The adriamycin-iron(III) complex is a potent inhibitor of protein kinase C

Using Triton X-100/lipid mixed micellar methods, we observed that the adriamycin-iron(III) complex was a potent inhibitor of protein kinase C while uncomplexed adriamycin itself was a poor inhibitor in the absence of heavy metal contaminants. The 3:1 adriamycin-iron complex was more potent than 2:1, 1:1, and 1:0 complexes. Inhibition of protein kinase C was reversible, and 50% inhibition occurred at 13 microM (adriamycin)3Fe3+. Both the catalytic and the regulatory domain of protein kinase C were affected by adriamycin-iron(III). Adriamycin-iron(III) was a competitive inhibitor of the catalytic domain of protein kinase C with respect to MgATP but not with respect to magnesium (IC50 350 microM). The predominant interaction of adriamycin-iron(III) with native protein kinase C was as a competitive inhibitor with respect to diacylglycerol. Inhibition was not competitive with respect to phosphatidylserine, calcium, magnesium, MgATP, or histone. Interaction with the regulatory domain was demonstrated by the ability of adriamycin-iron(III) to inhibit phorbol dibutyrate binding. Other adriamycin transitional metal complexes showed little inhibition of protein kinase C activity. Acetylation of the amine on the daunosamine moeity of adriamycin did not preclude the formation of a ferric complex but resulted in total loss of inhibitory activity. These results suggest that the presence of free amines in a highly structured adriamycin-iron complex is necessary for inhibition. The implications of inhibition of protein kinase C by adriamycin-iron(III) are discussed.     

Organothallium(III) reagents for modification of biomacromolecules: interaction of thallium derivatives with transfer RNA

As an extension of work on the inhibition of enzymes by arylthallium(III) reagents, the thallium analogues of the organomercurials, we have studied the interactions of these molecules with transfer RNA. In contrast to thallous acetate, thallium(III) derivatives (thallic trifluoroacetate, p-methylphenylthallium(III) bis-trifluoroacetate (MPT) and o-carboxyphenylthallium(III) bis-trifluoroacetate) bound to Escherichia coli tRNA. The interaction was fully reversible upon Sephadex G-25 gel filtration, and binding constants and stoichiometries were evaluated by a number of procedures. The likely site of interaction was shown to be the thiouridine residue (s4U8) based on changes induced by MPT on the absorbance around 330 nm. No changes in stacking interactions could be detected from the absorption or circular dichroic spectra. The detailed structure of the groups on thallium(III) affected the interaction with tRNA. Thalliation at s4U8 affects the absorbance at 335 nm and the amino-acid uptake capacity of E. coli tRNAPhe in parallel, the latter being progressively inhibited by increasing amounts of MPT. In a model nucleoside system, uridine disulphide is probably formed from reduced thiouridine by the oxidative action of the Tl(III) reagents. No evidence of cross-linking of E. coli tRNA molecules under gel electrophoretic conditions was obtained in contrast to the model nucleoside. The easily reversible interaction of MPT with sulphur sites in E. coli tRNA contrasts with the stable (to gel filtration) bonds formed between MPT and (thiol) sites in enzymes.     

Organothallium(III) reagents for modification of biomacromolecules: irreversible labelling of phosphoglycerate kinase from rabbit muscle

Organothallium(III) reagents, by analogy with organomercurials, have been found to rapidly label phosphoglycerate kinase from rabbit muscle. By use of a radio-labelled version of p-methylphenylthallium(III) bis-trifluoroacetate (MPT) the inhibition was shown to be irreversible by the criterion of gel filtration desalting. The rate of labelling was shown to depend on the temperature, enzyme and thallium reagent concentrations, and the presence or absence of the various substrates of the enzyme. The structure and oxidation state of the thallium reagent used affected the extent of modification by the compounds MPT, o-carboxyphenylthallium(III) bis-trifluoroacetate, thallic trifluoroacetate and thallous acetate. A number of pieces of evidence implicate cysteine residues in the labelling, including changes in the free thiol titre of the enzyme on thalliation, model studies on the interaction of thiols (e.g. glutathione) with thallium(III) and thallous materials, the lack of inactivation of phosphoglycerate kinase from yeast (which has only one thiol residue distant from the active site), and the partial restoration of enzymic activity by treatment of thalliated enzyme with sulphydryl reducing agents. Substrate protection studies showed that modification of rabbit muscle phosphoglycerate kinase by MPT was fully prevented by 3-phosphoglycerate and partially by MgATP. The latter protected only against the fast phase of thallic modification, the slower phase being unaffected. The presence of MgADP potentiated the labelling by MPT. No evidence of an MgADP-induced conformational change in the enzyme could be obtained from fluorescence or circular dichroic spectroscopies, although changes of the native spectra were noted on thalliation by MPT alone. The cross-linking potential of these arylthallium(III) reagents is discussed along with conformational changes required to trigger the hinge-movement between the N- and C-domains of the protein.     

