New fluorescent cytidine 5''-phosphate derivatives.

Interaction of cytidine 5'-phosphate with chloroacetone or p-tosyloxyacetone leads to 2-methyl-5,6-dihydro-5-oxo-6-(5-0-phospho-beta-D-ribofuranosyl)-imidazo/1,2-c/pyrimidine (2-methylethenocytidine 5'-phosphate) whereas analogous reaction with phenacyl bromide produces similar 2-phenyl-derivative. The bicyclic nucleotides obtained showed significant UV absorption at long wavelength where common nucleotides and proteins exhibited no absorption. These derivatives are highly fluorescent when heterocyclic ring is protonated. The absorption and fluorescent properties of the substituted ethenocytidine 5'-phosphoate derivatives seem to be suitable for their use as fluorescent probes or labels in biochemical studies.     

Buffer catalysis of amino proton exchange in compounds of adenosine, cytidine and their endocyclic N-methylated derivatives

The use of buffer catalysts having a wide range of pK (dissociation) values (4-12) provides the first estimates of two generally useful empirical parameters of amino proton exchange in compounds of adenine and cytosine. These are a nucleobase amino group dissociation constant (pKD) and the 'encounter frequency' for proton transfer (kD), which can be used to predict amino proton exchange rates. Values of amino pKD fall in the range 8.6-9.4 for the unsubstituted nucleobases and their endocyclic N-methylated derivatives. Similar values of kD are obtained for all nucleobases (1 X 10(8) M-1 s-1). These constants were obtained from a statistical fit of second-order catalytic rate constants for amino proton exchange, measured by amino 1H-NMR lineshape at varying field frequencies (100, 300 and 360 MHz). These results confirm the requirement for buffer conjugate base formation and nucleobase protonation, but point to a different mechanism of exchange at low pH; most probably direct amino protonation for adenine, but not for cytosine compounds. Anionic buffer conjugate bases (phosphate and acetate) show a greater catalytic effect than neutral (nitrogen) bases, especially with cytosine compounds. The use of high concentrations of sodium perchlorate to sharpen amino 1H resonances of 1-methyladenosine is examined, with respect to chemical and rotational exchange and NMR line broadening.     

Catabolism of adenine nucleotides in adenosine deaminase deficient erythrocytes

$\text{In vitro}$ incubation studies using fluoride and iodoacetate as glycolytic inhibitors have been carried out on red cells of the two subjects with adenosine deaminase deficiency. For comparison, similar studies have also been carried out on red cells from a normal subject and from a child with severe combined immunodeficiency with normal adenosine deaminase activity. The adenosine formed in the adenosine deaminase deficient red cells is a measure of adenosine 5′-phosphate breakdown initiated by 5′-nucleotidase, whereas inosine 5′-phosphate, inosine and hypoxanthine formation is a measure of adenosine 5′-phosphate breakdown initiated by adenylate deaminase. With fluoride as inhibitor, nearly all of the adenosine 5′-phosphate breakdown proceeded by way of adenylate deaminase, while with iodoacetate as inhibitor, 20–30% of the adenosine 5′-phosphate breakdown was initiated by 5′-nucleotidase acting on adenosine 5′-phosphate. In addition, significant amounts of adenine were produced in adenosine deaminase deficient red cells in the presence of the glycolytic inhibitors. Possible explanations for the findings noted in this study are discussed and related to recent studies on the properties of the pertinent purine nucleotide catabolic enzymes.     

Independent fluctuations of cytidine and adenosine diphosphate reductase activities in cultured Chinese hamster fibroblasts.

