Search PubMed⌕ Search

Biomedical subjects

R R Eady

Publications and source records attributed to R R Eady.

88 records · Page 5Linked to original sources
PubMed1978

Nitrogenase synthesis in Klebsiella pneumoniae: comparison of ammonium and oxygen regulation.

Rates of nitrogenase synthesis by Klebsiella pneumoniae were measured by pulse-labelling organisms with a mixture of 14C-labelled amino acids followed by sodium dodecyl sulphate gel electrophoresis and autoradiography. Populations from an NH4+-repressed, SO42--limited chemostat (0.46 mg dry wt ml-1), when released from NH4+ repression, simultaneously synthesized detectable quantities of the three nitrogenase polypeptides 45 min before acetylene-reducing activity was observed. Exposure of populations synthesizing nitrogenase to air or NH4+ (200 microgram N ml-1) repressed synthesis of both component proteins simultaneously, the rate initially decreasing by half in 11 to 12 min; in the presence of NH4+ a second slower phase with an approximate half-life of 30 min was observed. With 5% O2 in N2 the half-lives for the decreases in the rates of synthesis were 30 min for the Fe protein and 33 min for the Mo-Fe protein. Oxygen also repressed nitrogenase in a glutamine synthetase constitutive derivative of K. pneumoniae (strain SK24) which escapes NH4+ repression. Regulation of nitrogenase by O2 may therefore be independent of glutamine synthetase.

Ammonia↗
PubMed1977

Nitrogenase of Klebsiella pneumoniae. Distinction between proton-reducing and acetylene-reducing forms of the enzyme: effect of temperature and component protein ratio on substrate-reduction kinetics.

Non-linear rates of acetylene reduction and concomitant H2 evolution were observed for the nitrogenase of Klebsiella pneumoniae at 10 degrees C. A lag phase of 1-4 min, dependent on the ratio of Mo-Fe protein to Fe protein present, occurred before linear rates of acetylene reduction were achieved. A complementary burst phase for concomitant H2 evolution in the presence of acetylene was also observed. When the proton was the only reducible substrate present, linear rates of H2 evolution were observed. N2 was a poor substrate under these conditions. Similar lag and burst phases occurred at 30 degrees C, but only when a large molar excess of Mo-Fe protein with respect to Fe protein was present. The results at 10 degrees C show that the binding of acetylene to the enzyme stimulates electron flow, but that these electrons, which initially reduce protons, can only reduce acetylene after a lag phase that cannot be accommodated in the turnover time calculated under steady-state conditions.

Acetylene↗
PubMed1976

Nitrogenases from Klebsiella pneumoniae and Clostridium pasteurianum. Kinetic investigations of cross-reactions as a probe of the enzyme mechanism.

In combination with the Mo-Fe protein of nitrogenase from Klebsiella pneumoniae, the Fe protein of nitrogenase from Clostridium pasteurianum forms an active enzyme with novel properties different from those of either of the homologous nitrogenases. The steady-state rates of reduction of acetylene and H+ are 12% of those of the homologous system from C.pasteurianim. Acetylene reductase activity exhibited an approx. 10min lag at 30 degrees C before the rate of reduction became linear, consistent with a once-only activation step being necessary for acetylene reduction to occur. No such lag was observed for H2 evolution. The activity with N2 as a reducible substrate was very low, implying that acetylene reductase activity is not necessarily an accurate indication of nitrogen-fixing ability. This is of particular relevance to studies on mutant and agronomically important organisms. Stopped-flow spectrophotometric studies showed unimolecular electron transfer from the Fe protein to the Mo-Fe protein to occur at the same rate (k2 = 2.5 X 10(2)s-1) and with the same dependence on ATP concentration (apparent KD = 400 muM) as with the homologous Klebsiella nitrogenase. However, an ATP/2e ratio of 50 was obtained for H2 evolution, indicating that ATP hydrolysis had been uncoupled from electron transfer to substrate. These data indicate that ATP has at least two roles in the mechanism of nitrogenase action. The combination of the Mo-Fe protein of nitrogenase of C.pasteurianim and the Fe protein of K.pneumoniae were inactive in all the above reactions, except for a weak adenosine triphosphatase activity, 0.5% of that of the homologous K.pneumoniae system.