Comparative kinetic study of D-glucose oxidation by ruthenium(III) compounds catalyzed by FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase

The comparative kinetic study of two glucose oxidizing enzymes, FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase, is presented in the artificial electron transfer mediator system based on ruthenium(III) compounds. It is demonstrated that FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase follow Michaelis kinetics in the D-glucose/ruthenium(III) system. PQQ-dependent glucose dehydrogenase is more active than FAD-dependent glucose oxidase in the process of D-glucose oxidation by ruthenium(III) compounds, this being due to the different catalytic mechanisms of these enzymes.     

Cobalt(III) affinity-labeled aspartokinase. Formation of substrate and inhibitor adducts.

The kinase active site of the aspartokinase-homoserine dehydrogenase enzyme complex of Excherichia coli has been affinity labeled both with substrates aspartate and adenosine triphosphate and feedback inhibitor threonine. Co(III) exchange-inert adducts of aspartokinase and inhibitor or substrates were produced in situ by oxidation of Co(II) with H2O2. Emzyme-Co(III)-adenosine 5'-triphosphate (ATP), enzyme-Co(III)-aspartate, and enzyme-Co(III)-threonine ternary adducts were produced in this manner. The formation of the enzyme-Co(III)-threonine adduct leads us to conclude that threonine inhibits the kinase activity of this enzyme complex by binding in the first coordination sphere of the catalytic metal ion cofactor, a conclusion which is consistent with evidence derived from previous nuclear magnetic resonance data obtained in this laboratory. The quaternary adducts formed by H2O2 oxidation in the presence of aspartokinase, Co(II), ATP, aspartate, and threonine comprised a mixture of both ezyme-Co(III)-ATP-aspartate and enzyme-Co(III)-ATP-threonine adducts. The formation of the quaternary aspartate-containing adduct was unexpected, since the presence of threonine was expected to prevent access of the aspartate to the active site; most significantly however, the the sum of the numbers of aspartate plus threonine molecules incorporated per active site is one. We believe that this shows direct steric overlap between the metal-adjacent binding sites for aspartate and threonine. Aspartate or threonine can not occupy the kinase active site simultaneously; this conclusion is consistent with the direct competitive inhibition of aspartate by threonine observed in steady-state kinetic studies.     

The in situ inhibition of prothrombinase-formed human alpha-thrombin and meizothrombin(des F1) by antithrombin III and heparin

The inhibition of thrombin by antithrombin III (AT III) and heparin has been studied in pure systems to determine the kinetics of inhibition during human prothrombin activation. The present study shows that prothrombinase-catalyzed prothrombin activation resulted in the generation of thrombin and meizothrombin(des F1). In the absence of heparin the second-order rate constants of the inactivation of both thrombin and meizothrombin(des F1) formed in the reaction mixture appeared to be identical, k = 3.7 X 10(5) M-1 min-1. The rate constant of inhibition of purified thrombin was 6.5 X 10(5) M-1 min-1. In the presence of heparin the decay of the amidolytic activity was biexponential and could be modeled by a four-parameter equation to determine the pseudo first-order rate constants of inhibition as well as the composition of the reaction with respect to the levels of thrombin and meizothrombin(des F1). The ratio of thrombin over meizothrombin(des F1) varied with the initial prothrombin concentration. Heparin catalyzed the AT III inhibition of thrombin but not meizothrombin(des F1) formed during the prothrombin activation. Thrombin, generated by (Xa-Va-phospholipid-Ca2+) was inhibited by AT III/heparin more slowly than purified thrombin, and the saturation kinetics of the inhibition with respect to AT III differed from those found with purified thrombin.     

