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W J Stec

Publications and source records attributed to W J Stec.

At least 73 records · Page 4Linked to original sources
PubMed1984

Investigations on stimulation of lac transcription in vivo in Escherichia coli by cAMP analogues. Biological activities and structure-activity correlations.

The ability of 24 systematically modified analogues of adenosine 3',5'-monophosphate (cAMP) to enhance the synthesis of beta-galactosidase in glucose-repressed Escherichia coli strains KNBL 1001 and cpd- Crookes has been investigated. The properties of the analogues in comparison with cAMP are, with only two exceptions, alike in both strains. Two analogues, 7-deazaadenosine 3',5'-monophosphate (i.e. tubercidin 3',5'-monophosphate) and (Rp)-adenosine 3',5'-monothionophosphate, exhibit higher biological activity than cAMP. The latter analogue is 50-fold more active in both strains. Three analogues showed activities comparable to cAMP, four analogues were less active and 12 analogues were unable to antagonize catabolite repression. Structure-activity correlations showed that the 2'OH-, 3'O-, 5'O-, the negative charge and the 6-amino group cannot be modified without losing biological activity in vivo, while the N-1 and N-7 in adenine are not essential. The interaction with the catabolite gene activator protein is stereoselective for an unmodified axial exocyclic oxygen. The results are compared to those obtained with cAMP analogues in E. coli in vitro and those obtained with the same analogues in protein-kinase systems and Dictyostelium species. The model of McKay et al. [McKay, D.B., Weber, J.T. and Steitz, T.A. (1982) J. Biol. Chem. 257, 9518-9524] proposed for distinct chemical interactions of cAMP with the catabolite gene activator protein is discussed and supplemented by additional hydrogen bond interactions.

Cyclic AMP↗
PubMed1983

Inhibition of glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate.

The diastereomeric forms of adenosine cyclic 3',5'-phosphorothioate, Rp cAMPS and Sp cAMPS, were studied in isolated hepatocytes from fed rats for their ability to interact with the intracellular cAMP-dependent protein kinase and to affect the phosphorylase kinase-phosphorylase glycogenolytic cascade. Incubation of the cells with increasing concentrations of Sp cAMPS produced a concentration-dependent activation of cAMP-dependent protein kinase with a concomitant increase in the glycogenolytic rate. Half-maximal and maximal velocities of glycogenolysis were reached at 8 X 10(-7) and 1 X 10(-5) M Sp cAMPS, respectively. Incubation of the cells with 10(-9) to 10(-4) M Rp cAMPS had no effect on basal glucose production or on cAMP-dependent protein kinase activity. Incubation of the cells simultaneously with 3 X 10(-6) M Sp cAMPS and increasing concentrations of Rp cAMPS produced half-maximal inhibition of glycogenolysis at 1 X 10(-5) M Rp cAMPS and maximal inhibition at 1 X 10(-4) M. The concentrations of Sp cAMPS required for half-maximal and maximal activation of glycogenolysis were increased 10-fold when 1 X 10(-5) M Rp cAMPS was present. These data imply that Sp cAMPS is a cAMP-agonist while Rp cAMPS is a cAMP-antagonist.

Animals↗
PubMed1983

Transport and metabolism of N6- and C8-substituted analogs of adenosine 3',5'-cyclic monophosphate and adenosine 3'5'-cyclic phosphorothioate by the isolated perfused rat kidney.

The clearance and metabolism of N6-substituted (N6-dimethyl-), C8-substituted (8-bromo-, 8-p-chlorophenylthio- (PCPT-)), and exocyclic oxygen substituted phosphorothioate diastereomers (cAMPS(Sp)) and cAMPS (Rp)) of adenosine 3':5'-monophosphate (cyclic AMP, cAMP) has been studied in an isolated perfused rat kidney. The N6- and C8-substituted analogs of cyclic AMP (10-100 microM) were not cleared as rapidly as exogenous cyclic AMP and were metabolized: N6- and C8-substituted analogs of adenosine accumulated in perfusate and urine. All analogs exhibited net transtubular secretion, i.e. their urinary excretion rate greater than glomerular filtration rate. Probenecid (0.9 mM) included in the perfusate abolished transtubular secretion and inhibited the metabolism of PCPT-cyclic AMP, suggesting that cyclic AMP analogs, like cyclic AMP itself, penetrate the renal cell at the peritubular membrane by an organic acid transport system. The phosphorothioate diastereomers of cyclic AMP: cAMPS(Sp) and cAMPS(Rp) were cleared as rapidly from the perfusate as cyclic AMP, were extensively secreted (urinary excretion/ glomerular filtration greater than or equal to 10) and exhibited no metabolism. The latter analog would seem most suitable as an intracellular agonist for cyclic AMP-mediated phenomena in the rat kidney.

Animals↗
PubMed1983

Stereospecific synthesis of chiral metabolites of ifosfamide and their determination in the urine.

