Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617.

The aerobic respiratory system of the hydrocarbonoclastic marine bacterium Pseudomonas nautica 617 ends with a single terminal oxidase. It is a heme-containing membranous protein which has been demonstrated only to reduce molecular oxygen to hydrogen peroxide [Denis, M., Arnaud S. & Malatesta, F. (1989) FEBS Lett. 247, 475-479]. The purification of this oxidase was achieved in a single step through by DEAE-Trisacryl chromatography. SDS/PAGE showed the presence of four subunits. The pI was found to be 4.45 and a Mr of 130,000 was determined by gel filtration. The amino acid composition of the purified terminal oxidase has been determined. About 52% of the residues are hydrophobic, strengthening the membranous nature of this bacterial oxidase. Room temperature optical spectra are typical of heme b with a 560-nm band for the reduced form in the alpha range. The prosthetic group is made of two hemes b, one high-spin (S = 5/2, gl = 5.9, g parallel approximately 2.0), the other low-spin (S = 1/2, gz = 2.94, gy = 2.27). No other metal centre was detected by EPR. The two hemes remained unresolved in optical spectra, even at low temperature, and throughout redox titration. They behaved potentiometrically like a one-electron, single redox couple, with Em = 87 +/- 10 mV at pH 7.2 and 293 K. The purified oxidase did not oxidize ferrocytochrome c, but displayed quinol oxidase activity both with the native quinone (2419 nmol O2.min-1.mg protein-1 and commercially available coenzyme (101.74 nmol O2.min-1.mg protein-1). Exposure of the reduced enzyme to CO induced the collapse of alpha and beta bands as occurred during reoxidation. In contrast, NaCN and NaN3 fully inhibited the oxidase activity. Results are discussed with respect to other purified quinol oxidases.     

Identification by deletion analysis of an inducible protein required for plasmid pSC101-mediated tetracycline resistance.

Plasmids containing small deletions within a tetracycline (Tc) resistance gene(s) of plasmid pHA121 were isolated. Plasmid pHA121 was formed by ligating the EcoRI-digested Tc resistance plasmid pSC101 and similarly digested mini-ColE1 plasmid pHA105. The DNA deletion plasmids were constructed by digesting plasmid pHA121 DNA with the restriction endonucleases BamH1 and Sal1 and, in addition, λ exonuclease. Two plasmids, designated pJT131 and pJT133, had small deletions of approximately 0.64 to 0.8 kb and a comparison of the radioactive polypeptides synthesized in plasmid-containing minicells revealed that a 34-kdal polypeptide was not specified by either pJT131 or pJT133. We conclude that the 34-kdal polypeptide is required for the expression of Tc resistance and that its structural gene probably maps in the deleted region of pSC101 DNA.     

Temperature dependence of the electronic spin-lattice relaxation time in a 2-iron-2-sulfur protein.

The ferredoxins are characterized by a strong temperature dependence of the electronic spin-lattice relaxation time T1. The measurement of this dependence above the liquid nitrogen temperature has been presented in earlier work [1] for the 2-iron-2-sulfur ferredoxin of the blue green alga Spirulina maxima. The different relaxation mechanisms which could be efficient in this range were briefly discussed. In the present paper, we extend the measurement of the temperature dependence of T1 to the low temperature range 1.25 to 30 K. From 1.25 K to 13 K, T1 is obtained by the saturating pulse method, whereas the continuous saturation method is used from 8 K to 30 K. The experimental conditions concerning these methods are discussed. The analysis of the temperature dependence curve over the whole range 1.25 K to 133 K shows clearly that different regions must be distinguished. For each region the possible relaxation processes and the corresponding vibrational modes are discussed.     

A novel L-lactate-selective biosensor based on flavocytochrome b2 from methylotrophic yeast Hansenula polymorpha

A novel amperometric biosensor highly selective to L-lactate has been developed using L-lactate-cytochrome c oxidoreductase (flavocytochrome b2) isolated for the first time from thermotolerant methylotrophic yeast Hansenula polymorpha as biorecognition element. Different immobilization methods and low-molecular free-diffusing redox mediators have been tested for optimising the electrochemical communication between the immobilized enzyme and the electrode surface. Moreover, the possibility of direct electron transfer from the reduced form of FCb2 to carbon electrodes has been evaluated. The bioanalytical properties of FCb2-based biosensors, such as signal rise time, dynamic range, dependence of the sensor output on the pH value, the temperature and the storage stability were investigated, and the proposed biosensor demonstrated a very fast response and a high sensitivity and selectivity for L-lactate determination.     

Characterization of the soluble hydrogenase from Desulfovibrio africanus

The soluble hydrogenase from Desulfovibrio africanus has been isolated and characterized. The enzyme consists of two subunits of 65 kDa and 27 kDa. Its absorption spectrum is typical of an iron-sulfur protein. The protein contains 12 iron atoms, 10 labile sulfur atoms and 0.9 nickel atom per molecule. D. africanus hydrogenase is rapidly activated under reducing conditions and exhibits a specific activity of 570 mumoles H2 evolved/min/mg. The EPR spectrum of the oxidized enzyme shows no Ni(III) signals. Upon reduction under hydrogen, the protein sample exhibits signals due to nickel with g values at 2.21, 2.17 and 2.01 correlating with the active state of the enzyme.     

Cultured hepatoma cells for the study of enzyme regulation: induction of ornithine decarboxylase by insulin and asparagine

The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combination of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10(-5) M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present.     

A theoretical interpretation of the variations of some physical parameters within the [2Fe-2S] ferredoxin group.

A model is proposed to explain the variation of some physical parameters within the reduced [2Fe-2S] ferredoxin group. According to this model, the main effects result from a variable mixing of some d orbitals of the Fe2+ ion owing to rhombic distortion of the active site having the same geometrical character, but different in intensity, for each protein. Some peculiar experimental results such as the axial electron paramagnetic resonance spectra of adrenal ferredoxin and Pseudomonas putida ferredoxin and the electric field gradient tensor of P. putida ferredoxin are explained without assuming properties drastically different from those of the other ferredoxins, as had been suggested in the literature.     

The chromate resistance phenotype of some yeast mutants correlates with a lower level of Cr(V)-species generated in the extra-cellular medium

The paper describes the selection of chromate-resistant mutants of the yeast Pichia guilliermondii with a higher chromate-reducing activity and reports the EPR-study of Cr(V)-generation in the extra-cellular medium during the reduction of chromate by the yeast culture. It is shown that the reduction of chromate to Cr(III) species runs through the extra-cellular generation of Cr(V)-intermediate(s), thus supporting the assumption about the existence of an extra-cellular pathway of Cr(VI)-reduction. Furthermore, it is demonstrated that the chromate-resistance phenotype of tested mutants correlates with a lower stationary level of Cr(V)-species in the medium. It is thus suggested that isolated mutants can be used as sources of Cr(III)-biocomplexes due to their ability to effectively reduce chromate to Cr(III)-chelates with potential pharmacological applications.     

Chromate-resistant mutants of the yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis>: Selection and properties

Chromate-resistant mutants of the non-conventional yeast Pichia guilliermondii L2 were selected by different methods. The isolated mutants were capable of better growth and higher biomass yield at toxic (1.8–2.4 mM) chromate concentrations than the parent strain. The capacity of the mutants for extracellular chromate reduction and chelation of Cr(III) in the culture liquid was demonstrated. The effectiveness of these processes was shown to correlate with the resistance of P. guilliermondii strains to chromate. Extracellular metabolites of the yeast cells cultivated without chromate were shown to be capable of reducing chromate and forming stable soluble Cr(III)-biocomplexes.