Montmorillonite: A multifunctional mineral catalyst for the prebiological formation of phosphate esters

Reaction of diiminosuccinonitrile (DISN) with 3'-AMP in the presence of alkali- and alkaline earth-montmorillonites results in the formation of 2',3'-cAMP in aqueous solution. Little or no 2', 3'-cAMP is produced when metal ion concentrations equivalent to that of the metal ion associated with the homoionic clays are used instead of mobntmorillionite. Yields comparable to those obtained with DISN are obtained when diaminomaleonitrile (DAMN) is used in place of DISN as the condensing agent. DAMN, a compound which is more stable than DISN in aqueous solution, is oxidized to DISN on the surface of the clay by Fe+3 in the clay lattice. DISN, the true condensing agent, is thus generated in the presence of the bound 3'-AMP on the montmorillonite surface. The montmorillonite catalyzes the DISN-mediated formation of 2', 3'-cAMP and this product, which binds much less strongly than does the 3'-AMP, is desorbed from the clay surface. This research established that the montmorillonite performs four different functions in its role as catalyst: (1) Binding one of the substrate molecules (3'-AMP) (2) Activating the second substrate (DAMN) (3) Catalyzing the formation of 2', 3'-cAMP (4) Releasing the reaction product so another substrate molecules can bind to the montmorillonite.     

Effect of Inhibitors on the Montmorillonite Clay-Catalyzed Formation of RNA: Studies on the Reaction Pathway

The Langmuir adsorption isotherms of the phosphoroimidazolides of adenosine (ImpA) and uridine (ImpU), dA^5'ppdA and N⁶, N⁶-dimethyladenine binding on montmorillonite are consistent with their forming a monolayer on the clay surface. This suggests the condensation of ImpA and ImpU to oligomers proceeds on the surface of the clay and not in groups of monomers stacked on the clay surfaces. The binding and reactions of ImpU and ImpA on montmorillonite are blocked by N⁶, N⁶-dimethyladenine and dA^5'ppdA. dA^5'ppdA is a better inhibitor of oligomer formation than N⁶, N⁶-dimethyladenine because both adenine rings of dA^5'ppdA bind to the clay surface and block adjacent catalytic sites. An upper limit of 5–10 × 10¹⁵catalytic sites on 50 mg of clay was estimated from the binding of ImpU and the inhibition of oligomer formation by dA^5'ppdA.     

Molecular recognition of cAMP by an RNA aptamer

Two classes of RNA aptamers that bind the second messenger adenosine 3',5'-cyclic monophosphate (cAMP; 1) were isolated from a random-sequence pool using in vitro selection. Class I and class II aptamers are formed by 33- and 31-nucleotide RNAs, respectively, and each is comprised of similar stem-loop and single-stranded structural elements. Class II aptamers, which dominate the final selected RNA population, require divalent cations for complex formation and display a dissociation constant (K(D)) for cAMP of approximately 10 microM. A representative class II aptamer exhibits substantial discrimination against 5'- and 3'-phosphorylated nucleosides such as ATP, 5'-AMP, and 3'-AMP. However, components of cAMP such as adenine and adenosine also are bound, indicating that the adenine moiety is the primary positive determinant of ligand binding. Specificity of cAMP binding appears to be established by hydrogen bonding interactions with the adenine base as well as by steric interactions with groups on the ribose moiety. In addition, the aptamer recognizes 8,5'-O-cycloadenosine (2) but not N(3), 5'-cycloadenosine (3), indicating that this RNA might selectively recognize the anti conformation of the N-glycosidic bond of cAMP.     

Clay Catalysis of Oligonucleotide Formation: Kinetics of the Reaction of the 5'-Phosphorimidazolides of Nucleotides with the Non-Basic Heterocycles Uracil and Hypoxanthine