Kinetic studies on the interaction of gold (III) with nucleic acids. IV. RNA-Au (III) system.

The kinetics of the interaction of Au(III) with whole yeast RNA has been studied using UV-spectrophotometry. The reaction is second order with respect to the nucleotide unit of RNA and first order with respect to Au(III) in the respective stoichiometry of 2 : 1. The effects of initial composition, temperature, ionic strength, pH and chloride ion on the kinetics have been studied. Activation energy is found to be 11.5 kcal/mol. Effect of ionic strength indicates that both the positively charged and neutral species of Au(III) take part in the rate limiting step, the former being dominant at low ionic strength. A plausible mechanism has been proposed which involves the interaction of two nucleotide units of RNA with one species of Au(III) in the rate limiting step.     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Kinetics of Cr(III) and Cr(VI) removal from water by two floating macrophytes

The aim of this work was to compare Cr(III) and Cr(VI) removal kinetics from water by Pistia stratiotes and Salvinia herzogii. The accumulation in plant tissues and the effects of both Cr forms on plant growth were also evaluated. Plants were exposed to 2 and 6 mg L^?1 of Cr(III) or Cr(VI) during 30 days. At the end of the experiment, Cr(VI) removal percentages were significantly lower than those obtained for Cr(III) for both macrophytes. Cr(III) removal kinetics involved a fast and a slow component. The fast component was primarily responsible for Cr(III) removal while Cr(VI) removal kinetics involved only a slow process. Cr accumulated principally in the roots. In the Cr(VI) treatments a higher translocation from roots to aerial parts than in Cr(III) treatments was observed. Both macrophytes demonstrated a high ability to remove Cr(III) but not Cr(VI). Cr(III) inhibited the growth at the highest studied concentration of both macrophytes while Cr(VI) caused senescence. These results have important implications in the use of constructed wetlands for secondary industrial wastewater treatment. Common primary treatments of effluents containing Cr(VI) consists in its reduction to Cr(III). Cr(III) concentrations in these effluents are normally below the highest studied concentrations in this work.     

Inhibition of the bovine branched-chain 2-oxo acid dehydrogenase complex and its kinase by arylidenepyruvates

A novel class of inhibitors for the branched-chain 2-oxo acid dehydrogenase (BCOAD) complex has been synthesized and studied. The sodium salts of arylidenepyruvates: e.g., furfurylidenepyruvate (compound I), 4-(3-thienyl)-2-oxo-3-butenoate (compound II), cinnamalpyruvate (compound III) and 4-(2-thienyl)-2-oxo-3-butenoate (compound IV) inhibit the overall and kinase reactions of the BCOAD complex from bovine liver. Inhibitions of the overall reaction occur at the decarboxylase (E1) step as determined by a spectrophotometric assay with 2,6-dichlorophenolindophenol as an electron acceptor. Inhibition of the E1 reaction by compound I (Ki = 0.5 microM) is competitive, whereas inhibitions by compounds II (Ki = 150 microM) and III (Ki = 500 microM) are non-competitive with respect to the substrate 2-oxoisovalerate. The Km value for 2-oxoisovalerate is 6.7 microM as measured by the E1 assay. Inhibition of the E1 step by compounds I, II and III are reversible at low inhibitor concentrations based on the Michaelis-Menten kinetics observed. By comparison, compound I does not significantly inhibit pyruvate and 2-oxoglutarate dehydrogenase complexes. The arylidenepyruvates (compounds I, II and IV) inhibit the BCOAD kinase reaction in a manner similar to the substrate 2-oxo acids. The inhibition of the kinase reaction by compound I is non-competitive with respect to ATP, with an apparent Ki value of 4.5 mM. The results suggest that arylidenepyruvates may be useful probes for elucidating the reaction mechanisms of the BCOAD complex and its kinase.     

Crystal structures of hen egg-white lysozyme complexes with gadolinium(III) and gadolinium(III)-N-acetyl-D-glucosamine.

