Vanadium K-edge X-ray-absorption spectroscopy of the functioning and thionine-oxidized forms of the VFe-protein of the vanadium nitrogenase from Azotobacter chroococcum.

Vanadium K-edge X-ray-absorption spectra were collected for samples of thionine-oxidized, super-reduced (during enzyme turnover) and dithionite-reduced VFe-protein of the vanadium nitrogenase of Azotobacter chroococcum (Acl*). Both the e.x.a.f.s and the x.a.n.e.s. (X-ray-absorption near-edge structure) are consistent with the vanadium being present as part of a VFeS cluster; the environment of the vanadium is not changed significantly in different oxidation states of the protein. The vanadium atom is bound to three oxygen (or nitrogen), three sulphur and three iron atoms at 0.215(3), 0.231(3) and 0.275(3) nm respectively.     

Competition in the gradostat: the role of the communication rate

In this paper we study a mathematical model of competition between two species of microorganisms for a single limiting nutrient in a laboratory device called a gradostat. A gradostat consists of several (we consider only two) chemostats (CSTR's) connected together so that material can flow between the vessels in such a way that a nutrient gradient is established. Our model is a slightly modified version of one considered recently by Jäger et al. [3], in that the rate of exchange of material between the two vessels (the communication rate) is allowed to differ from the dilution rate. The outcome of competition turns out to be surprisingly sensitive to variation of the communication rate. We identify several coexistence regimes in parameter space and describe a method for obtaining operating diagrams for given pairs of competing microorganisms.Research supported in part by NSF Grant DMS 8521605     

Energetic and rate effects on methanogenesis of ethanol and propionate in perturbed CSTRs

Energetic and reaction-rate interactions between hydrogenic (hydrogen-producing) and hydrogenotrophic (hydrogen-consuming) bacteria were investigated in five perturbation experiments performed on steady-state, mixed-culture methanogenic CSTRs receiving ethanol, propionate, or both hydrogenic substrates. When a large quantity of propionate was suddenly added to a propionatefed CSTR, P(H(2) ) increased to 10(-4) atm and propionate oxidation remained energetically favorable. When ethanol was added to a CSTR receiving ethanol, P(H(2) ) rose to 6.3 x 10(-3) atm within 5 h. In both perturbations, P(H(2) ) remained at levels such that oxidation of the hydrogenic substrate remained energetically favorable throughout the transient. Sudden increase in ethanol concentration in the ethanol- and propionate-fed CSTR resulted in an increase in P(H(2) ) such that propionate oxidation became energetically unfavorable and was blocked. Propionate utilization resumed when the added ethanol was depleted and P(H(2) ) returned to its previous steady-state levels. Ethanol perturbation of ethanol- and propionate-fed CSTRs led to the formation of reduced products, including n-propanol and four-through seven-carbon n-carboxylic acids, when P(H(2) ) was elevated; these products disappeared after P(H(2) ) returned to previous, steady-state levels. The transformations were consistent with reaction energetics. Reduced product formation may have been a sink for reducing equivalents, as an alternative to oxidation for propionate utilization, as indicated by an electron equivalents balance over the time course of experiments.     

Selective inhibition of [3H]nitrendipine binding to brain and cardiac membranes by bacitracin

The effects of bacitracin were investigated on [3H]nitrendipine binding to rat brain and cardiac membranes in a low ionic strength (5 mM Tris-HCl) buffer. Bacitracin inhibited [3H]nitrendipine binding to rat brain and cardiac membranes with IC50 values of 400 +/- 100 and 4600 +/- 400 micrograms/mL, respectively. Scatchard analysis in brain membranes revealed that bacitracin inhibited [3H]nitrendipine binding primarily by reducing the Bmax but also by producing a small increase in the Kd. In brain membranes, Na+ (100 mM) and Ca2+ (2 mM) reduced the potency of bacitracin to inhibit [3H]nitrendipine binding by approximately sixfold with IC50 values of 2600 +/- 300 and 2100 +/- 400 micrograms/mL observed for bacitracin in the presence of 100 mM Na+ and 2 mM Ca2+, respectively. The EC50 values for the effects of Na+ and Ca2+ were 800 +/- 200 microM and 25 +/- 5 mM. K+, Mg2+, choline, and increasing the assay buffer of Tris-HCl to 50 mM also decreased the inhibition of [3H]nitrendipine binding by bacitracin. These results suggest that bacitracin specifically modulates [3H]nitrendipine binding in a cation-dependent manner and that brain and cardiac dihydropyridine binding sites are either biochemically different or exist in a different membrane environment.     