The montmorillonite clay catalyzed condensation of activated monocleotides to oligomers of RNA is a possible first step in the formation of the proposed RNA world. The rate constants for the condensation of the phosphorimidazolide of adenosine were measured previously and these studies have been extended to the phosphorimidazolides of inosine and uridine in the present work to determine if substitution of neutral heterocycles for the basic adenine ring changes the reaction rate or regioselectivity. The oligomerization reactions of the 5'-phosphoromidazolides of uridine (ImpU) and inosine (ImpI) on montmorillonite yield oligo(U)s and oligo(I)s as long as heptamers. The rate constants for oligonucleotide formation were determined by measuring the rates of formation of the oligomers by HPLC. Both the apparent rate constants in the reaction mixture and the rate constants on the clay surface were calculated using the partition coefficients of the oligomers between the aqueous and clay phases. The rate constants for trimer formation are much greater than those dimer synthesis but there was little difference in the rate constants for the formation of trimers and higher oligomers. The overall rates of oligomerization of the phosphorimidazolides of purine and pyrimidine nucleosides in the presence of montmorillonite clay are the same suggesting that RNA formed on the primitive Earth could have contained a variety of heterocyclic bases. The rate constants for oligomerization of pyrimidine nucleotides on the clay surface are significantly higher than those of purine nucleotides since the pyrimidine nucleotides bind less strongly to the clay than do the purine nucleotides. The differences in the binding is probably due to Van der Waals interactions between the purine bases and the clay surface. Differences in the basicity of the heterocyclic ring in the nucleotide have little effect on the oligomerization process.     

Molecular basis for P-site inhibition of adenylyl cyclase

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.     

Specific recognition of the 3'-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes

Ribosomes from Escherichia coli possess, in addition to A and P sites, a third tRNA binding site, which according to its presumed function in tRNA release during translocation has been termed the exit site. The exit site exhibits a remarkable specificity for deacylated tRNA; charged tRNA, e.g. N-AcPhe-tRNAPhe, is not bound significantly. To determine the molecular basis of this discrimination, we have measured the exit site binding affinities of a number of derivatives of tRNAPhe from E. coli, modified at the 3' end. Binding to the exit site of the tRNAPhe derivatives was measured fluorimetrically by competition with a fluorescent tRNAPhe derivative. We show here that removal of the 2' and 3' hydroxyl groups of the 3'-terminal adenosine decreases the affinity of tRNAPhe for the exit site 15 and 40-fold, respectively. Substitutions at the 3' hydroxyl group (aminoacylation, phosphorylation, cytidylation) as well as removal of the 3'-terminal adenosine (or adenylate) of tRNAPhe lower the affinity below the detection limit of 2 x 10(5) M-1, i.e. more than 100-fold. Modification of the adenine moiety (1,N6-etheno adenine) or replacement of it with other bases (cytosine, guanine) has the same dramatic effect. In contrast, the binding to both P and A sites is virtually unaffected by all of the modifications tested. These results suggest that a major fraction (at least -12 kJ/mol, probably about -17 kJ/mol) of the free energy of exit site binding of tRNAPhe (-42 kJ/mol at 20 mM-Mg2+) is contributed by the binding of the 3'-terminal adenine to the ribosome. The binding most likely entails the formation of hydrogen bonds.     

Tyrosine 65 is photolabeled by 8-azidoadenine and 8-azidoadenosine at the NAD binding site of diphtheria toxin

8-Azidoadenine and 8-azidoadenosine, two photoactivatable derivatives of adenine and adenosine, are competitive inhibitors of diphtheria toxin of similar potency with respect to their parent compounds. On irradiation, the two tritium-labeled photoactivatable azidoadenines bind covalently and specifically to an enzymic fragment of diphtheria toxin that is known to bind to NAD. This photolabeling is protected by the enzyme substrate NAD. The radiolabeled protein was fragmented, and the radioactive fragments were sequenced. Tyr-65 is labeled specifically by both photoreagents, and its labeling was reduced strongly when NAD was present during irradiation. Labeling is also reduced strongly by adenine, adenosine, and nicotinamide. These results suggest that Tyr-65 is at the NAD binding site of diphtheria toxin and that the competitive inhibitors adenine, adenosine, and nicotinamide bind to the same portion of the catalytic center of the toxin.     

