Imidazole Buffer Research Articles

Arylsulfatases: Colorimetric and fluorometric assays

This chapter discusses the colorimetric and fluorometric assays of arylsulfatases. The assay of arylsulfatases is simple in principle, and any of the reaction products may be determined. The general method is simple and requires only that the reaction mixture, after incubation with the enzyme, be made alkaline so that the liberated phenol can be determined, as the phenoxide, either spectrophotometrically or fluorometrically. With crude enzyme preparations deproteinization with, for example, ethanol or trichloroacetic acid may be necessary but any acid treatment should be as mild and as brief as possible because all aryl sulfates are acid-labile. In some cases, the spectra of the phenol and its sulfate ester differ sufficiently at the pH of arylsulfatase activity for the formation of the former to be followed directly so that continuous spec trophotometric assays are possible. Unhydrolyzed substrate can be determined spectrophotometrically. The lysosomal sulfatases A and B have pH optima at about 5, and acetate buffers, or formate buffers at lower pH, are generally suitable for their assay. These enzymes are inhibited by phosphate. The microsomal sulfatase C of mammalian tissues, and the arylsulfatases of mollusks and of microorganisms, have pH optima in the neutral or slightly alkaline region so that imidazole or Tris buffers may be used in their assay. Arylsulfatases may be quite sensitive to changes in the salt or buffer concentration of the reaction mixture so that careful control of the assay conditions is essential. Sulfatases A are hysteretic enzymes that undergo a substrate-induced inactivation with a half-time of a few minutes. The chapter also discusses the substrates for the arylsulfatases.

Purification and properties of 3?,5?-cyclic nucleotide phosphodiesterase from Rhizobium fredii MAR-1

Cyclic nucleotide phosphodiesterase was purified from Rhizobium freddi MAR-1. The enzyme had a molecular weight of approximately 59,000 and was composed of a single subunit. The pH optimum of the enzyme was approximately 7.0 in both Tris-Cl and Imidazole buffers. However Imidazole buffer was shown to produce a two-fold stimulation of enzyme activity without affecting the pH optimum. Cyclic nucleotide phosphodiesterase from R. fredii MAR-1 was stable at 28°C for at least 10 days. The enzyme did not require any metal ion for activity although Cu2+ at 2.5 mM stimulated the enzyme. EDTA up to 2.5 mM did not inhibit the enzyme. Both NAD and NADP as well as FAD inhibited the enzyme with FAD showing maximum inhibition. The enzyme was able to hydrolyse both 3′,5′-cyclic AMP and 3′,5′-cyclic GMP but did not hydrolyse 2′,3′-cyclic AMP. The apparent Km of the enzyme for cAMP was approximately 0.1 μM.

Ethanol‐induced Inhibition of Mouse Brain Adenosine Triphosphatase Activities: Lack of Interaction with Norepinephrine in Vitro

The in vitro effects of ethanol on C57BL/6J mouse forebrain ATPase activities were investigated in the presence and absence of norepinephrine. Three enzyme activities (Na + K-ATPase, Mg-ATPase, and low affinity Ca-ATPase) were studied in forebrain homogenates using a colorimetric assay. The effect of norepinephrine on ethanol inhibition of Sprague-Dawley rat brain Na + K-ATPase activity was examined in selected experiments for direct comparison with the results obtained using mouse brain. Ethanol (250-2000 mM) inhibited all ATPase activities in a concentration-dependent manner. In each case the IC50 was well beyond what would be a lethal concentration in vivo. Ethanol inhibition of mouse forebrain Na + K-ATPase activity was competitive with regards to K+ ion concentration, but was uncompetitive for inhibition of Mg-ATPase and Ca-ATPase activities. Norepinephrine (0.1 mM) did not sensitize mouse brain Na + K-ATPase activity to ethanol-induced inhibition. In contrast, norepinephrine sensitized rat brain Na + K-ATPase to ethanol inhibition when tested simultaneously with mouse brain under identical conditions. These results cannot be explained by differences in assay conditions and suggest that the interaction between norepinephrine and ethanol inhibition of Na + K-ATPase activity may be species specific. Norepinephrine alone stimulated mouse and rat brain Na + K-ATPase activity when assayed in imidazole buffer, but not when assayed in tris buffer. Furthermore, 0.1 mM norepinephrine slightly antagonized the inhibitory effect of ethanol on mouse brain Mg-ATPase activity, but did not affect ethanol-induced inhibition of Ca-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium and buffer cations inhibit dephosphorylation of (Na + + K +)-ATPase

