Vanadium chemistry and biochemistry of relevance for use of vanadium compounds as antidiabetic agents

The stability of 11 vanadium compounds is tested under physiological conditions and in administration fluids. Several compounds including those currently used as insulin-mimetic agents in animal and human studies are stable upon dissolution in distilled water but lack such stability in distilled water at pH7. Complex lability may result in decomposition at neutral pH and thus may compromise the effectiveness of these compounds as therapeutic agents; Even well characterized vanadium compounds are surprisingly labile. Sufficiently stable complexes such as the VEDTA complex will only slowly reduce, however, none of the vanadium compounds currently used as insulin-mimetic agents show the high stability of the VEDTA complex. Both the bis(maltolato)oxovanadium(IV) and peroxovanadium complexes extend the insulin-mimetic action of vanadate in reducing cellular environments probably by increased lifetimes under physiological conditions and/or by decomposing to other insulin mimetic compounds. For example, treatment with two equivalents of glutathione or other thiols the (dipicolinato)peroxovanadate(V) forms 9dipicolinato)oxovanadate(V) and vanadate, which are both insulin-mimetic vanadium(V) compounds and can continue to act. The reactivity of vanadate under physiological conditions effects a multitude of biological responses. Other vanadium complexes may mimic insulin but not induce similar responses if the vanadate formation is blocked or reduced. We conclude that three properties, stability, lability and redox chemistry are critical to prolong the half-life of the insulin-mimetic form of vanadium compounds under physiological conditions and should all be considered in development of vanadium-based oral insulin-mimetic agents.Abbreviations ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - ADP-V adenosine 5-diphosphate-vanadate - bpV bis(peroxo)oxovanadium(V) - (bpV)2 bis(peroxo)oxovanadium(V) dimer - bpVpic bis(peroxo)picolinatooxovanadate(V) - ¹³C carbon-13 - EDTA ethylenediaminetetraacetic acid - EPR electron paramagnetic resonance - EXSY exchange spectroscopy - ¹H proton - HSG glutathione - NAD -nicotinamide adenine dinucleotide - NADP -nicotinamide adenine dinucleotide phosphate - NADV -nicotinamide adenine dinucleotide vanadate - NMR nuclear magnetic resonance (also referred to as magnetic resonance imaging) - pVdipic (dipicolinato)peroxovanadate(V) - Vcit (citrato)dioxovanadate(V) - VEDTA (ethylenediaminetetraacetato)dioxovanadate(V) - Vmalto bis(maltolato)-oxovanadium(IV) - Voxal bis(oxalato)dioxovanadate(V) - ⁵¹V vanadium-51 - V₁ vanadate monomer - V₂ vanadate dimer - V₄ vanadate tetramer - V₅ vanadate pentamer - UV-vis spectroscopy ultraviolet-visible spectroscopy     

A new heterobinuclear FeIIICuII complex with a single terminal FeIII–O(phenolate) bond. Relevance to purple acid phosphatases and nucleases

A novel heterobinuclear mixed valence complex [Fe^IIICu^II(BPBPMP)(OAc)₂]ClO₄, 1, with the unsymmetrical N₅O₂ donor ligand 2-bis[{(2-pyridylmethyl)aminomethyl}-6-{(2-hydroxybenzyl)(2-pyridylmethyl)}aminomethyl]-4-methylphenol (H₂BPBPMP) has been synthesized and characterized. A combination of data from mass spectrometry, potentiometric titrations, X-ray absorption and electron paramagnetic resonance spectroscopy, as well as kinetics measurements indicates that in ethanol/water solutions an [Fe^III–()OH–Cu^IIOH₂]⁺ species is generated which is the likely catalyst for 2,4-bis(dinitrophenyl)phosphate and DNA hydrolysis. Insofar as the data are consistent with the presence of an Fe^III-bound hydroxide acting as a nucleophile during catalysis, 1 presents a suitable mimic for the hydrolytic enzyme purple acid phosphatase. Notably, 1 is significantly more reactive than its isostructural homologues with different metal composition (Fe^IIIM^II, where M^II is Zn^II, Mn^II, Ni^II, or Fe^II). Of particular interest is the observation that cleavage of double-stranded plasmid DNA occurs even at very low concentrations of 1 (2.5 M), under physiological conditions (optimum pH of 7.0), with a rate enhancement of 2.7×10⁷ over the uncatalyzed reaction. Thus, 1 is one of the most effective model complexes to date, mimicking the function of nucleases.Electronic Supplementary Material Supplementary material is available for this article at .     