Isolation of two novel adenosine binding proteins from bovine brain

Adenosine plays many significant roles both as a metabolic precursor and cell communicator. This report describes the preliminary characterization of two adenosine binding proteins isolated from bovine brain membranes. By using N6-9-aminononane adenosine labeled Sepharose 4B two major affinity bound proteins were purified having apparent molecular weights of 16 and 35 kDa. Either or both of the proteins could be selectively eluted from the affinity column with N6-9-aminononane adenosine, adenosine, cAMP, AMP, ADP, ATP, R-/S-phenylisopropyladenosine and NAD(H). By contrast, no proteins were eluted with caffeine, adenine, deoxyadenosine, 2',3'-AMP, inosine, IMP, xanthine, XMP, GMP, GTP or 5'-N-ethylcarboxamideadenosine. The selectivity of elution and lack of apparent enzymatic activity suggests that these proteins are novel membrane bound adenosine binding proteins.     

Interaction of [3H]AMP with liver cells and their plasma membranes

Specific binding of [3H]AMP to rat hepatocytes and their plasma membranes was studied. It was shown that the time course of this binding reached a maximum within the first 15 seconds. An equilibrium binding study revealed the presence of a single class of binding sites with Kd of 20 microM both in hepatocytes and in plasma membranes. The [3H]AMP binding sites were inactivated by treatment with trypsin as well as by heating. 5'-Phosphorylated derivatives of adenosine (ATP, ADP) effectively competed with [3H]AMP for the binding sites, while adenosine, beta-glycerophosphate and 3'-AMP were inactive. The binding of [3H]AMP increased by 400% in the presence of concanavalin A, a specific inhibitor of plasma membrane 5'-nucleotidase. It was concluded that the catalytic center of 5'-nucleotidase is a receptor for adenine nucleotides.     

The biodegradability of nucleic acid bases adsorbed on inorganic and organic soil components

Summary The biodegradability of nucleic acid bases (guanine, adenine, cytosine, thymine and uracil) adsorbed on montmorillonite, illite, kaolinite, soil, gibbsite, goethite and a fulvic acid (FA)-montmorillonite complex was investigated. Each material was mixed with sand, inoculated with a soil suspension and incubated in a Warburg vessel. Lag periods in O₂ uptake were observed at pH 4 and 6 but not at pH 8. Following the lag periods, adsorbed nucleic acid bases were degraded rapidly at linear rates until these levelled off. The cessation of O₂-uptake was shown to be due to the formation of excessive amounts of gaseous NH₃, which not only inhibited microbial respiration by raising the pH to 8 and higher, but also by killing bacteria and actinomycetes. The rate of biodegradation was found to depend on the type of clay or oxide, the dominant cation and the pH. Contribution No 1181     

The binding site for the adenosyl group of coenzyme B12 in diol dehydrase

The binding of cob(II)alamin (CblII) and 5'-deoxyadenosine to diol dehydrase was studied spectroscopically and with [U-14C]5'-deoxyadenosine. CblII was bound to this enzyme forming a tight 1:1 complex which was resistant to oxidation by O2 even in the presence of CN-. An irreversible 1:1:1 ternary complex was formed between enzyme, CblII, and 5'-deoxyadenosine, when the enzyme was incubated first with the nucleoside and then with CblII. When this order of addition of the constituents was reversed, no 5'-deoxyadenosine was bound to the enzyme-CblII complex. Hydroxocobalamin could also bind to the enzyme together with the nucleoside, while other cob(III)alamins bearing a bulkier Co beta ligand displaced the nucleoside upon binding to the enzyme. The binding of [U-14C]5'-deoxyadenosine was strongly inhibited by unlabeled 5'-deoxy-ara-adenosine, 4',5'-anhydroadenosine, adenosine, adenine, and 5',8-cyclic adenosine, in this order, but not by 5'-deoxyuridine. These results constitute direct evidence for the presence of the binding site for the adenosyl group of adenosylcobalamin, which is spatially limited to and highly specific for adenine nucleosides. The binding of 5'-deoxyadenosine to the apoenzyme was reversible.     

Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. [3H]FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of [3H]FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by [14C]DCCD [Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.     

