Identification of the tautomeric form of formycin A in its complex with Escherichia coli purine nucleoside phosphorylase based on the effect of enzyme-ligand binding on fluorescence and phosphorescence.

Fluorescence and phosphorescence emission spectroscopy were employed to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitor, formycin A (FA), a close structural analogue of adenosine (natural substrate), in the absence and presence of phosphate (P(i), substrate). Formation of enzyme-FA complexes led to marked quenching of enzyme tyrosine intrinsic fluorescence and phosphorescence, with concomitant increases in fluorescence and phosphorescence of FA. Fluorescence resonance energy transfer from the protein Tyr160 residue to the FA base moiety was identified as a major mechanism of protein fluorescence quenching, increased by addition of P(i). The effects of enzyme-FA interactions on the nucleoside excitation and emission spectra for fluorescence and phosphorescence revealed shifts in the tautomeric equilibrium of the bound FA, i.e. from the N(1)-H tautomer (predominant in solution) to the N(2)-H form, enhanced by the presence of P(i). The latter was confirmed by enzyme-ligand dissociation constant ( K(d)) values of 5.9+/-0.4 and 2.1+/-0.3 microM in the absence and presence of P(i), respectively. Addition of glycerol (80%, v/v) led to a lower enzyme affinity ( K(d) approximately 70 microM), without changes in binding stoichiometry. Enzyme-FA complex formation led to a higher increase of the fluorescence than the phosphorescence band of the ligand, consistent with the fact that the N(2)-H tautomer is characterized by a weaker phosphorescence than the N(1)-H tautomeric form. These results show, for the first time, the application of phosphorescence spectroscopy to the identification of the tautomeric form of the inhibitor bound by the enzyme.

[Coagulation activity in the airways of asthmatic patients].

Fibrin deposition in the mucus plugs of asthmatic patients has long been known, and asthmatic sputum has been held to be important in the pathogenesis of bronchial obstruction. We examined the coagulation activity in the airways of asthmatic patients. Albumin as an index of plasma leakage into the bronchial lumen, thrombin antithrombin III complex (TAT), tissue factor, FDP, D-dimer and the TAT/D-dimer ratio as indices of coagulation and fibrinolytic markers were determined in expectorated or hypertonic saline-induced sputum from patients with acute and stable asthma, and with chronic bronchitis, and from normal control subjects. Patients with acute asthma, in comparison with patients with stable asthma or chronic bronchitis and normal control subjects, had significantly higher levels of albumin, TAT and TAT/D-dimer. The fibrin antigen was more positively stained immunohistochemically in sputum from acute asthmatics than in other sputa. In both patients with acute asthma and those with stable asthma, there was a significant positive correlation between albumin and TAT or albumin and TAT/D-dimer in the sputum. However, in normal control subjects, there was no correlation between these markers. These results suggest that the coagulation system in the airways of acute asthmatic patients is activated, that this favors fibrin deposition in the bronchial lumen and that coagulation pathways in the bronchial compartment and the degree of plasma exudation into the airways are dependently regulated in patients with asthma but not in normal control subjects.

Interpretation of fluorescence decays using a power-like model.

A power-like decay function, characterized by the mean excited-state lifetime and relative variance of lifetime fluctuation around the mean value, was applied in analysis of fluorescence decays measured with the aid of time-correlated single photon counting. We have examined the fluorescence decay, in neutral aqueous medium, of tyrosine (L-tyrosine and N-acetyl-L-tyrosinamide), and of the tyrosine residues in a tryptophan-free protein, the enzyme purine nucleoside phosphorylase from Escherichia coli in a complex with formycin A (an inhibitor), and orthophosphate (a co-substrate). Tryptophan fluorescence decay was examined in neutral aqueous medium for L-tryptophan, N-acetyl-L-tryptophanamide, and for two tryptophan residues in horse liver alcohol dehydrogenase. To detect solvent effect, fluorescence decay of Nz-acetyl-L-tryptophanamide in aqueous medium was compared with that in dioxan. Hitherto, complex fluorescence decays have usually been analyzed with the aid of a multiexponential model, but interpretation of the individual exponential terms (i.e., pre-exponential amplitudes and fluorescence lifetimes), has not been adequately characterized. In such cases the intensity decays were also analyzed in terms of the lifetime distribution as a consequence of an interaction of fluorophore with environment. We show that the power-like decay function, which can be directly obtained from the gamma distribution of fluorescence lifetimes, is simpler and provides good fits to highly complex fluorescence decays as well as to a purely single-exponential decay. Possible interpretation of the power-like model is discussed.