The activities of CDP and ADP reductases were determined throughout the cell cycle of Chinese hamster fibroblasts synchronized by partial deprivation of isoleucine. Both enzyme activities were increased in S phase as compared to G1 phase. CDP reductase increased about 8-fold while ADP reductase increased about 2.5-fold. The ratio of CDP to ADP reductase was 0.26 at the G1 and early S phases of the cell cycle; the ratio was increased to 0.83 by late S phase. Addition of actinomycin D or cycloheximide to cell cultures in G1 phase prevented the increase of both CDP and ADP reductases activities in the latter part of the cell cycle, but the ratio of the two activities was not affected. The ratio of CDP to ADP reductase activities varied from 0.8 to 3 in different populations of exponentially growing DON cells. These results show that CDP and ADP reductase activities vary independently in growing cells. The independent variation with cell growth of CDP and ADP reductases suggests important individual functions of the deoxynucleotides during the cell cycle apart from their common role as precursors of DNA synthesis.     

Competition between the hydrolysis and deamination of cytidine and its 5-substituted derivatives in aqueous acid.

The monocations of a few 5-substituted cytidines have been shown to undergo competitive deamination to the corresponding uridines and hydrolysis to 5-substituted cytosines and D-ribose. The first-order rate constants measured at different temperatures indicate that the proportion of the hydrolysis is considerably increased with the increasing temperature. Electron-withdrawal by a polar substituent at C5 appears to facilitate the hydrolysis to a larger extent that the deamination. The ionic strength has practically no influence on the rate of either reaction.     

Catabolism in mollicutes.

The small genome size of mollicutes, and particularly mycoplasmas and ureaplasmas, precludes their possession of the extensive range of metabolic activities present in most other bacterial groups. Demonstrated catabolic activities appear primarily to be associated with energy generation, rather than the provision of substrates for synthetic pathways, and anabolism is largely dependent upon extracellular sources of amino acids, nucleic acid precursors and lipids. However, the pathways of energy generation in mollicutes are diverse and specialized, and may in vivo be dependent upon the presence of a single amino acid (arginine) or urea. Even in those species that utilize carbohydrates the range of substrates is restricted, and while Ac. laidlawii has both EMP and PP pathways and is able to oxidize pyruvate to acetate plus CO2, many mycoplasmas possess only a part of these activities. Such specialization and the infrequent demonstration of inducible enzyme activity in mollicutes implies adaptation to specific habitats in host species, and suggests that differences in the catabolic activities of mollicute strains may be significant in terms of their ecology and pathogenicity. The demonstrated energy-generating pathways of mollicutes produce low ATP yields. Thus, mollicute growth will generate relatively large quantities of metabolic end-products and may deplete host tissues of substrates. Arginine depletion may be of particular importance in pathogenesis and the close physical association between mollicutes and host cells will enhance the potential significance of NH4+ production from the hydrolysis of arginine and urea, and of H2O2 and superoxide formation during carbohydrate metabolism. In addition, lipid and protein catabolism may be associated with virulence where extracellular or membrane-bound enzyme activities exist. Membrane-bound DNAase and RNAase activities have also been demonstrated in mycoplasmas and Ac. laidlawii (Pollack et al., 1965) and U. urealyticum (Romano & La Licata, 1978). Many aspects of mollicute catabolism, including energy conservation in some groups, is poorly understood. Also, while substantial catabolic diversity has been demonstrated within mollicutes and new species are continually being isolated, metabolism has been studied in relatively few species, and even in these only single strains or small groups of strains have been used. In this review, therefore, an attempt to avoid generalizations concerning mollicute behaviour has been made. The lack of much basic knowledge concerning mollicute metabolism has also necessitated the widespread use of 'may be' and other equally vague terms.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mass spectrometry of uronic acid derivatives. Part V. Fragmentation of methyl derivatives of methyl 4-deoxy-beta-L-threo-hex-4-ehopyranosiduronic-acid.

The basic fragmentation mechanism of methyl(methyl 4-deoxy-2,3-di-O-methyl-β-l-threo-hex-4-enopyranosid)uronate has been deduced by deuteromethylation analysis, metastable transition measurements, and by interpreting the spectra of weakly excited foregoing molecules. The differences in fragmentation of partially methylated derivatives of methyl 4-deoxy-β-l-threo-hex-4-enopyranosiduronic acid compared to that of the fully methylated substance are discussed in detail and the criteria are proposed for identification of the compounds concerned by mass spectrometry.     