Analysis at 0.25 nm resolution of the crystal structures of lysozyme-Gd(III) and lysozyme-Gd(III)-N-acetyl-D-glucosamine (GlcNac), prepared by diffusion methods, show that there are two main binding positions for Gd(III), one of which is close to glutamic acid-35 and the other close to aspartic acid-52. The two sites are 0.36 nm part. There is no evidence for the weak binding of Gd(III) to any of the eight other carboxy groups of lysozyme. In the presence of Gd(III), the binding of GlcNac is similar to that observed for the binding of the beta-anomer in subsite C. There are numerous small conformational changes in the protein on binding (Gd(III) and the sugar, and these have been quantified to a first approximation by real-space refinement. These changes are similar in both structures, and involve, among other small movements, shifts of one of the disulphide bridges by up to 0.05 nm. The movement of residues 70--74 observed in the binary complex of lysozyme-GlcNac [Perkins, Johnson, Machin & Phillips (1978) Biochem. J. 173-617] is not observed in the ternary complex of lysozyme-Gd(III)-GlcNac. The nature of the lysozyme-Gd(III) complex is discussed in the light of evidence from other crystallographic studies and n.m.r. solution studies. Preliminary findings for a lysozyme-Gd(III) complex prepared by co-crystallization methods are reported.     

Reactions of Azotobacter vinelandii nitrogenase using Ti(III) as reductant

Nitrogenase-catalyzed reactions using Ti(III) were examined under a wide variety of conditions to determine the suitability of Ti(III) to serve as a general nitrogenase reductant. Solutions prepared from H2-reduced TiCl3, aluminum-reduced TiCl3, TiCl2, evaporated TiCl3 from an HCl, solution, and TiF3 were evaluated as reductants. Three general types of reactivity were observed. The first showed that, below Ti(III) concentrations of about 0.50 mM, nitrogenase catalysis utilized Ti(III) in a first-order reaction. The second showed that, above 0.50 mM, the rate of nitrogenase catalysis was zero order in Ti(III), indicating the enzyme was saturated with this reductant. Above 2.0-5.0 mM, nitrogenase catalysis was inhibited by Ti(III) depending on the titanium source used for solution preparation. This inhibition was investigated and found to be independent of the buffer type and pH, while high salt and citrate concentrations caused moderate inhibition. [Ti(IV)] above 2.0-3.0 mM and [Ti(III)] above about 5.0 mM were inhibitory. ATP/2e values were 4-5 for [Ti(III)] at or below 1.0-2.0 mM, 2.0 from 5.0 to 7.0 mM Ti(III) where nitrogenase is not inhibited, and 2.0 above 7.0 mM Ti(III) where severe inhibition occurs. For nitrogenase-catalyzed reactions using Ti(III) as reductant, the potential of the solution changes with time as the Ti(III)/Ti(IV) ratio changes. From the change in the rate of product formation (Ti(III) disappearance) with change in solution potential, the rate of nitrogenase catalysis was determined as a function of solution potential. From such experiments, a midpoint turnover potential of -480 mV was determined for nitrogenase catalysis with an associated n = 2 value.     

Inhibition of several enzymes by gold compounds. II. beta-Glucuronidase, acid phosphatase and L-malate dehydrogenase by sodium thiomalatoraurate (I), sodium thiosulfatoaurate (I) and thioglucosoaurate (I)

Bovine liver beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32), wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) and bovine liver L-malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) were inhibited by a series of gold (I) complexes that have been used as anti-inflammatory drugs. Both sodium thiosulfatoaurate (I) (Na AuTs) and sodium thiomalatoraurate (NaAuTM) effectively inhibited all three enzymes, while thioglucosoaurate (I) (AuTG) only inhibited L-malate dehydrogenase. The equilibrium constants (K1) ranged from nearly 4000 microM for the NaAuTM-beta-glucuronidase interaction to 24 microM for the NaAuTS-beta-glucuronidase interaction. The rate of covalent bond formation (kp) ranged from 0.00032 min-1 for NaAuTM-beta-glucuronidase formation to 1.7 min-1 for AuTG-L-malate dehydrogenase formation. The equilibrium data shows that the gold (I) drugs bind by several orders lower than the gold (III) compounds, suggesting a significantly stronger interaction between the more highly charged gold ion and the enzyme. Yet the rate of covalent bond formation depends as much on the structure of the active site as upon the lability of the gold-ligand bond. It was also observed that the more effective the gold inhibition the more toxic the compound.     

Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles.

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.     

