Non-typical nucleoside analogs as fluorescent and fluorogenic indicators of purine-nucleoside phosphorylase activity in biological samples.

Several novel non-typical nucleoside analogs were examined as potential fluorescent indicators of purine-nucleoside phosphorylase (PNP) activity in human blood. The substrates included N7-riboside of 8-aza-2,6-diaminopurine, N6-riboside of 1,N6-etheno-adenine and N2-riboside of N2,3-etheno-2-aminopurine. Reaction rates and apparent Michaelis' constants were determined in 1000-fold blood lysates and compared with those for reference compounds, guanosine and 7-methylguanosine. It was concluded that the most promising for assaying human PNP in biological material was N6-riboside of 1,N6-etheno-adenine and N2-riboside of N2,3-etheno-2-aminopurine was optimal for the E. coli PNP, both offering at least 10-fold improvement in sensitivity relative to conventional assays. Other potential applications of this approach are discussed.

Kinetics of C. elegans DcpS cap hydrolysis studied by fluorescence spectroscopy.

DcpS (scavenger decapping enzyme) from nematode C. elegans readily hydrolyzes both monomethyl- and trimethylguanosine cap analogues. The reaction was followed fluorimetrically. The marked increase of fluorescence intensity after the cleavage of pyrophosphate bond in dinucleotides was used to determine K(m) and V(max)values. Kinetic parameters were similar for both classes of substrates and only slightly dependent on pH. The hydrolysis was strongly inhibited by methylene cap analogues (m(7)Gp(CH(2))ppG and m(7)Gpp(CH(2))pG) and less potently by ARCA (m(7,3' O)GpppG).

Alcohol and aldehyde dehydrogenase activity in the stomach and small intestine of rats poisoned with methanol.

The activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) were measured with fluorogenic naphthaldehydes in the stomach and small intestine homogenates of rats dosed with 6 g methanol/kg bw after 6, 12, 24 h and 2, 5, 7 days. After intoxication with a sublethal dose, the ADH activity measured with these naphthaldehydes and ALDH activities in the stomach and small intestine were significantly decreased. This inhibition is stronger in the stomach and probably depends on cell damage and protein denaturation. We conclude that the activity measured with 6-methoxy-2-naphthaldehyde (MONAL-62) may be due to the activity of rat ADH-1 isoenzyme, and the activity detected with 4-methoxy-1-naphthaldehyde (MONAL-41) to the activity of rat ADH-2 isoenzyme.

Aromatic aldehydes as fluorogenic indicators for human aldehyde dehydrogenases and oxidases: substrate and isozyme specificity.

Substrate properties of a number of potentially fluorogenic aromatic aldehydes of naphthalenes, phenanthrenes and anthracenes and of some coumarin aldehydes towards various forms of the human and rat aldehyde oxidase and dehydrogenase were examined using absorption and emission spectroscopy. It was demonstrated that recombinant human class 1 aldehyde dehydrogenase (ALDH-1) readily oxidizes naphthalene (except for those ortho-substituted), phenanthrene and coumarin aldehydes, whereas the class 3 enzyme (ALDH-3) from human saliva is active only towards 2-naphthaldehyde derivatives. The observed reaction rates in both cases are comparable to those of the best known substrates, and the Km values are typically in the sub-micromolar range. Aldehyde oxidases (AlOx), which are present in mammalian liver, reveal much broader substrate specificity, oxidizing nearly all the compounds examined, including those of the anthracene series, with maximum activity in the micromolar range of substrate concentration. In rat liver, nearly all AlOx activity was located in the cytosolic fraction.

Formycin A and its N-methyl analogues, specific inhibitors of E. coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme-->ligand fluorescence resonance energy transfer.

Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz. formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m(1)FA), N(2)-methylformycin A (m(2)FA) and N(6)-methylformycin A (m(6)FA), in the absence and presence of phosphate (P(i)). Complex formation led to marked quenching of enzyme tyrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m(6)FA, independently of the presence of P(i). Fluorescence of m(1)FA in the complex increased only in the presence of P(i), while the weak fluorescence of FB appeared unaffected, independently of P(i). Analysis of the emission, excitation and absorption spectra of enzyme-ligand mixtures pointed to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m(6)FA base moieties, as a major mechanism of protein fluorescence quenching. With the non-inhibitor m(2)FA, fluorescence emission and excitation spectra were purely additive. Effects of enzyme-FA, or enzyme-m(6)FA, interactions on nucleoside excitation and emission spectra revealed shifts in tautomeric equilibria of the bound ligands. With FA, which exists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H is shifted to N(2), independently of the presence of P(i). Complex formation with m(6)FA in the absence of P(i) led to a shift of the amino-imino equilibrium in favor of the imino species, and increased fluorescence at 350 nm; by contrast, in the presence of P(i), the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a higher affinity for the enzyme, with a dissociation constant K(d)=0.5+/-0.1 microM, two orders of magnitude lower than that for m(6)FA in the absence of P(i) (K(d)=46+/-5 microM). The latter was confirmed by analysis of quenching of enzyme fluorescence according to a modified Stern-Volmer model. Fractional accessibility values (f(a)) varied from 0.31 for m(1)FA to 0.70 for FA, with negative cooperative binding of m(1)FA and FB, and non-cooperative binding of FA and m(6)FA. For all nucleoside ligands, the best model describing binding stoichiometry was one ligand per native enzyme hexamer. Fluorescence decays of PNP, FA and their mixtures were best fitted to a sum of two exponential terms, with average lifetimes () affected by their interactions. Complex formation resulted in a 2-fold increase in of FA, and a 2-fold decrease in of enzyme fluorescence. The amplitude of the long-lifetime component also increased, confirming the shift of the tautomeric equilibrium in favor of the N(2)-H species. The findings have been examined in relation to enzyme-nucleoside binding deduced from structural studies.

Interactions of calf spleen purine nucleoside phosphorylase with antiviral acyclic nucleoside phosphonate inhibitors: kinetics and emission studies.

Association between calf spleen purine nucleoside phosphorylase and a series of phosphonylalkoxyalkyl derivatives of purine bases was studied by inhibition kinetics and fluorimetric titrations. Dissociation constants, determined by fluorimetric titration in phosphate-free conditions, were lower than inhibition constants in 1 mM phosphate, and inhibition was still weaker in 50 mM phosphate, in accord with the postulated bisubstrate analogue character of this class of inhibitors.

Hydrolysis of some mRNA 5'-cap analogs catalyzed by the human Fhit protein--and lupin ApppA hydrolases.

Hydrolysis of the following four cap analogs: m7G(5')ppp(5')A, m7G(5')ppp(5')m6A, m7G(5')ppp(5')m2'OG and m7G(5')ppp(5')2'dG catalyzed by homogeneous human Fhit protein and yellow lupin Ap3A hydrolase has been investigated. The hydrolysis products were identified by HPLC analysis and the K(m) and Vmax values calculated based on the data obtained by the fluorimetric method.

Aldehyde dehydrogenase isoenzymes in tumours--assay with possible prognostic value for oxazaphosphorine chemotherapy.

A novel fluorimetric assay, allowing independent measurement of the activities of two principal cytosolic forms of human aldehyde dehydrogenase, ALDH-1 and ALDH-3 (known as a tumour-associated ALDH) was applied to estimate the activities of these isoenzymes in human liver and thyroid tumours. The assay is based on two artificial substrates, 6-methoxy-2-naphthaldehyde (MONAL-62) and 7-methoxy-1-naphthaldehyde (MONAL-71), exhibiting excellent substrate properties toward various forms of human ALDH (see Wierzchowski et al., 1997, Anal. Biochem. 245, 69-78). We have found significant differences in ALDH activities between malignant and non-malignant tissue fragments, particularly in cancerous livers. Out of 16 tumours examined, only 4 exhibited ALDH-1 activities comparable to that found in the tumour-free tissue (0.5-2.5 U/g), while in the remaining 12 this activity was at least 10-fold lower. The ALDH-3 activity was detectable in about 40% of both tumour and tumour-free liver samples (maximum value 1.5 U/g). Comparison of 13 pathological thyroid fragments revealed ALDH activities in the range of 0.02 to 0.35 U/g, with two malignant samples showing activities of 0.27 and 0.18 U/g. Both substrate specificity and kinetic behaviour of the thyroid ALDH (Km values for the fluorogenic naphthaldehydes as well as propanal inhibition profile) were similar to those of the purified ALDH-1. In 5 thyroid samples traces of ALDH-3 activity was detected, using MONAL-62 and NADP+ as substrates (maximum value 0.04 U/g). Possible prognostic value of the foregoing measurements for cyclophosphamide chemotherapy is discussed.

The activity of enzymes oxidizing alcohols in the liver of rats after methanol intoxication measured with fluorogenic substrates.

The activities of alcohol dehydrogenase isoenzymes (class ADH I and ADH II) and aldehyde dehydrogenase (ALDH) were measured using fluorogenic substrates in the liver of rats. Animals were dosed with 6 g of methanol/kg b.w. after 6, 12, 24 hours and 2, 5 and 7 days. Liver ADH I and ADH II activities were gradually increased after 6 h and up to 7 days after intoxication. ALDH activity in the liver had the highest elevation at 6 h, and then it decreased but was higher than the control value. It is concluded that subacute administration of methanol to rats leads to induction of hepatic enzymes involved in alcohols metabolism and that tested fluorogenic substrates are useful for these measurements.

