90% of the rigidity of the dT_n–dA_n homopolymer derives from base pair stacking effects, with phosphate–phosphate interactions contributing relatively little to net helix rigidity at moderate salt concentrations.     

Characterization of ammonium and nitrate uptake and assimilation in roots of tea plants

It has been pointed out that tea (Camellia sinensis (L.) O. Kuntze) prefers ammonium (NH ₄ ⁺ ) over nitrate (NO ₃ ^? ) as an inorganic nitrogen (N) source. ¹⁵N studies were conducted using hydroponically grown tea plants to clarify the characteristics of uptake and assimilation of NH ₄ ⁺ and NO ₃ ^? by tea roots. The total ¹⁵N was detected, and kinetic parameters were calculated after feeding ¹⁵NH ₄ ⁺ or ¹⁵NO ₃ ^? to tea plants. The process of N assimilation was studied by monitoring the dynamic ¹⁵N abundance in the free amino acids of tea plant roots by GC-MS. Tea plants supplied with ¹⁵NH ₄ ⁺ absorbed significantly more ¹⁵N than those supplied with ¹⁵NO ₃ ^? . The kinetics of ¹⁵NH ₄ ⁺ and ¹⁵NO ₃ ^? influx into tea plants followed a classic biphasic pattern, demonstrating the action of a high affinity transport system (HATS) and a low affinity transport system (LATS). The V _max value for NH ₄ ⁺ uptake was 54.5 nmol/(g dry wt min), which was higher than that observed for NO ₃ ^? (39.3 nmol/(g dry wt min)). K_M estimates were approximately 0.06 mM for NH ₄ ⁺ and 0.16 mM for NO ₃ ^? , indicating a higher rate of NH ₄ ⁺ absorption by tea plant roots. Tea plants fed with ¹⁵NH ₄ ⁺ accumulated larger amounts of assimilated N, especially glutamine (Gln), compared with those fed with ¹⁵NO ₃ ^? . Gln, Glu, theanine (Thea), Ser, and Asp were the main free amino acids that were labeled with ¹⁵N under both conditions. The rate of N assimilation into Thea in the roots of NO ₃ ^? -supplied tea plants was quicker than in NH ₄ ⁺ -supplied tea plants. NO ₃ ^? uptake by roots, rather than reduction or transport within the plant, seems to be the main factor limiting the growth of tea plants supplied with NO ₃ ^? as the sole N source. The NH ₄ ⁺ absorbed by tea plants directly, as well as that produced by NO ₃ ^? reduction, was assimilated through the glutamine synthetase-glutamine oxoglutarate aminotransferase pathway in tea plant roots. The ¹⁵N labeling experiments showed that there was no direct relationship between the Thea synthesis and the preference of tea plants for NH ₄ ⁺ .     

Reaction of nucleic acid bases with the mercury electrode: Determination of purine derivatives at submicromolar concentrations by means of cathodic stripping voltammetry

It was found that adenine, guanine, hypoxanthine, 8-hydroxyadenine, and a number of further purine derivatives react in alkaline media with mercury of the electrode charged to potentials close to zero V (against the saturated calomel electrode) and form sparingly soluble compounds. Formation of these compounds with mercury is manifested on the polarographic (voltammetric) curves by characteristic anodic waves (peaks) which can be exploited for analytical purposes. Differential pulse polarography renders it possible to determine bases at concentrations of 10^?5–10^?6m. Substantially higher sensitivity can be reached by cathodic stripping voltammetry (CSV). This method is based on a slow accumulation of the sparingly soluble compound at the electrode surface and its subsequent rapid cathodic stripping. A number of purine derivatives can be determined by CSV at concentrations as low as 10^?8m (the limit of adenine detection is about 2 × 10^?9m). As compared with sulphur-containing substances CSV analysis of the purine derivatives is limited to a narrower range of deposition potentials. It was shown that the presence of an excess of proteins or DNA does not interfere with determination of purine bases.     

Studies on Metabolic Pathway of NAD in Yeast

Quantitative studies on yeast 5′-nucIeotidase are presented.

K_m values for purine 5′-nucleotides were generally smaller than those for pyrimidine 5′-nucleotides and, among purine series, K_m value for 5′-AMP was the smallest, while their V values were almost same.

The enzyme activity was inhibited in the competitive type by bases, nucleosides, 3′- or 2′-nucleotides, and NMN and in the mixed type by NAD and NADP.