Inhibition of Rous sarcoma virus production by formycin.

The effect of formycin, an adenosine analog, on the growth of chick embryo fibroblasts and on Rous sarcoma virus (RSV) production was studied. An adverse effect on cell proliferation was observed in the presence of 10 microM formycin. Treatment with 5 microM formycin for 24 hr reduced by a factor of about 1000 the yield of infections progeny whereas the cell growth remained unaltered. Moreover the few particles released in the presence of formycin showed a markedly decreased ability to synthesize viral cDNA. This impairment was shown to be related to a nonfunctional primer tRNA.

Preparation and properties of formycin analogues methylated on the pyrazolo ring nitrogens and/or the ribose cis-hydroxyls.

1. Diazomethane treatment of formycin A in the presence or absence of SnCl2 as catalyst, was used for the preparation of the 2'-O-methyl, 3'-O-methyl, N1-methyl and N2-methyl derivatives. The four possible dimethylated derivatives, 2'(3")-O,N1(N2)-dimethylformycins, were obtained by controlled treatment of formycin with diazomethane in the presence of SnCl2, and subsequent column chromatography for product isolation. 2. All the foregoing products were characterized and identified by chromatography, ultraviolet absorption spectra, and proton magnetic resonance spectroscopy. Extensive u.v. spectral data, and spectrally determined pK values, for the various derivatives are presented. 3. N2-Methylformycin B was also prepared by enzymatic deamination of the parent N2-methylformycin A. 4. The sequence of elution of N1-methylformycin and N2-methylformycin on a strongly basic ion exchange column suggested that the latter is in the syn conformation. The susceptibility of N2-methylformycin to adenosine deaminase shows that this analogue may adopt the anti conformation on reaction with the enzyme. 5. The active species in the SnCl2-catalysed monomethylation of the 2'(3') cishydroxyls of ribonucleosides by diazomethane was shown to be an organo-tin product of the reaction of SnC2 with diazomethane. This product, not identified, contained no nitrogen or chlorine. 6. A simple column chromatographic procedure is described for the desalting of heterocyclic bases and their nucleosides with pK values for ring protonation down to about 0.

Hybridization of polymers of antibiotic C-nucleoside phosphates, poly(formycin phosphate) and poly(laurusin phosphate)

The ability of complex formation of poly-(formycin phosphate), poly(F), and poly(laurusin phosphate), poly(L), with the polymers of natural polynucleotides was examined mainly by mixing experiments in 0.1 M NaCl-0.05 M sodium cascodylate buffer (pH 7.0) at 2 degrees. Poly(F) formed complexes with poly(U) and poly(I) in the ratio of 1:1 and 1:2, respectively. Poly(L) formed complexes with poly(A) in 2:1 ration and poly(C) in 1:2 and 2:1 ratios in addition to a self-complex. Poly(F) and poly(L) also formed a 1:2 complex between them. Some of these complexes were assumed to contain novel types of base pairings using the 7-NH group. Thus it was concluded that poly(L) could form complexes with both, the oligomer of cycloadenylic acid (øcn-120 degrees) and polymers of natural nucleotides (øcn0degrees), showing flexibility of the torsion angle of the laurusin residue.