A fresh look into VO(salen) chemistry: synthesis, spectroscopy, electrochemistry and crystal structure of [VO(salen)(H2O)]Br · 0.5 CH3CN

The reaction of V^IVO(salen) with [Et₄N][SnBr₃] in air proceeds via an initial reduction to give a [V^III (salen)]⁺ intermediate, which is then oxidised to dark green [V^VO(salen)(H₂O)]Br, 1. As determined by X-ray crystallography, 1 in the solid state contains hexacoordinate vanadium. ⁵¹V NMR spectra indicate that dissociation of the aqua ligand occurs to give a pentacoordinated [V^VO(salen)] cation in methanol-d₄ solution, while in DMSO-d₆ solutions, coordination of the solvent occurs to give [V^VO(salen)(DMSO-d₆)]⁺. The colour of 1 can be accounted for by O_oxo → V^V and phenolate → V^V LMCTs. Results from this study have led to the re-assignment of LMCTs and V-N and V-O_phenolate stretching frequencies in the IR spectrum. Cyclic voltammetry of 1 indicates three redox processes. The first is typical of [VO(salen)]/[VO(salen)]⁺ couple and the other two are bromide oxidations.     

A new mouse T-cell receptorα chain variable region family

Sequence analysis of the rearranged T-cell receptor a chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribedV gene family. We have designated this familyV 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously describedV haplotypes:V ^a ,V ^b , andV ^c .     

Influence of structural and physical properties of the thylakoid membrane on QA - oxidation

Using isolated pea thylakoids, the relative rate of Q_A ^- oxidation has been estimated under various conditions, from the restoration of the induction curves following a dark period and from light 1-induced changes in modulated chlorophyll fluorescence excited by light 2.Alterations of _QinfA ^sup– oxidation rates were observed under conditions which affected the degree of thylakoid stacking, the lipid fluidity and the integrity of the membranes. The results are discussed in terms of the interactions between Q_A ^- and the plastoquinone pool with particular emphasis on lateral diffusion.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EDTA Ethylenediaminetetracetate - Hepes N-2-hydroxyethyl piperazine-N-2-ethanesulphonic acid - NADP nicotinamide adenine dinucleotide phosphate     

DNA cleavage activity of VO(acac)2 and derivatives

The DNA cleavage activity of several β-diketonate vanadyl complexes is examined. Vanadyl acetylacetonate, V^IVO(acac)₂, 1, shows a remarkable activity in degrading plasmid DNA in the absence of any activating agents, air and photoirradiation. The cleaving activity of several related complexes V^IVO(hd)₂ (2, Hhd = 3,5-heptanedione), V^IVO(acac-NH₂)₂ (3, Hacac-NH₂ = acetoacetamide) and V^IVO(acac-NMe₂)₂ (4, Hacac-NMe₂ = N,N-dimethylacetoacetamide) is also evaluated. It is shown that 2 exhibits an activity similar to 1, while 3 and 4 are much less efficient cleaving agents. The different activity of the complexes is related to their stability towards hydrolysis in aqueous solution, which follows the order 1∼2 ? 3∼4. The nature of the pH buffer was also found to be determinant in the nuclease activity of 1 and 2. In a phosphate buffered medium DNA cleavage by these agents is much more efficient than in tris, hepes, mes or mops buffers. The reaction seems to take place through a mixed mechanism, involving the formation of reactive oxygen species (ROS), namely OH radicals, and possibly also direct cleavage at phosphodiester linkages induced by the vanadium complexes.     