(1) Effects of various cations on the dephosphorylation of (Na + + K +)-ATPase, phosphorylated by ATP in 50 mM imidazole buffer (pH 7.0) at 22°C without added Na +, have been studied. (2) The dephosphorylation in imidazole buffer without added K + is extremely sensitive to K +-activation ( K m K + = 1 μ M ), less sensitive to Mg 2+-activation ( K m Mg 2+ = 0.1 mM ) and Na +-activation ( K m Na + = 63 mM ). (3) Imidazole and Na + effectively inhibit K +-activated dephosphorylation in linear competitive fashion ( K 1 imidazole 7.5 mM, K i Na + 4.6 mM). The K i for Na + is independent of the imidazole concentration, indicating different and non-interacting inhibitory sites for Na + and imidazole. (4) Imidazole inhibits Mg 2+-activated dephosphorylation just as effectively as K +-activated dephosphorylation, as judged from the K i values for imidazole in the two processes. (5) Tris buffer and choline chloride, like imidazole, inhibit dephosphorylation in the presence of residual K + (< 1 μM), but less effectively in terms of I 50 values and extent of inhibition. Tris inhibits to the same extent as choline. This indicates different inhibitory sites for Tris or choline and for imidazole. (6) These findings indicate that high steady-state phosphorylation levels in Na +-free imidazole buffer are due to the induction of a phosphorylating enzyme conformation and to the inhibition of (K + + Mg 2+)-stimulated dephosphorylation.

The adsorption and reaction of adenine nucleotides on montmorillonite.

The binding of AMP to Zn(2+)-montmorillonite was investigated in the presence of buffers and salts. Good's buffers, piperazine-N,N'-bis(2-ethanesulfonate) [PIPES] and morpholine-N-2-ethanesulfonate [MES], perturbed the exchangeable cations to a lesser extent (only 9% of Zn2+ displaced by 0.2 M buffer) than was observed with imidazole and lutidine buffers or NaCl and KCl salts (up to 80% of Zn2+ displaced). AMP adsorption isotherms measured in the presence of 0.2 M PIPES, MES, or Na2SO4 exhibited normal Langmuir-type behavior. The adsorption coefficient, KL, is 3-fold greater in the presence of HEPES or PIPES than it is in the absence of buffers. Basal spacings measured by X-ray diffraction for Zn(2+)-montmorillonite are 13 and 15 angstroms in the presence of PIPES, while a value of 12.8 angstroms was determined in the absence of PIPES. These data are interpreted in a model in which the adsorption of AMP is mediated by a Zn2+ complex of PIPES in different orientations in the interlamellar region of the montmorillonite. The type of exchangeable cation does not affect the ability of the lattice-bound Fe3+ in the montmorillonite to oxidize diaminomaleonitrile (DAMN). Exchangeable Cu2+ oxidizes DAMN, but exchangeable Fe3+ is nearly ineffective as an oxidant. The addition of DISN to 3'-AMP bound to Zn(2+)-montmorillonite in the presence of 0.2 M PIPES resulted in a higher yield of 2',3'-cAMP than is observed with a comparable concentration of Zn2+, a result which inplicates surface catalysis by the montmorillonite.

Crystallization of the gastric H,K-ATPase.

Crystalline arrays of the gastric H,K-ATPase were obtained in membrane preparations from hog and rabbit gastric mucosa. The lattice was formed rapidly in a medium containing K+, vanadate, Mg2+, and dimethyl sulfoxide at pH 6.0-6.9 in imidazole buffer from 4 to 22 degrees C. The crystal lattice exhibited P2 symmetry, and the unit cell dimension (a = 5.6, b = 11, and c = 10 nm) could accommodate 2 polypeptides of mass 116-129 kDa. In addition, the isolated preparation contained previously undescribed long cylindrical structures 16 nm thick. These structures consisted of a central core 6-7 nm wide from which particles spaced 5.5 nm apart protruded symmetrically.

