ABSTRACT
The effect of oxygen concentration on the regiospecificity of the soybean lipoxygenase-1 dioxygenation reaction was studied. At low oxygen concentrations (<5 microM), a dramatic change in the regiospecificity of the enzyme was observed with the hydroperoxy-octadecadienoic acid (HPOD) 13:9 ratio closer to 50:50 instead of the generally reported 95:5. This alteration of regiospecificity is not an isolated phenomenon, since it occurs during a reaction carried out under "classical" conditions, i.e. in a buffer saturated with air before the reaction. beta-carotene bleaching and electronic paramagnetic resonance findings provided evidence that substrate-derived free radical species are released from the enzyme. The kinetic scheme proposed by Schilstra et al. (Schilstra, M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1994) Biochemistry 33, 3974-3979) was thus expanded to account for the observed variations in specificity. The equations describing the branched scheme show two different kinetic pathways: a fully enzymatic one leading to a regio-isomeric composition of 13-HPOD:9-HPOD = 95:5, and a semienzymatic one leading to a regio-isomeric composition of 13-HPOD:9-HPOD = 50:50. The ratio between the two different pathways depends on oxygen concentration, which thus determines the overall specificity of the reaction.
Subject(s)
Glycine max/enzymology , Lipid Peroxides , Lipoxygenase/metabolism , Models, Chemical , Oxygen/pharmacology , Computer Simulation , Electron Spin Resonance Spectroscopy , Isomerism , Linoleic Acid/metabolism , Linoleic Acids/metabolism , Potentiometry , Substrate Specificity/drug effects , beta Carotene/metabolismABSTRACT
Rhodopseudomonas capsulata chromatophores were immobilized with a co-crosslinking method. Immobilization was used as a tool for a defined modification of the chromatophore environment to study ATP production over a long period of time. The light-induced phosphorylation of ADP as a function of time was studied with chromatophores under different conditions: (a) native chromatophores with and without the hexokinase ATP-trapping system; (b) immobilized chromatophores without hexokinase, with the enzyme added in the bulk solution and with the enzyme co-immobilized in the matrix. The overall amount of ATP produced as a function of ADP concentration was studied for native and immobilized chromatophores. The global phosphorylation performed was also studied as a function of the amount of biological material used. The results can be explained by an effect of the ATP/ADP ratio. The results given by the immobilization show that the important point is not the ATP/ADP ratio in the bulk solution but the ratio value in the microenvironment of the chromatophore itself.
Subject(s)
Bacterial Chromatophores/metabolism , Oxidative Phosphorylation , Rhodopseudomonas/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Electron Transport , Enzymes, Immobilized/metabolism , Hexokinase/metabolism , Kinetics , LightABSTRACT
One of the limiting steps in the further development of enzyme technology is the regeneration of cofactors, especially the pyridinic nucleotide cofactors. Immobilization of alcohol dehydrogenase and steroid dehydrogenase is described. In the last case stabilized enzymes could work in non aqueous solvents. Co-enzyme molecules are bound in the immediate vicinity of the active site of the enzyme. Cofactor regeneration was performed with an electron carrier (Phenazine methosulfate). Ageing phenomena were observed. The co-immobilization of superoxide dismutase gives rise to an increase of stability.
Subject(s)
Alcohol Dehydrogenase/chemistry , Enzymes, Immobilized/chemistry , Enzymes/chemistry , Binding Sites , Biochemistry/methods , Cross-Linking Reagents/chemistry , Dose-Response Relationship, Drug , Electrons , Methylphenazonium Methosulfate/chemistry , NAD/chemistry , Oxidoreductases/chemistry , Pseudomonas aeruginosa/metabolism , Solvents/chemistry , Superoxides/chemistry , Testosterone/chemistryABSTRACT
Lettuce thylakoïds were immobilized by the action of glutaraldehyde at subzero temperature in the presence of albumin. Foam structures with good mechanical properties were obtained. The activity yields for photosystem II and photosystems I + II were found equal to 71 per cent and 35 per cent respectively. The yield for ATP regeneration from ADP and Pi was 26 per cent. Increases of stability after immobilization were observed for all the functions of thylakoïds when stored and when continuously working. Spheroplasts and chromatophores from Rhodopseudomonas capsulata were immobilized with the same method; yields for ATP regeneration were found equal to 40 per cent and 70 per cent, respectively. An important increase of stability after immobilization was observed in both cases.