Accumulation of essential oils by Agrobacterium tumefaciens-transformed shoot cultures of Pimpinella anisum

Axenic transformed shoot cultures of Pimpinella anisum (anise) were established following inoculation of plant stems with the nopaline strain T37 of Agrobacterium tumefaciens. The stable incorporation of T-DNA in the transformed tissues was demonstrated by polymerase chain reaction. Total essential oil accumulated by transformed shoot cultures grown under continuous light was found to be 18% lower (per unit fresh weight of tissue) than that produced by untransformed shoot cultures incubated under similar conditions, but more than 89% lower than the yield of oil from the intact plant. The relative amounts of the principal components of the essential oil of the transformed shoot cultures, namely geraniol, -bisabolene, trans-pseudoisoeugenol-2-methylbutyrate and transanethole, were similar to those present in the parent plant, but significantly different from those of the untransformed shoot cultures.Abbreviations T-DNA transfer-DNA - MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - TY tryptone and yeast extract medium - t_D doubling time - GC-MS gas chromatography coupled with mass spectrometry - FID flame ionisation detector - PCR polymerase chain reaction - TE Tris-HCl, EDTA buffer - TBE Tris, borate, EDTA buffer     

Redox reactions of [VO(salen)] couple in acetonitrile: Volume analyses in relation to large chiral recognitions observed for electron self-exchange reactions of [VO(Schiff-Base)]

Kinetic measurements were carried out for the outer-sphere electron transfer reactions involving [VO(Schiff-Base)]^+/0 couples. Electron self-exchange rate constant for [VO(3-MeOsal-(RR)-chxn)]^+/0 couple was determined as k_ex = (5.2 ± 0.8) × 10⁶ kg mol⁻¹s⁻¹ at 25 °C. It was found that added water in acetonitrile solvent retarded the electron transfer reactions between [VO(salen)]⁺ and [Co(o-phen)₃]²⁺: the six-coordinate V(V) species, [VO(salen)(OH₂)]⁺ was found to be ca. 3.5 times less reactive compared with the five-coordinate species, [VO(salen)]⁺. This small difference between the reactivity of five- and six-coordinate species indicates that the reaction through the direct OV(V)-OV(IV) interaction is outer-sphere in nature, contrary to the previously proposed inner-sphere mechanism. Activation volumes corresponding to the electron self-exchange reactions for [VO(salen)]⁺/[VO(salen)]⁰ and [VO(salen)OH₂]⁺/[VO(salen)]⁰ couples were estimated from the volume profiles of the reduction reactions of [VO(salen)]⁺ by [Co(o-phen)₃]²⁺, with and without added water in acetonitrile: ΔV* = −3.4 ± 3.7 and −8.0 ± 4.0 cm³ mol⁻¹ for [VO(salen)]⁺/[VO(salen)]⁰ and [VO(salen)OH₂]⁺/[VO(salen)]⁰ couples, respectively. It was suggested that previously reported chiral recognitions in the redox reactions involving [VO(Schiff-Base)]^+/0 redox couples take place within the encounter complex through the outer-sphere mechanism by maximizing the direct coupling between the dπ orbitals of V(IV) and V(V) species (Scheme 2).     

Na+-independent,electrogenic Cl- uptake across the posterior gills of the Chinese crab (Eriocheir sinensis): Voltage-clamp and microelectrode studies