Reversible addition of bisulfite buffer to the cytidine ring system

The rate constants for the reversible addition of protons and sulfite to the 5,6 double bond of cytidine and 3-methylcytidine have been spectrophotometrically measured under conditions (25°C, μ = 1.0 m) where the deamination of 5,6-dihydrocytidine-6-sulfonate is minimal. Both the addition and the elimination of sulfite from the ring system are subject to general catalysis of proton transfer. For the reaction in either direction, plots of the pseudo-firstorder rate constants against increasing buffer concentration are biphasic and indicative of at least a two-step reaction pathway with both steps being subject to general acid-base catalysis. Kinetic hydrogen-deuterium isotope effects were measured for both buffer-catalyzed steps of sulfite elimination from 3-methyl-5,6-dihydrocytidine-6-sulfonate and sulfite addition to 3-methylcytidine. Both H 2O and D 2O were used as solvent. For both the addition and the elimination of SO 3 2− values of k 2 H k 2 D were 6.3–7.1 and 2.3–2.6 at low and high imidazole buffer concentration, respectively. The large isotope effects values in the range of 6–7 can be attributed to rate-determining proton transfer to carbon-5 of the cytidine ring system. The smaller values are more likely caused by proton transfer to a electronegative atom such as the oxygen on carbon-2 of the cytidine ring. The equilibrium constants for bisulfite buffer addition to 3-methylcytidine and cytidine at 25°C, μ = 1.0 m, pH 7.2, are 10.2 and 1.3 m −1, respectively.

Ionic strength effects on the electrophoretic mobility of casein-coated polystyrene latex particles

Electrophoretic mobilities of polystyrene particles coated with three casein proteins (α sl, β, and κ) have been measured at 25°C in 20 m M imidazole buffer (pH 7.5) as a function of ionic strength of added sodium chloride (0.01–0.15 mole dm −3) or calcium chloride (0.00003–0.09 mole dm −3), with protein concentrations sufficient to ensure saturation coverage as shown by measured adsorption isotherms. Particle mobilities were found to lie in the order α sl > κ ≳ β in NaCl and at low concentrations of CaCl 2, but in the order κ > α sl ≳ β at higher concentrations of CaCl 2, reflecting the different calcium-binding properties of the three proteins. Qualitatively similar results for bare and coated particles show that the large difference between sodium and calcium counterions is not due to specific calcium ion-binding on the adsorbed protein. For these biocolloidal particles, the two techniques of laser Doppler electrophoresis and particle microelectrophoresis give essentially identical results.

Inhibition of smooth muscle 5′-nucleotidase by imidazole and its reversal by magnesium

Imidazole, commonly used as an effective pH-buffering reagent in aqueous media maintained at pH 7–8, was found to depress the 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity of microsomal membrane fraction isolated from rat vas deferens smooth muscle in a dose-dependent manner in the absence of added Mg 2+. Such an inhibitory effect of imidazole on the smooth muscle 5′-nucleotidase was not dependent upon the purity or integrity of the membrane fractions used and could be fully reversed by the inclusoin of 5–10 mM Mg 2+ in the assay medium. Of the five different pH-buffering reagents tested, imidazole was specific in exerting inhibitory effect on the 5′-nucleotidase in the absence of Mg 2+ and this inhibition could not be accounted for by the impurities present in the imidazole. Differential effects of chelating reagents and other divalent metal ions on the 5′-nucleotidase activity were also observed in imidazole and Tris buffer solutions. The 5′-nucleotidase activity was not affected if the membranes were preincubated and washed with a large volume of 50 mM imidazole and subsequently assayed in 50 mM Tris in the absence of Mg 2+. Similar findings were obtained with EDTA treated membrane. These results suggest that imidazole does not act by removal of the activating metal ion but rather interacts directly with 5′-nucleotidase and alters the metal-enzyme interactions.

Characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human adrenal cortex.

Our study compares the properties of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from a human metastatic virilizing carcinoma and that from normal adrenal glands obtained from kidney donors. Optimal conditions for enzyme assay were obtained when a 50-mM imidazole buffer (pH 7.2) containing 5 mM EDTA, 250 mM NaCl, 1 mM phenyl-methylsulfonylfluoride, 0.1 mM leupeptin, and 5.5 mM dithiothreitol (DTT) was used. A 30-min preincubation period preceding addition of substrates enhanced reductase activity by 1.75-fold. In crude microsomal preparations, Km values were similar for both tumor and normal tissues and varied between 4 and 5 microM (S)HMG-CoA. The presence of NaF in homogenization and incubation media decreased the maximum velocity, but not the Km. A partially purified rat liver phosphorylase phosphatase preparation or a similar preparation from the carcinoma restored to maximal levels the reductase activity of microsomes prepared in the presence of NaF. A Km of 96 microM NADP was found for the carcinoma microsomal preparation. Preincubation of microsomes in the presence of monothioglycerol or DTT resulted in an increased reductase activity, suggesting a possible inactive enzyme precursor(s) consisting of disulfide-linked units. Reactions of the DTT-activated enzyme incubated in the presence of increasing amounts of NADPH showed sigmoidal kinetics. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed the presence of a 92.5K mol wt protein band that reacted with a rat antireductase antibody. Reductase activity in different regions of the carcinoma varied from 679-1763 pmol/mg protein, with an average of 1146 pmol. In three normal adrenal glands we found values of 23.4, 48.1, and 36 pmol. We concluded that the expression of HMG-CoA reductase activity was elevated in human adrenal carcinoma.