Inhibition of lymphocyte activation with anti-transferrin receptor Mabs: a comparison of three reagents and further studies of their range of effects and mechanism of action

Prior work has suggested that Mabs against the transferrin receptor (ATRAs) may function as selective inhibitors of lymphocyte activation and that T cell activation protocols may be more sensitive to ATRA-mediated inhibition than B cell activation protocols. New side-by-side functional comparisons of three ATRAs are presented. When these studies are considered with our prior work they demonstrate unambiguously that although one particular IgG ATRA consistently fails to inhibit LPS responses and although IgM ATRAs may be slightly more effective inhibitors than IgG ATRAs, ATRAs as a class consistently appear to abolish the MLR at submicrogram concentrations, essentially eliminate cytotoxic cell generation at concentrations between 1 and 5 micrograms/ml, and produce no more than about 50% inhibition of LPS responses at concentrations as high as 25 micrograms/ml. Therefore, an even stronger case can now be made for the idea that lymphocyte subsets differ in their dependence on transferrin receptor function during activation. This, in turn, makes an even stronger case for the idea that lymphocyte subsets differ in fundamental aspects of the management of their iron economies. New studies also show that IgG ATRAs appear to function by causing down-modulation of surface expression of the transferrin receptor in normal lymphocytes in a manner similar to that previously shown for tumor cells. It is clear that a sophisticated model will ultimately be required to account for all of the data arising from studies with ATRAs, and a new attempt at a more detailed construct is presented.     

Effect of acetaminophen on hepatic content and biliary efflux of glutathione disulfide in mice

The increased expiration of ethane and pentane by mice treated with hepatotoxic doses of acetaminophen suggests the possibility of oxidant mechanisms associated with the necrosis. However, studies in rats are not consistent with oxidant stress mechanisms causing the damage, because acetaminophen given to rats does not increase GSSG efflux, a sensitive index of intrahepatic oxidant stress. To compare the extent of oxidant stress generated by acetaminophen in mice versus rats, hepatic content and biliary efflux of GSSG and GSH in mice have been examined. Bile was collected from anesthetized male ICR mice before and after intraperitoneal administration of acetaminophen (325 mg/kg, 2.15 mmol/kg), t-butyl hydroperoxide (TBHP) (1.5 mmol/kg), diethyl maleate (400 mg/kg, 2.33 mmol/kg, in corn oil) or saline (control) and GSH and GSSG were measured by the enzymatic recycling method of Tietze. An increase in biliary GSSG efflux was produced by t-butyl hydroperoxide, but not by the other agents. Biliary GSH/GSSG ratios decreased in acetaminophen-treated animals, presumably reflecting the marked depletion of hepatic GSH, since a similar decrease was observed with non-hepatotoxic doses of diethyl maleate. The failure of acetaminophen to increase the hepatic content or biliary efflux of GSSG in ICR mice is not consistent with the view that oxidant stress mechanisms cause the damage, despite the increases in alkanes expired after acetaminophen administration in this specific animal model.     

Reaction of bovine-liver copper-zinc superoxide dismutase with hydrogen peroxide. Evidence for reaction with H2O2 and HO2- leading to loss of copper