Summary Single gill lamellae from posterior gills of Chinese crabs (Eriocheir sinensis) were isolated, separated into halves and mounted in a modified Ussing chamber. Area-related short-circuit current (I_sc) and conductance (G_tot) of this preparation were measured. Epithelial cells were impaled with microelectrodes through the basolateral membrane and cellular potentials (V_i under open- and V_sc under short-circuit conditions) as well as the voltage divider ratios (F_i, F_o) were determined.With NaCl salines on both sides an outside positive PD_te (22±2 mV) and an I_sc (-64±13 A·cm^-2) with a polarity corresponding to an uptake of negative charges (inward negative) were obtained. Trough-like potential profiles were recorded across the preparation under open- as well as short-circuit conditions (V_o=-101±5 mV, external bath as reference; V_i=-78±2 mV, internal bath as reference; V_sc=-80±2 mV, extracellular space as reference). The voltage divider ratios of the external (apical membrane plus cuticle) and internal (basolateral membrane) barrier were F_o=0.92±0.01 and F_i=0.08±0.01, respectively. To investigate a Cl^--related contribution to the above parameters, Na⁺-free solutions in the external bath (basolateral NaCl-saline) were used. Inward negative I_sc under these conditions almost completely depended on external Cl^-. Elimination of Cl^- in the external bath reversed I_sc, and G_tot decreased substantially. Concomitantly, V_sc depolarised and F_o increased. Cl^--dependent current and conductance showed saturation kinetics with increasing external [Cl^-]. Addition of 20 mmol·1^-1 thiocyanate to the external bath had similar, although less pronounced, effects as Cl^- substitution. Equally, external SITS (1 mmol·1^-1) inhibited the current and, concomitantly, G_tot decreased substantially. Addition of 1 mmol·1^-1 acetazolamide to, and omission of NaHCO₃ from, the basolateral bath resulted in a decrease of I_sc while G_tot remained unchanged. The Cl^--channel blocker DPC inhibited I_sc almost completely when added to the basolateral saline, whereas G_tot decreased moderately; however, V_sc depolarised without significant change of F_i. Ouabain had no influence on I_sc and G_tot. Increasing the basolateral [K⁺] resulted in a decrease in I_sc, while G_tot was not affected. At the same time V_sc largely depolarised and F_i decreased. Addition of the K⁺-channel blocker Ba⁺⁺ (5 mmol·1^-1) to the basolateral solution resulted in a two-step alteration of the transepithelial (I_sc, Gtot) and cellular (V_sc, F_i) parameters. The results are discussed with regard to (i) the mechanisms responsible for active transbranchial Cl^- uptake, and (ii) the technical improvement of being able to perform transport studies with crab gill preparations in an Ussing chamber.Abbreviations DMSO dimethylsulfoxide - DPC diphenylamine-2-carboxylate - F _{o, i} voltage divider ratio for external (o) and internal (i) barrier, respectively - G _Cl conductance related to the external [Cl^-] - G _tot total tissue conductance - I _Cl short-circuit current related to the external [Cl^-] - I _sc short-circuit current - PD _te transepithelial potential difference - R _ME resistance of the microelectrode - SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - V _{o, i} open-circuit voltage across the external (o) and internal (i) barrier, respectively - V _sc intracellular potential under short-circuit conditions     

Depolymerization of Hyaluronic Acid by D-Fructose 6-Phosphate

Depolymerization of hyaluronic acid obtained from Streptococcus zooepidemicus by D-fructose 6-phosphate was investigated for characterization of reducing sugar-mediated degradation of biopolymers under physiological conditions. The extent of depolymerization was monitored by the decrease of viscosity of a reaction mixture containing 1.0% hyaluronic acid, D-fructose 6-phosphate, and 1.0 × 10^?2 mM of Cu²⁺ in phosphate buffer, pH 7.4. It was found that the depolymerization of hyaluronic acid was dependent on the concentration of the reducing sugar and was specifically accelerated by the presence of Cu²⁺. The reaction was found to be significantly inhibited by catalase, superoxide dismutase (SOD), 1,2-dihy­ droxybenzene 3,5-disulfonic acid (Tiron), and chelating agents such as EDTA and diethylene triamine penta­ acetic acid (DETAPAC), although the inhibition by SOD was low. Almost the same depolymerization rates were observed in hyaluronic acid preparations of different molecular weight (1.1 × 10⁶, 8.8 × 10⁵, and 6.8 × 10⁵). The rates, however, were different for hyaluronic acids obtained from S. zooepidemicus, rooster comb, and umbilical cord. It was concluded that depolymerization of the polysaccharide was caused by active oxygen species generated by the autoxidation of D-fructose 6-phosphate in the presence of Cu²⁺, in a mechanism similar to that previously reported for the degradation of DNA and inactivation of virus in vitro.     