The separation and identification of enolase isozymes of brain and sciatic nerve by high-pressure liquid anion-exchange chromatography

A rapid technique for separating and quantitating the three enolase isozymes present in rodent brain and sciatic nerve was developed using high-pressure liquid anion-exchange chromatography. At pH 7.9, one cationic and two anionic enzyme forms were separated with baseline resolution in an imidazole buffer containing ethylenediaminetetraacetic acid (EDTA) and magnesium. The recovery of enolase activity was 90% or greater for brain and 85% for sciatic nerve. Chromatography of liver and axon-free (degenerated) sciatic nerve allowed the identification of non-neuronal, hybrid, and neuron-specific enolase isozymes. These enzyme forms, respectively, constituted 40%, 29% and 19% of total activity in brain, and 63%, 13% and 4% of total activity in normal sciatic nerve.

Isolation of human factor VIII:C by preparative high-performance size-exclusion chromatography

Human Factor VIII procoagulant protein (VIII:C) is a plasma protein that participates in the cascade of events leading to blood coagulation. It is absent or defective in patients with hemophilia A. In vivo Factor VIII:C associates with Von Willibrand factor and its multimers to form a high-molecular-weight particle that cna be dissociated into a lower-molecular-weight form in the presence of high concentrations of salt. We have been able to purify rapidly Factor VIII:C on a large scale by sequential high-performance size-exclusion chromatography (HPSEC) under conditions of first low salt and then high salt concentration. Reconstituted commercial Factor VIII:C concentrate was purified by chromatography on a preparative HPSEC column (Toyo Soda, 60 × 2.5 cm, 300ml) in 0.05 M imidazole buffer, (pH 7.0), containing 0.15 M sodium chloride. Factor VIII:C activity was eluted in the void volume in less than 20 min as a high-molecular-weight particle, well separated from low-molecular-weight contaminants. Purification was 20-fold, with a yield of 80%. Up to 4 g of Factor VIII concentrate could be purified at one time in this manner. This material was then concentrated and made 0.35 M in calcium chloride prior to re-chromatography on the same column in a buffer containing 0.30 M calcium chloride. Under these conditions, Factor VIII:C activity was eluted in the inner volume of the column at a position corresponding to a molecular weight of several hundred thousand in less than 1 h. It was well separated from both larger proteins and smaller peptide fragments. Analysis of the preparation with radiolabelled antibody to human Factor VIII:C antigen indicated that at least two molecular weight forms of Factor VIII:C were present.

Scrutinization of the Direct Assay Method for Plasma Cyclic AMP and Clinical Applications of Nephrogenous Cyclic AMP

Several problems in the measurement of plasma cyclic AMP (PcAMP) and nephrogenous cyclic AMP were studied using a YAMASA RIA Kit (YAMASA Shoyu, Choshi, Japan). In this assay method, cAMP in plasma is directly succinylated without prior deproteinization, and then it is bound to antibody in an imidazole buffer. So far as the blood samples were obtained with EDTA-4Na at least 5.0 mM in the final concentration, PcAMP was not reduced until 16 hours after the blood samples were drawn. Even without EDTA, the reductions in PcAMP were not detected within 1 hour after the blood samples were drawn. This assay method for PcAMP showed parallelism in the dilution curve. Recovery was almost complete. Intra- and interassay variations were low. When plasma was incubated at 37 degrees C for 24 hours, PcAMP became negligible. Furthermore, the values of PcAMP measured with this direct assay system almost agreed with those obtained after the purification by deproteinization and Dowex column chromatography through an anion-exchange resin. The normal values of PcAMP were 13.6 +/- 3.62 pmol/ml [mean +/- SD, n = 43]. Nephrogenous cAMP expressed as a function of GFR never did show any negative values in various clinical situations. From the data of basal levels and the oral calcium tolerance test, nephrogenous cAMP appeared to be more useful than total urinary cAMP in the diagnosis of parathyroid disorders, especially hyperparathyroidism.