Base-, ribose-, 3′- or 5′-phosphate moiety of nucleoside and nucleotide had some effects on binding with enzyme; especially the structure of base moiety characterizes the K_m or K_i value.

The enzyme activity was accelerated by Ni⁺⁺ or Co⁺⁺, which increases V value but never affects K_m value.

The relationship between the structure of substrate and its affinity towards enzyme is discussed.     

Microbial Synthesis of Purine Arabinosides and Their Biological Activity

A simple method for the synthesis of various purine arabinosides from purine bases and uracil arabinoside by microbial transarabinosylation is described. A wet cell paste of Enterobacter aerogenes AJ 11125 showed a wide substrate specificity range for purine bases. Not only naturally occurring purine bases such as adenine and hypoxanthine but also unnatural bases such as 6-thioguanine and 2-chlorohypoxanthine were catalyzed to give the corresponding purine arabinosides. The enzymatically synthesized purine arabinosides were isolated from the reaction mixtures and identified by physicochemical means. The biological activities of the compounds were investigated and it was found that thioguanine arabinoside and 2-methyladenine arabinoside have potent activity against Hela cells, and their ED₅₀ were 10.5 and 21.5 μg/ml, respectively.     

Thermodynamics and kinetics of the interaction of copper (II) ions with native DNA.

Based on equilibrium binding studies, as well as on kinetic investigations, two types of interactions of Cu²⁺ ions with native DNA at low ionic strength could be characterized, namely, a nondenaturing and a denaturing complex formation. During a fast nondenaturing complex formation at low relative ligand concentrations and at low temperatures, different binding sites at the DNA bases become occupied by the metal ions. This type of interaction includes chelate formation of Cu²⁺ ions with atoms N₍₇₎ of purine bases and the oxygens of the corresponding phosphate groups, chelation between atoms N₍₇₎ and O of C₍₆₎ of the guanine bases, as well as the formation of specific intestrand crosslink complexes at adjacent G°C pairs of the sequence dGpC. CD spectra of the resulting nondenatured complex (DNA–Cu²⁺)_nat may be interpreted in terms of a conformational change of DNA from the B-form to a C-like form on ligand binding. A slow cooperative denaturing complex formation occurs at increased copper concentrations and/or at increased temperatures. The uv absorption and CD spectra of the resulting complex, (DNA–Cu²⁺)_denat, indicate DNA denaturation during this type of interaction. Such a conclusion is confirmed by microcalorimetric measurements, which show that the reaction consumes nearly the same amount of heat as acid denaturation of DNA. From these and the kinetic results, the following mechanism for the denaturing action of the ligands is suggested: binding of Cu²⁺ ions to atoms N₍₃₎ of the cytosine bases takes place when the cytosines come to the outside of the double helix as a result of statistical fluctuations. After the completion of the binding process, the bases cannot return to their initial positions, and thus local denaturation at the G·C pairs is brought about. The probability of the necessary fluctuations occurring is increased by chelation of Cu²⁺ ions between atoms N₍₇₎ and O of C₍₆₎ of the guanine bases during nondenaturing complex formation, which loosens one of the hydrogen bonds within the G·C pairs, as well as by raising the temperature. The implications of the new binding model, which comprises both the sequence-specific interstand crosslinks and the described mechanism of denaturing complex formation, are discussed and some predictions are made. The model is also used to explain the different renaturation properties of the denatured complexes of Cu²⁺, Cd²⁺, and Zn²⁺ ions with DNA. In temperature-jump experiments with the nondenatured complex (DNA–Cu²⁺)_nat, a specific kinetic effect is observed, namely, the appearance of a lag in the response to the perturbation. The resulting sigmoidal shape of the kinetic curves is considered to be a consequence of the necessity of disrupting a certain number of the crosslinks existing in the nondenatured complex before the local unwinding of the binding regions (a main step of denaturing complex formation) may proceed.     

Effect of caffeine on mucus secretion and agonist-dependent Ca2+ mobilization in human gastric mucus secreting cells

Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca²⁺]_i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca²⁺]_i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [³H]glucosamine. Caffeine by itself failed to increase [Ca²⁺]_i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca²+]_i rise, resulting in inhibiting activation of Ca²⁺-dependent K⁺ current (I_K.Ca) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca²⁺]_i rise and activation of I_K.Ca induced by A23187 or inositol trisphosphate (IP₃). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca²⁺]_i rise in human gastric epithelial cells, probably through the blockade of receptor-IP₃ signaling pathway, which may affect the mucin secretion. © 1997 Elsevier Science B.V. All rights reserved.