Modification of pLL/DNA complexes with a multivalent hydrophilic polymer permits folate-mediated targeting in vitro and prolonged plasma circulation in vivo

Background

Gene delivery vectors based on poly(L ‐lysine) and DNA (pLL/DNA complexes) have limited use for targeted systemic application in vivo since they bind cells and proteins non‐specifically. In this study we have attempted to form folate‐targeted vectors with extended systemic circulation by surface modification of pLL/DNA complexes with hydrophilic polymers.

Methods

pLL/DNA complexes were stabilised by surface modification with a multivalent reactive polymer based on alternating segments of poly(ethylene glycol) and tripeptides bearing reactive ester groups. Folate moieties were incorporated into the vectors either by direct attachment of folate to the polymer or via intermediate poly(ethylene glycol) spacers of 800 and 3400 Da.

Results

Polymer‐coated complexes show similar morphology to uncoated complexes, their zeta potential is decreased towards zero, serum protein binding is inhibited and aqueous solubility is substantially increased. Intravenous (i.v.) administration to mice of coated complexes produced extended systemic circulation, with up to 2000‐fold more DNA measured in the bloodstream after 30 min compared with simple pLL/DNA complexes. In further contrast to simple pLL/DNA complexes, coated complexes do not bind blood cells in vivo. Folate receptor targeting is shown to mediate targeted association with HeLa cells in vitro, leading to increased transgene expression. We demonstrate for the first time that DNA uptake via the folate receptor is dependent on pEG spacer length, with the transgene expression relatively independent of the level of internalised DNA.

Conclusions

We show increased systemic circulation, decreased blood cell and protein binding, and folate‐targeted transgene expression using pLL/DNA complexes surface‐modified with a novel multireactive hydrophilic polymer. This work provides the basis for the development of plasma‐circulating targeted vectors for in vivo applications. Copyright © 2002 John Wiley & Sons, Ltd.

     

Complexes prepared from protein A and human serum,IgG, or Fcγ fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion

Summary Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of ¹²⁵I-protein A and human ¹³¹I-IgG alone or in serum, and ¹³¹1-Fc fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess ¹³¹I-IgG or ¹³¹1-Fc, all the ¹²⁵I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess ¹²⁵I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.Abbreviations SpA protein A of Staphyloccus aureus - VBS EDTA gel, 0.0055 M veronal buffered saline containing 0.01 M EDTA and 0.1% gelatin, pH 7.4 - PBS 0.01 M phosphate buffered saline, pH 7.4     

Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

Acidity and photolability of ruthenium salen nitrosyl and aquo complexes in aqueous solutions

The photochemical behavior of nitrosyl complexes Ru(salen)(NO)(OH₂)⁺ and Ru(salen)(NO)Cl (salen = N,N′-ethylenebis-(salicylideneiminato) dianion) in aqueous solution is described. Irradiation with light in the 350-450 nm range resulted in nitric oxide (NO) release from both. For Ru(salen)(NO)Cl secondary photoreactions also resulted in chloride aquation. Thus, in both cases the final photoproduct is the diaquo cation , for which pK_a’s of 5.9 and 9.1 were determined for the coordinated waters. The pK_a of the Ru(salen)(NO)(OH₂)⁺ cation was also determined as 4.5 ± 0.1, and the relative acidities of these ruthenium aquo units are discussed in the context of the bonding interactions between Ru(III) and NO.     

Methylation sensitivity of the restriction enzymes FnuDII and AccII

The restriction enzymes FnuDII and AccII are isoschizomers of the DNA sequence 5-CGCG-3. We have determined that 5-methylcytidine at either cytidine position in this recognition sequence inhibits DNA cleavage by FnuDII and AccII. A third isoschizomer, ThaI was previously shown to exhibit an identical methylation sensitivity. It is remarkable that 3 restriction enzymes derived from diverse microbiological sources exhibit this identical methylation sensitivity.Abbreviations bp base pair(s) - m_c 5-methylcytidine - Tris-borate buffer 89 mM Tris-base, 89 mM boric acid, 2.5 mM EDTA, pH 8.3     