Intracellular pH in hibernation and respiratory acidosis in the European hamster.

Intracellular pH was determined (DMO method) in European hamsters, in the spontaneously-occurring respiratory acidosis of hibernation, in hypercapnia due to breathing 12% CO2 in air in euthermy in spring, and in euthermicnormocapnic controls. From euthermy to hibernation, the temperature coefficient of pH was lowest in blood plasma and brain, intermediate in striated muscles (thigh muscles and diaphragm), and highest in heart and liver (Fig. 1). Correspondingly, the estimated dissociation ratio of the protein imidazole buffer groups, alpha Im, decreased markedly in plasma and brain, denoting an acid titration, but varied little in liver and heart. Striated muscles were intermediate (Fig. 2). Like in other mammals, intracellular responses to short-term euthermic respiratory acidosis were characterized by a partial metabolic compensation in the brain and a small metabolic acidification in striated muscles. In hibernation, a powerful metabolic compensation took place in liver and heart, nearly restoring alpha Im, but none occurred in brain (Figs. 3 to 5). The existence of an intracellular acidosis in brain and striated muscles during hibernation is in keeping with an inhibitory role of acidosis, whereas the homeostasis of intracellular alpha Im in liver and heart would subserve the eurythermal functioning of metabolic regulations in these organs, like in most organs of ectotherms.

Method for the preparation of human erythrocyte membrane with low basal calcium ATPase, responsive to stimulation

A simple procedure for preparing erythrocyte membranes with low basal Ca 2+ ATPase activity is described, which is stimulated several-fold by the addition of hemolysate in the incubation mixture. The cells are hemolyzed in hypotonic imidazole buffer and resulting membranes are washed with hypotonic phosphate buffer (pH 8.0) and the hemolyzing medium. The membrane preparations also have Mg 2+-stimulated and Na +K +-stimulated ATPase activities. The method allows the comparison of basal Ca 2+ ATPase as well as hemolysate- or calmodulin-stimulated Ca 2+ ATPase activities and thus may be useful in studying Ca 2+ ATPase activity in various physiopathological conditions.

Possible role of singlet molecular oxygen in the control of the phototactic reaction sign of Anabaena variabilis

In Anabaena variabilis the phototactic reaction sign at high photon fluence rates is reversed from negative to positive by the addition of 10 −3 M sodium azide. This effect has been interpreted to indicate that a hypothetical phototactic reaction sign reversal generator in the sensory transduction chain is controlled by the level of singlet oxygen which is quenched by azide. To confirm this hypothesis, experiments with other quenching agents, it various pH values, as well as in nitrogen and oxygen atmospheres, have been carried out. Experiments with a phosphate buffer at various pH values show that the transition point from positive to negative phototaxis is shifted from 5.7 W m −2 at pH 7 to about 15 W m −2 at pH 6. At pH 5.5 only positive responses occur even at 120 W m −2. At lower pH values movement ceases. However, at alkaline pH (8.0) the transition point is shifted to lower fluence rates (4.5 W m −2). When imidazole buffer (pH 6) is used, the reaction sign reversal is even more pronounced. l-histidine, a 1O 2 quencher, decreases negative phototaxis at 13.5 W m −2 with increasing concentration, but does not reverse the reaction sign. 1,4-diazabicyclo[2.2.2]octane reverses the reaction sign at 13.5 W m −2 from negative to slightly positive, but is cytotoxic at higher concentration. When the Petri dishes are gassed with nitrogen and oxygen, only positive reactions are observed up to 54 W m −2. These results support the hypothesis that in Anabaena the phototactic reaction sign is controlled by the 1O 2 concentration via a reaction sign reversal generator. This mechanism enables the Anabaena filaments to escape dangerous light conditions. 2 × 10 −2 M KI inhibits both positive and negative phototaxis.