Reduction of Fe(III) ions complexed to physiological ligands by lipoyl dehydrogenase and other flavoenzymes in vitro: implications for an enzymatic reduction of Fe(III) ions of the labile iron pool

Enzymatic reduction of physiological Fe(III) complexes of the "labile iron pool" has not been studied so far. By use of spectrophotometric assays based on the oxidation of NAD(P)H and formation of [Fe(II) (1,10-phenanthroline)3]2+ as well as by utilizing electron paramagnetic resonance spectrometry, it was demonstrated that the NAD(P)H-dependent flavoenzyme lipoyl dehydrogenase (diaphorase, EC 1.8.1.4) effectively catalyzes the one-electron reduction of Fe(III) complexes of citrate, ATP, and ADP at the expense of the co-enzymes NAD(P)H. Deactivated or inhibited lipoyl dehydrogenase did not reduce the Fe(III) complexes. Likewise, in the absence of NAD(P)H or in the presence of NAD(P)+, Fe(III) reduction could not be detected. The fact that reduction also occurred in the absence of molecular oxygen as well as in the presence of superoxide dismutase proved that the Fe(III) reduction was directly linked to the enzymatic activity of lipoyl dehydrogenase and not mediated by O2. Kinetic studies revealed different affinities of lipoyl dehydrogenase for the reduction of the low molecular weight Fe(III) complexes in the relative order Fe(III)-citrate > Fe(III)-ATP > Fe(III)-ADP (half-maximal velocities at 346-485 microm). These Fe(III) complexes were enzymatically reduced also by other flavoenzymes, namely glutathione reductase (EC 1.6.4.2), cytochrome c reductase (EC 1.6.99.3), and cytochrome P450 reductase (EC 1.6.2.4) with somewhat lower efficacy. The present data suggest a (patho)physiological role for lipoyl dehydrogenase and other flavoenzymes in intracellular iron metabolism.     

Properties of a complex of Fe(III)-soybean lipoxygenase-1 and 4-nitrocatechol

Fe(III)-soybean lipoxygenase-1 yields with 4-nitrocatechol a green coloured 1 : 1 complex, which shows at pH 7.0 absorption maxima at 385 nm and 650 nm. The formation of this complex is reversible. The circular dichroism spectrum of the complex of Fe(III)-lipoxygenase-1 and 4-nitrocatechol has a positive band at around 380 nm and a negative band at around 450 nm and is significantly different from that of the Fe(III)-enzyme as such. 4-Nitrocatechol can be displaced from the green complex by 13-L-hydroperoxy-cis-9, trans-11-octadecadienoic acid, resulting in the formation of the blue complex between the Fe(III)-enzyme and 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid both under aerobic and anaerobic conditions. Also linoleic acid competes with 4-nitrocatechol for the binding site on the Fe(III)-enzyme, as can be demonstrated under anaerobic conditions, ultimately leading to reduction of the Fe(III)-enzyme. The oxygenation of linoleic acid by Fe(III)-lipoxygenase-1 is inhibited by 4-nitrocatechol. From steady-state kinetics a non-competitive inhibition pattern is obtained. Probably it has to be considered as pseudo non-competitive because of the slow establishment of the complex equilibrium. An inhibition constant (K4NC) of 16.3 microM is found. On prolonged incubation of Fe(III)-lipoxygenase-1 and 4-nitrocatechol the green complex converts into a brown species. This conversion is found to be coupled with a change in the nature of the inhibition from reversible to irreversible. A complex between native lipoxygenase-1 and 4-nitrocatechol is found to be unlikely.     

Effects of chromium(III) on DNA replication in vitro

A number of metal compounds are important environmental carcinogens; however, the molecular mechanisms of metal-induced genotoxicity are not yet understood. Chromium, for example, is substantially mutagenic in vivo and has been shown to decrease the DNA replication fidelity in vitro. But the mechanism of chromium-induced mutagenesis is unkown and the role of replication fidelity in chromium-induced carciogenesis is unclear. We have used in vitro DNA replication assays to investigate the effects of chromium ions on DNA polymerase activity preliminary to studying their role in chromium-induced mutagenesis. Biologically active M13mp2 DNA was replicated with purified DNA polymerases in the presence of micromolar amounts of chromium with or without the normal divalent cation, magnesium. Nucleotide incorporation kinetics were determined and sequence specific pausing was analyzed by primer-extension. Our results have demonstrated an unexpected polymerase activation by low (0.5–5.0 μm) concentrations of chromium(III) although higher concentrations of chromium are increasingly inhibitory. The increased incorporation seem at low chromium(III) concentrations is the result of increased enzyme processivity and is not polymerase specific. The possible relationship between processivity and metal-ion mutagenesis is discussed.     