Fluorimetric detection of aldehyde dehydrogenase activity in human blood, saliva, and organ biopsies and kinetic differentiation between class I and class III isozymes.

Two highly fluorogenic aldehydes, 7-methoxy-1-naphthaldehyde (MONAL-71) and 6-methoxy-2-naphthaldehyde (MONAL-62), were examined as indicators of the aldehyde dehydrogenase (ALDH) activity in human tissue homogenates and accessible body fluids. Both compounds were previously found to be excellent substrates for the ALDH from erythrocytes and for the purified class I (cytosolic) ALDH from human liver. By contrast, only MONAL-62, but not the isomeric MONAL-71, was oxidized by class III ALDH present in human saliva. The apparent Km for the former compound reacting with salvia ALDH is 0.24 microM, with the reaction rate (Vmax) close to that of benzaldehyde oxidation. There is also a fully competitive inhibition of the fluorogenic oxidation of the MONAL-62 by benzaldehyde. Both NAD+ and NADP+ can be used as oxidants in this reaction, with comparable rates, a fact previously reported for the human class III aldehyde dehydrogenase. In human liver homogenate (cytosolic + microsomal fraction), the ALDH activity is easily detectable using either MONAL-71 or MONAL-62, with specific activities of approximately 2.5 and 3.2 units per gram of protein, respectively. The low apparent Km values, 0.85 and < 0.03 microM, respectively, together with the inhibition profile by propionic aldehyde (ID50 in the micromolar range) indicate that both compounds are oxidized primarily by the class I ALDH, further confirmed by low activity (0.4 U/g) with NADP+ as oxidant. By contrast, in human stomach, containing mostly class III ALDH, the activity measured with MONAL-71, 0.4 U/g, is much lower than that with MONAL-62 (5.1 U/g with NAD+ and 3.1 U/g with NADP+), the latter being virtually insensitive to 1 mM propionic aldehyde. Hence, in a stomach homogenate, class I and class III ALDH activities can be measured selectively with the two fluorogenic substrates described. In all experiments, the activity of aldehyde oxidase was at least 10-fold lower than that of the ALDH. Addition of 5 mM 4-methylpyrazole, a known inhibitor of the alcohol dehydrogenase, did not change the resultant ALDH activities by more than 10%, indicating lack of interference by the former enzyme. A preliminary screening of two liver tumour samples showed diminished class I ALDH activities (0.7 and 0.03 U/g), but no evidence for class III ALDH induction. The above observations are discussed in relation to the mechanism of detoxication of cyclophosphamide.

Nicotinamide riboside, an unusual, non-typical, substrate of purified purine-nucleoside phosphorylases.

Nicotinamide 1-beta-D-riboside (Nir), the cationic, reducible moiety of the coenzyme NAD+, has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The Km values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the Vmax values were comparable, 96% and 35% that for Ino. The pseudo first-order rate constants, Vmax/Km, were 1.1% and 2.5% for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP. Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with Ki values close to the Km for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7-methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E. coli PNP, with Ki values similar to those for inhibition of other natural substrates. The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates. The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme-inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.

Selective assay of the cytosolic forms of the aldehyde dehydrogenase in rat, with possible significance for the investigations of cyclophosphamide cytotoxicity.

Continuous fluorimetric assay for aldehyde dehydrogenase activity in rat tissue, based on oxidation of the fluorogenic 6-methoxy-2-naphthaldehyde, is described. The new assay is ca. 2 orders of magnitude more sensitive than the standard procedures and it is not subjected to interferences by other dehydrogenases, although occasionally the aldehyde dehydrogenase activity may be obscured by aldehyde oxidase. The new fluorimetric assay is highly selective for the cytosolic forms of the both rat and human aldehyde dehydrogenase, which are primarily responsible for cyclophosphamide inactivation in vivo. These forms can now be detected in crude homogenates of several rat organs without necessity of subcelluar fractionation.

Fluorescence emission properties of 8-azapurines and their nucleosides, and application to the kinetics of the reverse synthetic reaction of purine nucleoside phosphorylase.