The mechanism of in vitro clot lysis induced by vascular plasminogen activator

The contribution of vascular plasminogen activator (v-PA) to the lysis of whole blood and plasma clots was investigated. v-PA released into the circulation after infusion of deamino-D-arginine vasopressin (DDAVP) was shown to bind quantitatively to plasma clots. Its apparent molecular weight, determined by the SDS-PAGE fibrin-agarose underlay method, was approximately 68,000 daltons, and its activity was quenched by antibodies against human tissue plasminogen activator (t-PA). Clots prepared from post-DDAVP plasma or post-DDAVP whole blood, rich in v-PA, did not lyse when incubated in imidazole buffer or normal plasma, as determined by the release of 125I from radiolabeled clots. However, clots made of v-PA-poor plasma or whole blood, incubated in v-PA-rich plasma, underwent substantial lysis. The concentration of PA in clots incubated in v-PA-rich plasma progressively increased in relation to the initial concentration of v-PA in the surrounding plasma. The results suggest that, at low concentrations of circulating v-PA, a hemostatic plug will lyse at a very low rate. However, when the v-PA concentration in the clot environment is increased, v-PA will accumulate progressively onto fibrin and induce thrombolysis.

The Mechanism of In Vitro Clot Lysis Induced by Vascular Plasminogen Activator

Abstract The contribution of vascular plasminogen activator (v-PA) to the lysis of whole blood and plasma clots was investigated. v-PA released into the circulation after infusion of deamino-D-arginine vasopressin (DDAVP) was shown to bind quantitatively to plasma clots. Its apparent molecular weight, determined by the SDS-PAGE fibrin-agarose underlay method, was approximately 68,000 daltons, and its activity was quenched by antibodies against human tissue plasminogen activator (t-PA). Clots prepared from post-DDAVP plasma or post-DDAVP whole blood, rich in v- PA, did not lyse when incubated in imidazole buffer or normal plasma, as determined by the release of 125I from radiolabeled clots. However, clots made of v-PA-poor plasma or whole blood, incubated in v-PA-rich plasma, underwent substantial lysis. The concentration of PA in clots incubated in v-PA-rich plasma progressively increased in relation to the initial concentration of v-PA in the surrounding plasma. The results suggest that, at low concentrations of circulating v-PA, a hemostatic plug will lyse at a very low rate. However, when the v-PA concentration in the clot environment is increased, v-PA will accumulate progressively onto fibrin and induce thrombolysis.

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides of DL-alanine and N-(tert-butoxycarbonyl)-DL-alanine.

The aminoacylation of diinosine monophosphate (IpI) was studied. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-DL-alanine, a 40% enantiomeric excess of the L isomer was incorporated at the internal 2' site and the positions of equilibrium for the 2' in equilibrium with 3' migration reaction differed for the D and L enantiomers. The reactivity of the nucleoside hydroxyl groups decreased in the order 2'(3') greater than internal 2' greater than 5', and the extent of reaction was affected by the concentration of the imidazole buffer (pH 7.1). In contrast, reaction of IpI with the imidazolide of unprotected DL-alanine led to an excess of the D isomer at the internal 2' site, while reaction with the N-carboxy anhydride of DL-alanine proceeded without detectable stereoselection. The relevance of these results to the evolution of optical activity and the origin of genetically directed protein synthesis is discussed.

The investigation of the HCN derivative diiminosuccinonitrile as a prebiotic condensing agent. The formation of phosphate esters.

Diiminosuccinonitrile (DISN) is formed readily by the Fe3+ oxidation of diaminomaleonitrile, a tetramer of HCN. DISN effects the phosphorylation of uridine in 13% yield to a mixture of the isomers of UMP when the reaction is performed in dimethylformamide solution. A 4% yield of the UMP isomers is obtained in neutral aqueous solution using 2 times the DISN concentration and 7 times the phosphate concentration used in DMF. DISN did not effect the conversion of adenosine to AMP or 5'-AMP to 5'-ADP in aqueous solution. The cyclization of 3'-AMP and 3'-UMP to the corresponding 2'-3'-cyclic phosphates proceeds in yields as high as 40-50% at 60 degrees C in pH 6 aqueous solutions in the presence of divalent metal ions. Lower yields of the cyclic phosphate are observed when 2'-AMP is the starting material. Substitution of acetate buffer for imidazole buffer results in a decrease in the yield of cyclic phosphate, the extent of which depends on the metal ion used in the reaction. No 3'5'-cyclic AMP was detected as a reaction product with either 5'-AMP or 3'-AMP as the starting material except for a 2.4% yield from 3'-AMP in the presence of Zn2+. BrCN effects the conversion of 3'-AMP to the 2'-3'-cyclic AMP in 37-65% yield depending on the divalent cation used as catalyst. A mechanism has been proposed for these cyclization reactions and their potential significance to the prebiotic synthesis of ribonucleic acid derivatives is discussed.