Self-association of adenosine 5'-monophosphate (5'-AMP) as a function of pH and in comparison with adenosine, 2'-AMP and 3'-AMP

The concentration dependence of the chemical shifts for protons H-2, H-8, and H-1' of adenosine (Ado), 2'-AMP, 3'-AMP and 5'-AMP was measured in D2O at 27 degrees C under several degrees of protonation. All results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for Ado decrease with increasing protonation: Ado (K = 15 M-1) greater than D(Ado)+/Ado (6.0 M-1) greater than D(Ado)+ (0.9 M-1). In contrast, a maximum is observed with 5'-AMP: 5'-AMP2- (K = 2.1 M-1) less than D(5'-AMP)- (3.4 M-1) less than D2(5'-AMP) +/- /D(5'-AMP)- (5.6 M-1) greater than D2(5'-AMP) +/- (approximately 2 M-1) greater than D3(5'-AMP)+ (less than or equal to 1 M-1). Self-stacking is most pronounced here if 50% of the adenine residues are protonated at N-1; complete base protonation reduces the stacking tendency drastically. Comparing the self-association of 2'-, 3'- and 5'-AMP shows that there is no influence of the phosphate-group position in the 2-fold negatively charged species, i.e., K congruent to 2 M-1 for all three AMP2- species. More importantly, there is also no significant influence observed if the stacking tendency of the three D2(AMP) +/- /D(AMP)-1:1 mixtures is compared (K congruent to 6-7 M-1); moreover, the measured association constants are within experimental error identical with the constant determined for D(Ado)+/Ado (K = 6.0 M-1). This indicates that any coulombic contribution between the -PO3(H)- group and the H+ (N-1) unit of the adenine residue to the stability of the mentioned stacks in D2O is small. However, experiments in 50% (v/v) dioxane-D8/D2O with the D2(5'-AMP) +/- /D(5'-AMP)- 1:1 system reveal, despite its low solubility, that coulombic interactions contribute to the self-association in an environment with a reduced polarity (compared to that of water). The implications of these observations for biological systems are briefly indicated.     

Use of a fluorescent probe for the study of the active center of D-glyceraldehyde-3-phosphate dehydrogenase]

The effect of NAD on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to yeast glyceraldehyde-3-phosphate dehydrogenase has been studied using difference spectrophotometric and fluorescence techniques. Coenzyme addition causes the displacement of ANS from its complex with the dehydrogenase, as suggested by the effect of NAD on the fluorescence of the enzyme--ANS complex, as well as on the magnitude of the difference spectrum of the complex. Adenine containing NAD fragments, adenosine, 5'-AMP, and ADP were shown to compete with ANS for the common site on the enzyme using fluorimetric technique; in the case of adenosine and 5'-AMP a direct method of analytical ultracentrifugation was also employed. The results obtained by both methods suggest the dye binding at the adenine subsite of the dehydrogenase. The interaction with ANS causes no detectable conformational changes of the protein. The fluorescence of the dye-enzyme complex increases and the emission maximum shifts to shorter wavelengths on addition of nicotinamide mononucleotide. This suggest some conformational changes to occur in the microenvironment of the bound dye in response to the interaction with the ligand in the nicotinamide subsite. The participation of the nicotinamide subsite of the active center in determining the character of conformational transitions associated with coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase is discussed.     

Arapen: a secondary complex between platinum ethylenediamine dichloride and cytosine arabinoside. I. Preparations and reactivity towards nucleic acid constituents.

An equimolecular complex between platinum ethylenediamine dichloride (PtenCl2) and 1-beta-D-arabinofuranosyl cytosine (araC) has been prepared (arapen). This 1 : 1 complex in which both ethylenediamine and araC moieties carried a radioactive label was found to be unreactive towards cytosine and guanine derivatives, but to react to the extent of 30% with deoxyadenosine, adenosine 5' -monophosphate, polyadenylic acid, poly a(AT) and DNA at comparable rates. It is suggested that adenine compounds react with platinum complexes such as PtenCl2 in a bidentate manner, but are still able to react with arapen as monodentate ligands. However, hydrolysis of the last chloride ion from arapen may not be complete, and the incoming adenine nucleophile is only able to react to a limited extent.     

Binding of adenine and adenine-related compounds to the clay montmorillonite and the mineral hydroxylapatite

The first living things may have consisted of no more than RNA or RNA-like molecules bound to the surfaces of mineral particles. A key aspect of this theory is that these mineral particles have binding sites for RNA and its prebiotic precursors. The object of this study is to explore the binding properties of two of the best studied minerals, montmorillonite and hydroxylapatite, for possible precursors of RNA. The list of compounds investigated includes purines, pyrimidines, nucleosides, nucleotides, nucleotide coenzymes, diaminomaleonitrile and aminoimidazole carbox-amide. Affinities for hydroxylapatite are dominated by ionic interactions between negatively charged small molecules and positively charged sites in the mineral. Binding to montmorillonite presents a more complex picture. These clay particles have a high affinity for organic ring structures which is augmented if they are positively charged. This binding probably takes place on the negatively charged faces of these sheet-like clay particles. Additional binding sites on the edges of of these sheets have a moderate affinity for negatively charged molecules.Small molecules that bind to these minerals sometimes bind independently to sites on the minerals and sometimes bind cooperatively with favorable interactions between the bound molecules.     