DNA binding and nuclease activity of copper(II) complexes of tridentate ligands

Two copper(II) complexes, 1 and 2 with L₁ and L₂ [L₁ = 2-hydroxybenzyl(2-(pyridin-2-yl)ethylamine); L₂ = 2-hydroxybenzyl(2-(pyridin-2-yl)methylamine)] ligands, respectively, have been synthesized and characterized. The interaction of both the complexes with DNA has been studied to explore their potential biological activity. The DNA binding properties of the complexes with calf thymus (CT) DNA were studied by spectroscopic titration. The complexes show binding affinity to CT DNA with binding constant (K_b) values in the order of 10⁵ M⁻¹. Thermal denaturation and circular dichroism studies suggest groove binding of the complexes to CT DNA. Complexes also exhibit strong DNA cleavage activity in presence of reducing agents like 3-mercaptopropionic acid and β-mercaptoethanol. Mechanistic studies reveal the involvement of reactive hydroxyl radicals for their DNA cleavage activity.     

Bacterial Degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal–EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Co²⁺, Cd²⁺, Zn²⁺, Cu²⁺, or Fe³⁺. The metal–EDTA complexes (Me–EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10¹⁶ (log K < 16), such as Mg–EDTA, Ca–EDTA, and Mn–EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5–10 h of incubation. Me–EDTA complexes with log K above 16 (Zn–EDTA, Co–EDTA, Pb–EDTA, and Cu–EDTA) were not completely degraded during a 24-h incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd–EDTA or Fe(III)–EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

Ethylene oxidation by Nitrosomonas europaea

Incubation of whole cells of the nitrifying bacterium Nitrosomonas europaea with ethylene led to the formation of ethylene oxide. Ethylene oxide production was prevented by inhibitors of ammonium ion oxidation, and showed properties implying that ethylene is a substrate for the ammonia oxidising enzyme, ammonia monooxygenase. Endogenous substrates, hydroxylamine, hydrazine and ammonium ions were compared as sources of reducing power in terms of rates and stoichiometries of ethylene oxidation. The highest rates of ethylene oxide formation (15 mol h^-1 mg protein^-1) were obtained with hydrazine as donor. The data suggest that at high concentrations of ethylene the rate of oxidation is limited by the rate at which reducing power can be supplied to the monooxygenase, not by an intrinsic V _max. Ethylene had an inhibitory effect on the rate of ammonium ion utilisation; an approximate K _i of 80 M was derived, but the results deviated from simple competitive behaviour. Measurement of relative rates of ethylene oxide formation and ammonium ion utilization led to a k _cat/K _m value for ethylene of 1.1 relative to NH ₄ ⁺ , or 0.04 relative to the true natural substrate, NH₃. The effects of higher concentrations of ethylene oxide on oxygen uptake rates were also investigated. The results imply that ethylene oxide is also a substrate for the monooxygenase, but with a much lower affinity than ethylene.     

The major DNA polymerase in cultured plant cells: Partial purification and correlation with cell multiplication

A DNA polymerase activity was isolated from cells of Oryza sativa L. grown in suspension culture. Molecular mass ( 180,000), optimal requirements for pH (neutral), Mg²⁺ (5–10 mM), Mn²⁺ (1 mM), template preference (activated DNA), lack of activity with native or denatured DNA, and sensitivity to N-ethylmaleimide and ionic strength are similar to those of the vertebrate -polymerase. Like DNA polymerase , the DNA polymerase described in this work is the most abundant in proliferating cells of Oryza sativa L., Parthenocissus tricuspidata (Siebold et Zucc.) Planchon, Acer pseudoplatanus L., and Medicago sativa L. and responds to changes in the rate of cell multiplication. We therefore postulate that this -like DNA polymerase is the replicating enzyme of plant cells.Abbreviations BSA bovine serum albumin - EDTA ethylendiamino-tetracetic acid - DTT dithiothreitol - PTSF p-toluenesulfonyl fluoride