An extensive study has been made of the fluorescence emission properties of the neutral and ionic forms in aqueous medium of the azapurine nucleosides, 8-azaadenosine (8-azaAdo), 8-azainosine (8-azaIno), 8-azaguanosine (8-azaGuo), and their aglycons. The fluorescence of 8-azaGuo at pH 7 originates from its anionic species (pKa = 8.05, phi= 0.55), as is also the case for 8-azaIno (pKa = 8.0, phi = 0.02), whereas 8-azaAdo is a strong emitter (phi = 0.06) as the neutral species. By contrast the corresponding free 8-azapurines are only weakly fluorescent in aqueous medium, with the exception of 8-azaguanine (8-azaG). Examination of the emission properties of N-substituted 8-azaguanines demonstrated that the observed blue emission of the neutral form of 8-azaG (phi = 0.05 to 0.33, dependent on lambda exc) originates from a minor tautomer of the compound, the N(8)-H form, present to the extent of 10-15%; while the principal N(9)-H tautomer is virtually nonfluorescent. The 8-azapurines are substrates of purine nucleoside phosphorylase (PNP), leading to their irreversible conversion to the corresponding nucleosides in the synthetic pathway of this enzyme. The fluorescent properties of these compounds, together with spectrophotometric methods, were applied to determine the basic kinetic parameters for synthesis of 8-azapurine nucleosides by PNP from mammalian (calf spleen) and bacterial (Escherichia coli) sources. The fluorimetric method was also used to determine the kinetic parameters for the second substrate, alpha-D-ribose 1-phosphate, and for the analytical titration of the latter in solution. The pH optimum of the reverse synthetic PNP reaction with 8-azapurines as substrates is below pH 7, due to their enhanced acidity in comparison with natural purines. The 8-azapurine nucleosides, but not their aglycons, are reasonably good inhibitors of phosphorolysis of Ino and Guo by E. coli PNP. The most effective is 8-azaIno (Ki approximately 20 microM), also the only one to inhibit phosphorolysis by the calf spleen enzyme (Ki approximately 40 microM). The nature of this inhibition is apparently uncompetitive.

[Class I and II alcohol dehydrogenase isoenzymes as biochemical markers of alcoholism]. / Izoenzymy I i II klasy dehydrogenazy alkoholowej (ADH) jako biochemiczne wskazniki alkoholizmu.

The activity of class I and II of alcohol dehydrogenase isoenzymes was tested in the sera of alcoholics using specific and fluorogenic substrates. Almost 3-fold increase of the activity of class I isoenzymes was found in developed alcoholism.

Human serum alcohol dehydrogenase isoenzymes as a markers of liver injury during viral hepatitis.

We examined the activity of class I and II of alcohol dehydrogenase isoenzymes in the sera of patients with viral hepatitis using fluorogenic substrates, 4-methoxy-1-naphthaldehyde for class I and 6-methoxy-2-naphthaldehyde for class II. It was found that serum activities of class I and II of alcohol dehydrogenase isoenzymes during five weeks of hospitalisation were higher than that of control. The greatest increase in activities was found at the onset of disease, and exceeded the mean control value about 30 times for class I and 4 times for class II. These were lower than the aminotransferase activities but higher than the activity of lactate dehydrogenase, alkaline phosphatase and gamma-glutamyltransferase. In the following periods of investigation the activity of alcohol dehydrogenase isoenzymes gradually decreased, but did not reach the values of the control groups in the last period of the study. Activity of class I and II of alcohol dehydrogenase isoenzymes showed a good correlation with alanine and aspartate aminotransferase and lactate dehydrogenase in the first weeks of the illness. These results clearly demonstrate that particularly the activity of class I of alcohol dehydrogenase isoenzymes measured by a fluorimetric method can be a useful marker of liver cell damage in the course of viral hepatitis.

Activity of class I and II alcohol dehydrogenase in the sera of alcoholics.

The activity of class I and II alcohol dehydrogenases in the sera of alcoholics was estimated using specific and highly sensitive fluorogenic substrates. It was found that serum class I activity was increased about 2 fold above that of the control group. No such increase was observed in the activity of class II, and we did not find a significant difference in non-alcoholics. The total alcohol dehydrogenase activity measured by classical spectrophotometric methods at pH 9.2 confirmed the increase of class I alcohol dehydrogenase. Additionally, the activity of liver injury markers were detected. Only gamma-glutamyltransferase activity exceeded the normal range. Based on the results of the present study, we would suggest that the significant elevated serum activity of class I alcohol dehydrogenase may be derived not from liver cells, because they did not display the obvious enzymatic markers, but from different organs, especially the gastrointestinal tract. The tissue distribution of alcohol dehydrogenase isoenzymes and gamma-glutamyltransferase would help to confirm this possibility.

Determination of human serum alcohol dehydrogenase using isozyme-specific fluorescent substrates.