The stereospecific synthesis of two chiral metabolites of ifosfamide (2), 4-ketoifosfamide (5) and 2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3, 2-oxazaphosphorine 2-oxide (9), is reported. The absolute configuration of both compounds was assigned on the basis of chemical correlation. In addition, two other achiral metabolites of 2, carboxyifosfamide (6) and IPAM (7), were synthesized. These and other organophosphorus metabolites of ifosfamide were found, by 31P NMR, in the urine of patients to whom racemic 2 was administered. The measurements performed in the presence of optically active lanthanide shift reagent [Eu(tfc)3] showed considerable stereoselectivity of in vivo formation of some chiral metabolites of ifosfamide.

Cyclophosphamide↗
PubMed1983

Comparative study on the immunosuppressive and lympholytic activity of optical isomers of cyclophosphamide.

The influence of chirality of cyclophosphamide (Cy) on the reactivity of lymphoid cells in vivo was studied in Balb/c mice. It has been shown that (+) Cy inhibits the formation of direct PFC in Balb/c mice more effectively than (-) Cy. The augmentation of DTH--reaction against HuRBC in mice, measured by the foot-pad test and the elongation of graft survival time were noted when racemic or (-) Cy were applied. Investigated forms of Cy decreased the number of nucleated cells in mice spleens, but lympholytic effect of (+) Cy was lower than that of racemic and (-) Cy. The possible consequences of stereo-differentiated immunosuppressive activity of enantiomeric forms of cyclophosphamide in the experimental procedures and the therapy of "autoimmune" and malignant diseases are considered.

Animals↗
PubMed1982

A kinetic study of interactions of (Rp)- and (Sp)-adenosine cyclic 3',5'-phosphorothioates with type II bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase.

The stereoselectivity of the adenosine cyclic 3',5'-phosphate (cAMP) binding sites on the regulatory subunit of the type II bovine cardiac muscle cAMP-dependent protein kinase was investigated by examining the interactions of (Rp)- and (Sp)-adenosine cyclic 3',5'-phosphorothioates (cAMPS) with these sites. While activation of the holoenzyme and binding to the regulatory subunit of the type II kinase were observed for both of these diastereomers, there were significant differences between the interactions of the cAMPS isomers with the enzyme. In particular, the Sp isomer is more potent than the Rp species not only in the activation of reconstituted, as well as directly isolated, holoenzyme but also in the inhibition of [3H]cAMP binding to the regulatory subunit. A marked preference for the binding of the Sp isomer to site 2 in the regulatory subunit exists. Hydrogen bonding of a functional group on the regulatory subunit with preferential orientation toward the exocyclic oxygen rather than the sulfur of the thiophosphoryl residue may be involved in the observed selectivity of cAMPS binding and activation. In addition to our findings on the stereoselectivity of the binding of cAMPS to cAMP-dependent protein kinase, we have established a method for the reconstitution of holoenzyme from the purified subunits without subjecting the regulatory protein to denaturing conditions.

Binding Sites↗
PubMed1982

Stereochemical course of nucleotidyl transfer catalyzed by bacteriophage T7 induced DNA polymerase.

The bacteriophage T7 induced DNA polymerase, consisting of the phage specified gene 5 protein associated with Escherichia coli thioredoxin, catalyzes the copolymerization of SP-dATP alpha S with dTTP, producing the alternating of polymer poly[dTs-A)] by a mechanism involving inversion of configuration at P alpha. Degradation of poly[d(5s-A)] by the nucleolytic action of E. coli DNA polymerase produced the dinucleotide pdTps-dA, whose configuration at the phosphorothioate diester was assigned as R by comparison of the phosphorus-31 nuclear magnetic resonance chemical shift (55.0 ppm downfield from H3PO4) with that of an authentic sample. Further degradation by alkaline phosphatase to Rp-dTps-dA (55.6 ppm downfield from H3PO4) confirmed the configuration. The stereochemistry provides no evidence of a double displacement mechanism.

DNA-Directed DNA Polymerase↗
PubMed1982

A stereochemical investigation of the hydrolysis of cyclic AMP and the (Sp)-and (Rp)-diastereoisomers of adenosine cyclic 3':5'-phosphorothioate by bovine heart and baker's-yeast cyclic AMP phosphodiesterases.

Bovine heart cyclic AMP phosphodiesterase, which has a requirement for Mg2+, hydrolyses cyclic AMP with inversion of configuration at the phosphorus atom, but only the (Sp)-diastereoisomer of adenosine cyclic 3':5'-phosphorothioate is hydrolysed by this enzyme. By contrast, the low-affinity yeast cyclic AMP phosphodiesterase, which contains tightly bound Zn2+, hydrolyses both the (Sp)- and the (Rp)-diastereoisomers of adenosine cyclic 3':5'-phosphorothioate, the (Rp)-diastereoisomer being the preferred substrate under V max. conditions. Both of the diastereoisomers of adenosine cyclic 3':5'-phosphorothioate, as well as cyclic AMP, are hydrolysed with inversion of configuration at the phosphorus atom by the yeast enzyme. It is proposed that, with both enzymes, the bivalent metal ion co-ordinates with the phosphate residue of the substrate, and that hydrolysis is catalysed by a direct "in-line' mechanism.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Interaction of cAMP derivatives with the 'stable' cAMP-binding site in the cAMP-dependent protein kinase type I.