The reaction of hydrogen peroxide with the copper-zinc bovine-liver superoxide dismutase at low molar ratios (0.2-20.0) of H2O2/active site between pH 7.3-10.0 leads to the loss of native enzyme as a distinct form monitored by electrophoresis. The pH dependence of the loss of native enzyme between 7.3 and 9.0 indicates the involvement of a conjugate base on the enzyme of pKa of 8.7 +/- 0.1. The rate of loss of the native enzyme is first order with respect to the concentration of both enzyme and hydrogen peroxide between pH 7.3 and 9.0 with no evidence for binding of peroxide. A second-order rate constant of 3.0 +/- 1.0 M-1 s-1 is obtained from these data. At pH 10.0 the reaction is first order with respect to enzyme concentration but saturable in H2O2. All data are consistent with the interpretation that H2O2 reacts with the enzyme at the lower pH where the reaction is dependent upon the conjugate base of a functional group on the enzyme. At the higher pH, the data are consistent with the reaction of HO2- and H2O2 with the dismutase. The dissociation constant for HO2- calculated from the kinetic data at pH 10.0 is between 25-50 microM and the rate constant for the breakdown of the HO2- dismutase complex is 1.10 + 0.05 x 10(-2) s-1. The change in the electrophoretic pattern at all pH values is accompanied by the loss of the ability of the enzyme to bind copper. Weakly bound or free copper can be detected using bathocuproine disulfonate. Furthermore copper-defficient forms of the enzyme can be detected by staining gels of the peroxide-treated dismutase with diethyldithiocarbamate.     

Solubilisation of methane monooxygenase from Methylococcus capsulatus (Bath)

The membrane-bound (particulate) form of methane monooxygenase from Methylococcus capsulatus (Bath) has been solubilised using the non-ionic detergent dodecyl-beta-D-maltoside. A wide variety of detergents were tested and found to solubilise membrane proteins but did not yield methane monooxygenase in a form that could be subsequently activated. After solubilisation with dodecyl-beta-D-maltoside, enzyme activity was recovered using either egg or soya-bean lipids. Attempts to further purify the solubilized methane monooxygenaser protein into its component polypeptides were unsuccessful and resulted in complete loss of enzyme activity. The major polypeptides present in the solubilised enzyme had molecular masses of 49 kDa, 23 kDa and 22 kDa which were similar to those seen in crude extracts [Prior, S. D. & Dalton H. (1985) J. Gen. Microbiol. 131, 155-163]. Studies on substrate and inhibitor specificities indicated that the membrane-associated and solubilised forms of methane monooxygenase were quite similar to each other but differed substantially from the well-characterised soluble methane monooxygenase found in cells grown in a low copper regime and synthesised independently of the particulate methane monooxygenase.     

Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis. The chloroplast enzyme hydroxymethylbilane synthase (porphobilinogen deaminase) is synthesised with a very long transit peptide in Euglena

A cDNA expression library was constructed from light-grown Euglena gracilis poly(A)-rich RNA in lambda gt11. Antibodies to Euglena hydroxymethylbilane synthase, the third enzyme in the porphyrin biosynthetic pathway, were used to screen the library and a clone encoding part of the sequence of hydroxymethylbilane synthase was identified. This was used to rescreen the library and a full-length clone was isolated, which encoded not only the entire mature protein (Mr 36,927), but also an N-terminal extension of 139 amino acids. The deduced Mr of the whole polypeptide is 51,744, which corresponds to the size of the protein immunoprecipitated from the translation products of Euglena poly(A)-rich RNA. The mature protein is 60-70% similar to hydroxymethylbilane synthase from human erythrocytes and Escherichia coli. The sequence of the N-terminal extension has similarities to both the transit peptides of chloroplast proteins and those for the endoplasmic reticulum. This is the first report both of a cDNA clone for an enzyme of the chlorophyll biosynthetic pathway and of a putative transit peptide for a nuclear-encoded Euglena protein.     

A G4-DNA/B-DNA junction at codon 12 of c-Ha-ras is actively and asymmetrically methylated by DNA(cytosine-5)methyltransferase

Oligodeoxynucleotides spanning codon 12 of the human c-Ha-ras gene were found to be exceptionally good substrates for de novo methylation by human DNA(cytosine-5)methyltransferase. In the complex formed by two complementary 30mers, only the C-rich strand was methylated by the enzyme. Guanines at the 3' end of the G-rich strand of the complex could not be completely modified by dimethyl sulfate [corrected] suggesting tetrameric bonding at these G-residues. An eight-stranded structure, composed of four duplex DNAs at one end, joined to a G4-DNA segment at the other with the junction between the two DNA forms at codon 12, can account for our results.