Both class I and class II alcohol dehydrogenase (ADH) activities are present in human serum. The contribution of each class can be measured using two class-specific, fluorogenic substrates, 4-methoxy-1-naphthaldehyde and 6-methoxy-2-naphthaldehyde. The former is highly selective for class I isozymes, especially those containing alpha or gamma subunits, whereas class II (pi) ADH preferentially reduces the latter. Selective inhibition of class I ADH by 4-methylpyrazole further increases the specificity. Specificity, accuracy, and precision of the assay for serum measurements have been determined. The activity of class I ADH in normal human serum is below the limit of detection of this method, i.e., less than 1.0 nM/min. The activity of class II ADH in normal individuals is 15 +/- 5 nM/min. In some patients values as high as 2100 nM/min are observed for class I, but in all instances, the amount of class II found was higher than that of class I ADH.

Fluorometric assays for isozymes of human alcohol dehydrogenase.

Two new fluorogenic substrates for human alcohol dehydrogenase (ADH), 4-methoxy-1-naphthaldehyde (IA) and 6-methoxy-2-napthaldehyde (IIA), are described. The 2-naphthaldehyde derivative fluoresces in aqueous media with a quantum yield of 0.22 with an emission maximum at 450 nm, but the 1-naphthaldehyde shows only weak fluorescence. The corresponding alcohol reduction products, 4-methoxy-1-naphthalenemethanol (IB) and 6-methoxy-2-naphthalenemethanol (IIB), exhibit fluorescence in the near uv region with quantum yields of 0.36 and 0.26, respectively. The Km values for the individual homogenous class I ADH isozymes, with the above naphthaldehydes as substrates, range from 0.35 to 11.5 microM. The kappa cat values range from 70 to 610 min-1 and are thus comparable to those for the best ADH substrates. Except for the beta 1 beta 1 isozyme, IA is the preferred substrate for class I ADH isozymes while IIA is rapidly reduced by class II (pi-ADH). The sensitivity and specificity of the enzymatic assay with IA as substrate are demonstrated and provide the basis for the determination of class I ADH activity in human serum.

Properties of two unusual, and fluorescent, substrates of purine-nucleoside phosphorylase: 7-methylguanosine and 7-methylinosine.

The properties of two unusual substrates of calf spleen purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1), 7-methylguanosine and 7-methylinosine, are described. The corresponding bases, 7-methylguanine and 7-methylhypoxanthine, are neither substrates in the reverse, synthetic reaction, nor inhibitors of the phosphorolysis reaction. Both nucleosides exhibit fluorescence, which disappears on cleavage of the glycosidic bond, providing a new convenient procedure for continuous fluorimetric assay of enzymatic activity. For 7-methylguanosine at neutral pH and 25 degrees C, Vmax = 3.3 mumol/min per unit enzyme and Km = 14.7 microM, so that Vmax/Km = 22 X 10(-2)/min per unit as compared to 8 X 10(-2) for the commonly used substrate inosine. The permissible initial substrate concentration range is 5-100 microM. Enzyme activity may also be monitored spectrophotometrically. For 7-methylinosine, Vmax/Km is much lower, 2.4 X 10(-2), but its 10-fold higher fluorescence partially compensates for this, and permits the use of initial substrate concentrations in the range 1-500 microM. At neutral pH both substrates are mixtures of cationic and zwitterionic forms. Measurements of pH-dependence of kinetic constants indicated that the cationic forms are the preferred substrates, whereas the monoanion of inosine appears to be almost as good a substrate as the neutral form. With 7-methylguanosine as substrate, and monitoring of activity fluorimetrically and spectrophotometrically, inhibition constants were measured for several known inhibitors, and the results compared with those obtained with inosine as substrate, and with results reported for the enzyme from other sources.

Sensitive fluorimetric assay for adenosine deaminase with formycin as substrate; and substrate and inhibitor properties of some pyrazolopyrimidine and related analogues.

The nucleoside antibiotic formycin, 7-amino-3-(beta-D-ribofuranosyl)pyrazolo(4,3-d)pyrimidine, a structural analogue of adenosine, is deaminated about 10-fold faster by adenosine deaminase than adenosine itself, and is therefore a superior substrate for both routine assays and kinetic studies with the purified enzyme. The luminescence properties of formycin have been profited from to develop a fluorimetric assay for adenosine deaminase which is considerably more sensitive than the spectrophotometric procedure widely employed with adenosine as substrate. Examples are presented of its application to routine assays of adenosine deaminase levels in cellular extracts, as well as to kinetic studies with the purified enzyme, including the properties of some pyrazolopyrimidine and purine substrates and inhibitors.