cAMP binding to the 'stable' cAMP-binding sites in the regulatory subunit of the cAMP-dependent protein kinase type I was investigated using a set of 18 selected derivatives. All the tested analogues were competitive with [3H]cAMP and inhibitor constants from 12 nM to 20 microM with the free regulatory subunit were determined. The cAMP molecule seemed to be bound by these specific hydrogen bonds to the 5' and 3' oxygen, the 2' hydroxyl, and an ion pair interaction between the negative charge in equatorial position and a positively charged amino acid side chain. The adenine base is rather unspecifically bound with no hydrogen bonds involved. This binding specificity of the 'stable' site is similar to the requirement for dissociation as determined by the activation of the kinase by a respective analogue. This indicates that occupation of the 'stable' sites leads to activation of the protein kinase. The presence of the catalytic subunit reduced the affinity of most analogues. The binding of one derivative with the negative charge fixed in the axial position is not influenced by the addition of the catalytic subunit and ATP. A plausible model for a conformational change during the activation process in the 'stable' site is discussed.

Adenosine Triphosphate↗
PubMed1981

Specificity of cyclic GMP activation of a multi-substrate cyclic nucleotide phosphodiesterase from rat liver.

Cyclic nucleotide derivatives have been used as a tool to investigate the existence of distinctive activating and hydrolytic sites on the phosphodiesterase from rat liver activated by cGMP (guanosine 3',5'-monophosphate). This positively cooperative enzyme was stimulated up to 30-fold by 3 microM cGMP when 3 microM cAMP (adenosine 3',5'-monophosphate) was used as substrate. All analogues were less potent activators than cGMP. Most cAMP derivatives were inactive, with two exceptions: 7-deazaadenosine 3',5'-monophosphate and 3'-amino-3'-deoxy-adenosine 3',5'-monophosphate. Benzimidazole ribonucleoside 3',5'-monophosphate, where the two atoms of nitrogen of the pyrimidine ring are missing was a better stimulator than the intact purine-related cyclic derivative. When cAMP and cGMP with identical chemical ligands substituted at the same position were compared, the cGMP analogue was always the more potent activator suggesting that the activating site is sensitive to a guanine-type cyclic nucleotide structure. Degradation of the derivatives by the enzyme was measured by high-performance liquid chromatography: no relation could be established between hydrolysis and effectiveness of activation. In addition, there was no parallelism between inhibitory and activating potency for ten cyclic nucleotide derivatives. Since the chemical interactions between the analogues at the activating site on the one hand and at the catalytic site on the other, are different, it is proposed that the sites are distinct. Consequently, it is suggested that the enzyme operates in steps. In the first activating step, cGMP is fixed by at least two hydrogen bonds at a specific binding site of the enzyme. This is followed by a conformational change of the protein and subsequently a change of the kinetic parameters. In a rather unspecific process and in a second hydrolytic step, several purine-related cyclic nucleotides are converted to the corresponding 5' nucleotides.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Stereochemistry of hydrolysis of adenosine 3':5'-cyclic phosphorothioate by the cyclic phosphodiesterase from beef heart.

Adenosine 3':5'-cyclic phosphorothioate, Sp-diastereomer was hydrolyzed by cyclic phosphodiesterase from beef heart in the presence of [18O]water to [18O]adenosine 5'-phosphorothioate. This was phosphorylated by myokinase and pyruvate kinase to [18O]adenosine 5'-(1-thiotriphosphate),Sp-diastereomer. The position of 18O was determined to be in a nonbridging position. This result indicates that the hydrolysis proceeded with inversion of configuration at phosphorus.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Comparative metabolism of 2-[bis(2-chloroethyl)amino]tetrahydro-2-H-1,3,2-oxazaphosphorine-2-oxide (cyclophosphamide) and its enantiomers in humans.

The comparative metabolism of the enantiomers of cyclo phosphamide and of the racemate has been studied in humans. Four patients were each given, sequentially, the racemate, the (+)-enantiomer, and its (-)-antipode. The plasma levels of parent drug and the urinary output (24 hr) of unchanged drug and of two enzymatically produced metabolites, 4-ketocyclophosphamide and carboxyphosphamide, were determined using mass spectrometry-stable isotope dilution. There was no significant difference between the three forms of cyclophosphamide with respect to plasma half-life (beta phase) or in the urinary outputs of the drug or of carboxyphosphamide. The output of 4-ketocyclophosphamide after administration of (+)-cyclophosphamide was significantly greater than that produced from the racemate. Cyclophosphamide recovered from the urine of patients given the racemate was either racemic or only slightly enriched in the (-)-enantiomer. The two enantiomers were almost equally bound to plasma protein. Based on these metabolic studies alone, there is little reason to predict that the enantiomers will differ from each other or from the racemate in their therapeutic effects in humans, but there are other factors, e.g., stereoselective uptake of the intermediary 4-hydroxylated metabolites by neoplastic cells, which could elicit such differences.

Aged↗