Acetylene↗
PubMed1976

The molybdenum--iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide 'mapping'.

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide 'maps' after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.

Amino Acids↗
PubMed1975

Nitrogenases of Klebsiella pneumoniae and Azotobacter chroococum. Complex formation between the component proteins.

1. Sedimentation velocity analyses of mixtures of highly purified component proteins of Azotobacter chroococcum are consistent with the formation of a tight 1 : 1 complex in the absence of Na2 S2 O4. 1 : 1 complex formation between complementary proteins from A. chroococcum and Klebsiella pneumoniae was also observed. The addition of 5 mM Na2 S2 O4 weakened the interaction between the A. chroococcum proteins and also the interaction between complementary proteins of A. chroococcum and K. pneumoniae. 2. Steady-state kinetic data for acetylene reduction at low protein concentrations have been used to calculate association constants at 30 degrees C for the 1 : 1 protein complexes of nitrogenase proteins from A. chroococcum, K. pneumoniae and mixtures of complementary proteins from both organisms. Values centered around 3 - 10(7) M-1 were obtained. 3. The temperature dependence of the association constant for the complex formed by the K. pneumoniae proteins exhibited a sharp break at 17 degrees C with deltaH = 0 and deltaH = 418 kJ - mol-1 above and below 17 degrees C, respectively. 4. The Arrhenius plot for acetylene reduction by the complex formed by the K. pneumoniae proteins was linear over the range 12-40 degrees C with deltaH = 80 kJ - mol-1.

Azotobacter↗
PubMed1974

Nitrogenase.

Explore the source record for details and available documents.

Adenosine Triphosphate↗
PubMed1973

Nitrogenase of Klebsiella pneumoniae: evidence for an adenosine triphosphate-induced association of the iron-sulphur protein.

The effect of various nucleotides on the Fe-containing component of nitrogenase of Klebsiella pneumoniae was investigated by ultracentrifugation and thiol-group reactivity towards 5,5'-dithiobis-(2-nitrobenzoate). In the absence of Na(2)S(2)O(4), ATP and ADP produced changes in sedimentation behaviour and thiol-group reactivity consistent with association of the protein.

Adenosine Diphosphate↗
PubMed1973

Nitrogenase of Klebsiella pneumoniae. Interaction of the component proteins studied by ultracentrifugation.

Sedimentation-velocity analyses of mixtures of the component proteins of nitrogenase of Klebsiella pneumoniae at a 1:1 molar ratio, showed a single peak of sedimentation coefficient (12.4S) considerably greater than that obtained for the larger (Fe+Mo-containing) protein centrifuged alone (10.4S). When the ratio exceeded 1:1 (the smaller Fe-containing protein in excess) an additional peak corresponding in sedimentation coefficient (about 4.5S) to free Fe-containing protein appeared. When proteins, which had been inactivated by exposure to air were used, no interaction occurred. Na(2)S(2)O(4) at 2mm both reversed and prevented interaction between the two proteins; sedimentation coefficients corresponded to those of the proteins when centrifuged alone. These results demonstrate the formation of a complex between the nitrogenase proteins, and, together with data of activity titration curves, are consistent with the formulation of the nitrogenase complex of K. pneumoniae as (Fe-containing protein)-(Fe+Mo-containing protein).

Bacterial Proteins↗
PubMed1972

Nitrogenase of Klebsiella pneumoniae. Purification and properties of the component proteins.

