To investigate the temperature-dependence of red cell aggregation 20 blood samples of normal donors and 20 blood samples of patients with venous ulcers of the leg were examined by photometric aggregometry at 3 degrees C, 10 degrees C, 20 degrees C, 30 degrees C and 37 degrees C. With decreasing temperature red cell aggregates become more resistant to hydrodynamic dispersion and they become more prone to growing under low shear stress. It is concluded that a decrease in temperature causes an increase in adsorptive energy of red cell aggregation, which is most likely due to an increase in molecular adsorption stress. Red cell aggregate formation as an overall process is retarded by a decrease in temperature, which is primarily due to an increase in plasma viscosity causing increased damping of aggregate formation. Accordingly the rate constant of aggregate formation corrected for plasma viscosity increases with decreasing temperature. The temperature-dependence of the kinetic parameters can be explained by a theoretical model that suggests the increase in contact area between aggregating red blood cells as the rate-limiting step of red cell aggregation. As a whole red cell aggregation is favoured by lowering of temperature.
NADH oxidase was extracted from the membranes of Acholeplasma laidlawii with buffer containing 3% Triton X‐100 and subsequently purified by several chromatographic steps. The final preparation was essentially homogeneous as judge by gel electrophoresis under nondenaturing conditions.
The enzyme appears to be a copper‐containing iron‐sulfur flavoprotein (FMN:Cu:Fe:labile S = 1:1:6:6). The enzyme, containing a high fraction of hydrophobic amino acids, is composed of three subunits of molecular weight 65000, 40000 and 19000.
When oxygen is used as electron acceptor the purified enzyme demonstrates a specific activity of 58.0 IU/mg of protein and catalyzes the formation of H2O2in nearly stoichiometric amount. The apparent Km value for NADH is estimated to be 0.4 mM (pH 7.4). NADPH cannot serve as a substrate for the enzyme. In additionto the NADH oxidase activity, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (ferricyanide, dichloroindophenol, cytochrome c). Metal‐chelating agents and mercurials are shown to inhibit the activity of the enzyme.
From electron paramagnetic resonanace and optical absorption measurements evidence was obtained that the flavin semiquinone radical in the NADH oxidase has a high air‐stability, and that the flavin shuttles between the fully reduced and the semiquinone state upon electron transport from NADH to the electron acceptors. Inhibition of the NADH oxidoreductase activities by superoxide dismutase indicaaataaes that O−2 serves as an intermediate in the electron transfer from NADH to all electron acceptors used in this work. In addition to electron transfer via the superoxide radical O−2, an alternative pathway probably involving Fe‐S centers is operative.From these results and literature date we present a reaction schme for electron transport from NADH to the various electron acceptors.
From the prokaryotic microorganismMycoplasma capricolum an FAD-containing NADH oxidase has been purified by preparative FPLC to homogeneity, as judged by polyacrylamide gel electrophoresis. The apparent molecular mass of the enzyme was found to be 72.5 kDa, with an isoelectric point of 5.2, and no detectable subunits. No rion, copper, manganese or molybdenium could be detected. On the basis of a minimum molecular mass of 72.5 kDa a ratio of FAD/protein of 1 :1 could be derived. Its amino-acid composition, the light absorption and the fluorescence spectra are presented.
Reinigung und Eigenschaften einer FAD enthaltenden NADH-Oxidase aus Mycoplasma capricolumZusammenfassung: Aus dem prokaryotischen Mikroorganismus Mycoplasma capricolum ist eine FAD enthaltende NADH-Oxidase durch präparative FPLC in reiner Form isoliert worden. Als Reinheitskriterium diente die Polyacrylamidgel-Elektrophorese. Die scheinbare Molekularmasse des Enzyms beträgt 72.5 kDa, es besitzt einen isoelektrischen Punkt von 5.2.Es konnten keine Untereinheiten nachgewiesen werden. Weder Eisen, Kupfer, Mangan noch Molybdaen konnten nachgewiesen werden. Auf der Grundlage einer minimalen Molekularmasse von 72.5 kDa konnte ein Verhältnis FAD/Protein von l: l bestimmt werden. Die Aminosäure-zusammensetzung sowie das Lichtabsorptionsund Fluoreszenzspektrum werden beschrieben.
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