Aluminium(III), chromium(III) and iron(III) complexes with 5-iodouracil and 5-iodouracil-histidine and their antitumour activity

Complexes of the type [Al(HL)(OH)Cl(2)], [M(HL)(OH)(2)Cl] and [M'(HL)(L')(OH)Cl], where HL = 5-iodouracil; HL' = histidine; M = Cr(III), Fe(III) and M' = Al(III), Cr(III), Fe(III), were synthesized and characterized. The complexes are polymeric showing high decomposition points and are insoluble in water and common organic solvents. The mu(eff) values, electronic spectral bands and ESR spectra suggest a polymeric 6-coordinate spin-free octahedral stereochemistry for the Cr(III) and Fe(III) complexes. 5-Iodouracil acts as a monodentate ligand coordinating to the metal ion through the O atom of C((4)) = O while histidine through the O atom of -COO(- ) and the N atom of -NH(2) group. In vivo antitumour effect of 5-iodouracil and its complexes was examined on C(3)H /He mice against P815 murine mastocytoma. As evident from their T/C values, Cr(III) and Fe(III) complexes display significant and higher antitumour activity compared to the 5-iodouracil ligand. The in vitro results of the complexes on the same cells indicate that Cr(III) and Fe(III) complexes show higher inhibition on (3)H-thymidine and (3)H-uridine incorporation in DNA and RNA replication, respectively, at a dose of 5 microg/mL.     

Paradoxical activation of Leptomonas NAD-linked alpha-glycerophosphate dehydrogenase by ethidium and antrycide.

Antrycide and ethidium bromide — 2 cationic trypanocides — inhibited NAD-linked α-glycerophosphate dehydrogenase from $\text{Leptomonas}$ sp. The kinetics of enzyme inhibition was determined by Lineweaver-Burk, Dixon, or direct linear plots. Inhibition by Antrycide was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. With dihydroxyacetone phosphate at saturation, Antrycide inhibition was also noncompetitive with respect to Mg²⁺ (K_i = 115 μM) and spermidine (K_i = 85 μM). Inhibition by ethidium in the presence of saturating dihydroxyacetone phosphate, was noncompetitive for Mg²⁺ (K_i = 400 μM) but mixed for spermidine (K_i = 495 μM); inhibition was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. Rabbit-muscle α-glycerophosphate dehydrogenase was inhibited at all concentrations of Antrycide and ethidium tested, but the $\text{Leptomonas}$ enzyme was stimulated up to 3.5-fold by low concentrations of inhibitors in the absence of polyamine. New chemotherapeutic possibilities may thus be opened and an evolutionary distinction between trypanosomatid and mammalian enzyme.     

Enzymic synthesis of lignin precursors. Further studies on cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures

Isoenzyme 2 of cinnamyl-alcohol dehydrogenase from soybean suspension cultures was purified about 3800-fold to apparent homogeneity by an improved purification procedure involving biospecific elution of the enzyme from a NADP+-agarose column. On sodium dodecylsulfate gels the dehydrogenase showed only one protein band with Mr 40 000 +/- 500. The enzyme is strongly inhibited by thiol reagents. Various metal chelators as well as the nonchelating 7,8-benzoquinoline also inhibited enzyme activity. Inhibition by 10 mM 1,10-phenanthroline could be partially reversed by addition of Zn2+. 1,10-Phenanthroline and 7,8-benzoquinoline are non-competitive inhibitors with respect to NADP+. The presence of zinc in the dehydrogenase was proved by atomic absorption spectroscopy and by specific incorporation of 65Zn into the enzyme. In steady-state kinetics inhibition patterns were obtained which are consistent with an ordered bi-bi mechanism in which NADP(H) is the first substrate to bind and the last product released. The cinnamyl-alcohol dehydrogenase belongs to the A-specific dehydrogenases and removes the pro-R hydrogen from coniferyl alcohol. The enzyme shows many similarities with alcohol dehydrogenases from horse and rat liver and from yeast.