1. Nitrogenase from the facultative anaerobe Klebsiella pneumoniae was resolved into two protein components resembling those obtained from other nitrogen-fixing bacteria. 2. Both proteins were purified to homogeneity as shown by the criteria of disc electrophoresis and ultracentrifugal analysis. 3. The larger component had a mol.wt. of 218000 and contained one Mo atom, 17Fe atoms and 17 acid-labile sulphide groups/mol; it contained two types of subunit, present in equal amounts, of mol.wts. 50000 and 60000. All the common amino acids were present, with a predominance of acidic residues. The apparent partial specific volume was 0.73; ultracentrifugal analysis gave s(0) (20,w)=11.0S and D(0) (20,w)=4.94x10(-7)cm(2)/s. The specific activities (nmol of product formed/min per mg of protein) when assayed with the second nitrogenase component were 1500 for H(2) evolution, 380 for N(2) reduction, 1200 for acetylene reduction and 5400 for ATP hydrolysis. The reduced protein showed electron-paramagnetic-resonance signals at g=4.3, 3.7 and 2.015; the Mössbauer spectrum of the reduced protein consisted of at least three doublets. The u.v. spectra of the oxidized and reduced proteins were identical. On oxidation the absorbance increased generally throughout the visible region and a shoulder at 430nm appeared. The circular-dichroism spectra of both the oxidized and reduced proteins were the same, consisting mainly of a negative trough at 220nm. 4. The smaller component had mol.wt. 66800 and contained four Fe atoms and four acid-labile sulphide groups in a molecule comprising two subunits each of mol.wt. 34600. All common amino acids except tryptophan were present, with a predominance of acidic residues. The apparent partial specific volume calculated from the amino acid analysis was 0.732, which was significantly higher than that obtained from density measurements (0.69); ultracentrifugal analysis gave s(0) (20,w)=4.8S and D(0) (20,w)=5.55x10(-7)cm(2)/s. The specific activities (nmol of product formed/min per mg of protein) were 1050 for H(2) evolution, 275 for N(2) reduction, 980 for acetylene reduction and 4350 for ATP hydrolysis. The protein was not cold-labile. The reduced protein showed electron-paramagnetic-resonance signals in the g=1.94 region. The Mössbauer spectrum of the reduced protein consisted of a doublet at 77 degrees K. The u.v. spectra of reduced and O(2)-inactivated proteins were identical, and inactivation by O(2) generally increased the absorbance in the visible region and resulted in a shoulder at 460nm. The circular-dichroism spectra exhibited a negative trough at 220nm and inactivation by O(2) decreased the depth of the trough. 5. The reduction of N(2) and acetylene, and H(2) evolution, were maximal at a 1:1 molar ratio of the Fe-containing protein to the Mo-Fe-containing protein; excess of the Mo-Fe-containing protein was inhibitory. All reductions were accompanied by H(2) evolution. The combined proteins had no ATP-independent hydrogenase activity.

Amino Acids↗
PubMed1971

Microbial oxidation of amines. Spectral and kinetic properties of the primary amine dehydrogenase of Pseudomonas AM1.

1. An improved procedure is reported for purification of the amine dehydrogenase from methylamine-grown Pseudomonas AM1 which yielded a product homogeneous by sedimentation and disc-electrophoretic analysis, with molecular weight of 133000. 2. The purified enzyme had absorption maxima at 280 and 430nm. On aging, a third peak appeared at 325nm, and the 430nm peak decreased in intensity. This spectrum was independent of pH. 3. Addition of 2.5mm-semicarbazide, phenylhydrazine, hydrazine or hydroxylamine produced modified spectra with maxima respectively at 400, 440, 395 and 425nm. 4. Aerobic addition of methylamine resulted in a bleaching of the 430nm peak and the appearance of a new one at 325nm. This spectral change was retained after removal of the methylamine by dialysis. The original spectrum could be restored on addition of phenazine methosulphate. 5. Addition of borohydride partially inactivated the enzyme and produced spectral changes similar to those observed with methylamine. Pre-treatment with methylamine prevented the inactivation by borohydride. The degree of inactivation could be increased by alternate phenazine methosulphate and borohydride treatments. 6. The addition of methylamine or borohydride each caused shifts in the fluorescence emission maximum from 348 to 380nm. 7. Lineweaver-Burk plots of reciprocal activity against reciprocal concentration of either of the substrates n-butylamine or phenazine methosulphate were consistent with a mechanism that involves interconversion of two free forms of the enzyme by the two substrates. 8. The enzyme, although spectrally modified, was not inactivated by dialysis against diethyldithiocarbamate, and contained about 0.27 g-atom of copper/mol, with small traces of cobalt, iron and zinc. 9. Conventional methods of resolution did not release the prosthetic group. Heat denaturation after treatment of the enzyme with methylamine liberated a yellow chromophore which did not reactivate resolved aspartate aminotransferase, and whose spectral, electrophoretic and fluorescence properties did not agree with any recognizable pyridoxal derivatives. 10. Despite the inconclusive results with the isolated chromophore, the observations on the enzyme suggest that it may contain a pyridoxal derivative bound as a Schiff's base which is converted into the pyridoxamine form on aerobic treatment with methylamine and reconverted into the pyridoxal form with phenazine methosulphate. 11. The copper detected is probably not involved in the enzyme mechanism, since most copper-chelating agents are not inhibitory, and since the enzyme does not react with oxygen.