Macromolecular derivatives of NAD+ in heart nuclei: poly adenosine diphosphoribose and adenosine diphosphoribose proteins.

Rates of poly (ADP-R) formation from NAD⁺ were determined in isolated pigeon heart and liver nuclei. In heart nuclei K_m for NAD⁺ was 330 μM. On a DNA basis rates were more than twice in heart nuclei than in liver nuclei. The polymer poly (ADP-R) was identified in both nuclear systems by isolation, digestion with snake venom phosphodiesterase and chromatographic separation of phosphoribosyl-AMP and AMP. ADP-R binds to macromolecular nuclear components to form ADP-R derivatives, which upon digestion with snake venom phosphodiesterase yield only AMP, distinguishing these ADP-R compounds from poly (ADP-R).     

Selective toxicity of the polyene antibiotics and their methyl ester derivatives.

The methyl ester hydrochlorides of amphotericin B and nystatin are less effective than the parent compounds in causing K⁺ release from human erythrocytes. The parent compounds and the derivatives are of comparable activity toward $\text{Candida albicans}$. The enhanced selective toxicity of polyene methyl ester salts for $\text{C. albicans}$ may mean that these antibiotics will be more effective therapeutic agents for systemic fungal infections.     

Transition state stabilization by deaminases: Rates of nonenzymatic hydrolysis of adenosine and cytidine

Nonenzymatic rates of hydrolytic deamination of adenosine and cytidine by acids and bases analogous to side chains of naturally occurring amino acids are compared with the rates of uncatalyzed deamination in water and with the rates of the hydroxide- and hydrogen ion-catalyzed reactions. For adenosine, hydroxide ion is an effective catalyst, with a second-order rate constant of 7.5 × 10⁻⁶ m⁻¹ s⁻¹ at 85°C and an energy of activation of 19.9 kcal/mol. Acid-catalyzed deamination of adenine proceeds with a second-order rate constant of 1.5 × 10⁻⁶ m⁻¹ s⁻¹ at 85°C. At concentrations of 1 m and at pH values corresponding to their respective pK_a values, dimethylamine, acetate, selenide, imidazole, phosphate, and zinc(II) do not enhance the rate of deamination of adenosine beyond that observed in water, and 2-mercaptoethanol produces only a modest rate enhancement. The uncatalyzed rate of adenosine deamination in water is 8.6 × 10⁻⁹ s⁻¹ at 85°C: extrapolation to 37°C and comparison with k_cat for rat hepatoma adenosine deaminase yield a rate enhancement by the enzyme of approximately 2 × 10¹²-fold. 1,6-Dimethyladenosine, the conjugate acid of which has a pK_a value much higher than that of adenosine, is not readily deaminated, suggesting that the uncatalyzed deamination of adenosine does not proceed by hydroxide ion attack on the rare protonated form of adenosine, but rather by attack on the neutral species. Deamination of cytidine is catalyzed most effectively by hydroxide ion, with a second-order rate constant of 4.5 × 10⁻⁴ m⁻¹ s⁻¹ at 85°C and an energy of activation of 28.5 kcal/mol. The uncatalyzed rate of deamination of cytidine in water, which also exhibits an energy of activation of 28.5 kcal/mol, is 8.8 × 10⁻⁸ s⁻¹ at 85°C. Comparison of the rate extrapolated to 25°C with k_cat for bacterial cytidine deaminase gives a rate enhancement for the enzyme of 4 × 10¹¹-fold. The C-5 proton of the pyrimidine ring of cytidine does not exchange with solvent during alkaline hydrolysis, suggesting that deamination under these conditions does not involve prior addition of water across the 5,6 double bond.