Analysis of peptides synthesized in the presence of SAz-1 montmorillonite and Cu(2+) exchanged hectorite.

We have investigated the synthesis of oligopeptides containing glycine and tyrosine in the presence of the clay minerals montmorillonite (non-exchanged, SAz-1) and Cu(2+) exchanged hectorite. In both cases, homopolymers of the two amino acids are formed, as are mixed peptides. In the case of Cu(2+) hectorite, mixed oligopeptides up to trimers are detected in small amounts. For montmorillonite, heterogeneous oligopeptides up to hexamers are detected. Our experiments indicate montmorillonite is more effective in promoting oligopeptide formation than Cu(2+) hectorite. Analysis of the oligopeptide sequences formed on the montmorillonite surfaces indicates preferential synthesis of certain Gly-Tyr sequences over others.     

Physical studies on the binding of cis-dichlorodiamine platinum (II) to DNA and homopolynucleotides.

The amount of cis-dichlorodiamine platinum (II) bound to DNAs of varying (dA + dT) content was assayed by both ultraviolet absorbance spectrophotometry and the use of the radioisotope 1 9 5 Pt. Radioisotope labeling indicates twice as much bound platinum as do optical measurements. The molar ratio of bound platinum r at saturation is approximately half the sum of the nearest-neighbor frequencies of all base-pairs that do not contain thymine. We therefore conclude that platinum does not bind to thymine in DNA. Chromatographic studies with (14C) purine-labeled DNA indicate preferential binding of platinum to guanine, followed by binding to adenine. The luminescence properties of DNA and of homopolynucleotides are strongly affected by bound platinum as a result of a heavy-atom effect. A plot of the fluorescence-to-phosphorescence ratio as a function of r gives a saturation binding curve similar to that obtained using 1 9 5 Pt. Ultraviolet irradiation of DNA treated with the platinum compound results in a 30% increase in the rate of formation of thymine homocyclobutadipyrimidine. When acetophenone sensitization is employed, platinum binding enhances cytosine homocyclobutadipyrimidine formation 10-fold presumably because the triplet level of cytosine complexed with platinum is lowered below that of acetophenone. The viscosity of DNA decreases sharply upon binding platinum, with half the change occuring when less that 6% of the bases are complexed. From the rate of reaction with formaldehyde, we conclude that binding of the platinum compound to DNA induces small denatured regions that unwind in the presence of formaldehyde with a rate about 40 times slower than that of a single-strand chain break.     

Intramolecular cyclization in irradiated nucleic acids: correlation between high-performance liquid chromatography and an immunochemical assay for 8,5'-cycloadenosine in irradiated poly(A)

A correlation between high-performance liquid chromatography (HPLC) analysis and an in situ enzyme-linked immunosorbent assay (ELISA) for 8,5'-cycloadenosine formation in irradiated poly(A) has been established. The correlation shows that the ELISA precisely reflects changes in the combined yield of R- and S-8,5'-cycloadenosine but that a correction factor must be applied to the ELISA values for accuracy. The HPLC analysis reveals that the intramolecular cyclization proceeds stereoselectively in irradiated poly(A) to preferentially produce the R isomer at pH 7.0 which is similar to the result for irradiated adenosine but in contrast to the result for 5'-AMP where the S isomer predominates at neutral pH. The HPLC analysis shows that two events originating in hydroxyl radical attack at the sugar phosphate backbone in poly(A); that is, adenine release and 8,5'-cycloadenosine formation have somewhat different dose-yield responses. The formation of 8-hydroxyadenosine was detected in the HPLC chromatograms of poly(A) irradiated under N2O at neutral pH, and the yield of this compound was similar to the yield observed in 5'-AMP or adenosine irradiated under similar conditions.