Amines↗
PubMed1971

Microbial oxidation of amines. Partial purification of a mixed-function secondary-amine oxidase system from Pseudomonas aminovorans that contains an enzymically active cytochrome-P-420-type haemoprotein.

1. Crude extracts of Pseudomonas aminovorans grown on methylamine, di-methylamine, trimethylamine or trimethylamine N-oxide contain an enzyme or enzyme system catalysing the NADH- or NADPH- and oxygen-dependent oxidation of dimethylamine to methylamine and formaldehyde. 2. The enzyme has been partially purified about five-fold. It is unstable, but can be stabilized by addition of 5% (v/v) ethanol. 3. The partially purified enzyme will utilize either NADH (K(m) 6.5mum) or NADPH (K(m) 13.2mum): The following secondary amines have been shown to be substrates: dimethylamine, ethylmethylamine, diethylamine, methyl-n-propylamine, ethyl-n-propylamine, n-butylmethylamine and N-methylethanolamine. The K(m) values and comparative reaction rates for each substrate have been determined. Where the alkyl groups are different, the aldehyde products are derived from both groups. 4. The enzyme system has a pH optimum of 6.8 and is inhibited by mercurials, thiol compounds, cyanide and carbon monoxide. 5. The partially purified preparation had a spectral maximum at 412nm with shoulders at 427 and 550nm. Reduction with dithionite or NAD(P)H bleached the 412nm peak, and the shoulder at 427nm became a peak. Additional peaks appeared at 550 and 580-588nm. Reduction of a preparation bubbled with carbon monoxide enhanced and sharpened the Soret peak and caused it to shift to 422nm. 6. Analysis of the preparation showed the presence of flavin, acid-extractable iron and non-acid-extractable iron in the proportion 1.1:1.9:1. On reduction with dithionite or NADPH the preparation showed an electron-paramagnetic-resonance signal at around g=1.946.

Amines↗
PubMed1968

Purification and properties of an amine dehydrogenase from Pseudomonas AM1 and its role in growth on methylamine.

1. Whole cells of Pseudomonas AM1 grown on methylamine oxidize methylamine, formaldehyde and formate. Crude extracts oxidize methylamine only if supplemented with phenazine methosulphate. 2. By using a spectrophotometric assay, the methylamine-oxidizing enzyme has been purified 20-fold in 31% yield. 3. The enzyme is a dehydrogenase, unable to utilize oxygen, NAD, NADP, flavines or menadione as electron acceptors, but able to utilize phenazine methosulphate, ferricyanide, cytochrome c or brilliant cresyl blue. 4. The enzyme is non-specific, readily oxidizing aliphatic monoamines and diamines, histamine and ethanol-amine. Secondary and tertiary amines, quaternary ammonium salts and aromatic amines are not oxidized. 5. The pH optima for methylamine, n-pentylamine and putrescine are respectively 7.6, 8.0 and 8.5. 6. The K(m) value for methylamine is 5.2mum and that for phenazine methosulphate 56mum. 7. The enzyme will withstand heating for 15min. at 80 degrees without loss of activity, but is inactivated at higher temperatures. It is not inactivated by any pH value between 2.6 and 10.6. 8. The dehydrogenase is inhibited by semicarbazide (K(i) 3.35mum), isoniazid (K(i) 1.17mum), cuprizone (K(i) 0.49mum), p-chloromercuribenzoate (K(i) 0.45mm) and quinacrine (K(i) 12.1mm). 9. The enzyme is absent from succinate-grown cells, and, during adaptation from succinate to methylamine, activity appears before growth on methylamine begins.

Amines↗