Protein damage, induced by small amounts of photodynamically generated singlet oxygen or hydroxyl radicals

The influence of limited oxidation of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and myoglobin by singlet oxygen and by hydroxyl radicals was investigated. The intrinsic fluorescence of glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase decreased rapidly during oxidation, indicating a conformational change of the protein molecules. The free energy of isothermal unfolding in urea solutions was increased by singlet oxygen, but decreased by hydroxyl radical attack. The velocity of refolding of the denatured protein after dilution of the denaturant was increased by exposure to either singlet oxygen or hydroxyl radicals, with one exception: the velocity of refolding of myoglobin, oxidized by singlet oxygen, was strongly decreased. Hydroxyl radicals produced covalently crosslinked protein aggregates and some fragmentation, whereas singlet oxygen produced only crosslinked aggregates with glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase. All oxidized proteins were more susceptible to proteolysis by elastase and proteinase K, as compared to the undamaged proteins. Singlet oxygen-induced crosslinked aggregates were degraded very rapidly by elastase. Hydroxyl radical-induced aggregates of glyceraldehyde-3-phosphate dehydrogenase were also degraded very rapidly by this enzyme, but hydroxyl radical-induced aggregates of alcohol dehydrogenase were resistent to enzymatic degradation. The results indicate that limited protein oxidation may have a pronounced influence on several properties of the protein. The effects vary, however, with varying proteins and with the oxidizing species.     

Photodynamic protein cross-linking

Exposure of spectrin to visible light in the presence of a photosensitizer results in photo-oxidation of sensitive amino acid residues and covalent cross-linking of the polypeptides. In a previous paper the cross-linking was ascribed to a secondary reaction between photo-oxidized histidine residues and amino groups. The following observations, described in this paper, are in accordance with this supposition. (1) During illumination of spectrin in the presence of a photosensitizer a pronounced photo-oxidation of histidine residues takes place. (2) Simultaneously a decrease of free amino groups is observed. (3) Semicarbazide protects against cross-linking and is bound to a histidine photo-oxidation product in spectrin. (4) The pH profile of histidine photo-oxidation and subsequent reaction with amino groups is similar to the pH profile of spectrin cross-linking. Amidination of NH₂ groups in spectrin does not inhibit cross-linking, as visualized by gel electrophoresis. On the other hand aminidation of denatured myoglobin causes a 50% inhibition of cross-linking. These observations support the notion of NH₂-involvement in cross-linking but also demonstrate, that other photodynamic cross-linking mechanisms exist.     

The influence of ATP on sugar uptake mediated by the constitutive glucose carrier of Saccharomyces cerevisiae

The glucose carrier of Saccharomyces cerevisiae transports the phosphorylatable sugars glucose, mannose, fructose and 2-deoxy-D-glucose (2-dGlc) and the non-phosphorylatable sugar 6-deoxy-D-glucose (6-dGlc). Reduction of the ATP concentration by, for example, incubating cells with antimycin A, results in a decrease in uptake of 2-dGlc and fructose. These uptake velocities can be increased again by raising the ATP level. These results establish a role of ATP in sugar transport. Transport of glucose and mannose is less affected by changes in the ATP concentration than 2-dGlc and fructose uptake, while the 6-dGlc transport is independent of the amount of ATP in the cells. Also, reduction of the kinase activity by incubation with xylose diminished transport of 2-dGlc and fructose, while the uptake of glucose and mannose remained unchanged. It is discussed that these results are due to transport-associated phosphorylation with ATP as substrate and the hexokinases and the glucokinase as phosphorylating enzymes.     

Acetaminophen protects human erythrocytes against oxidative stress

Acetaminophen protects human erythrocytes against various modes of oxidative stress. Protection against ozone-induced damage can be explained by a direct scavenging reaction between the drug and ozone. With t-butylhydroperoxide acetaminophen appeared to be an effective scavenger of radicals, generated in secondary reactions. The protection by acetaminophen against t-butylhydroperoxide- and hydrogen peroxide-induced lipid peroxidation and K+-leakage can be explained along these lines. In all cases the protective effect of acetaminophen was attended with covalent binding of acetaminophen to membrane proteins.     

The effect of abscisic acid on sugar levels in seedlings of Phaseolus vulgaris L. cv. redland pioneer

Abscisic acid (ABA) caused an increase in total-sugar and a 3-fold increase in reducing-sugar content in the roots of intact bean seedlings. The level of reducing sugars was also increased in the stem but total sugar levels remained unaffected by ABA. ABA also increased reducing-sugar content of the root in seedlings with cotyledons removed but in this case the reducing- and total-sugar contents of the leaf were reduced. However, ABA did not affect reducing- and total-sugar levels in excised bean root systems. The observed increase in sugar content of the root of intact bean seedlings appears to be the consequence of an ABA-induced stimulation of sugar transport from the shoot to the root. It is proposed that a relationship may exist between the effect of ABA on sugar transport and its effect on ion transport in excised root systems and in intact seedlings.     

Phase transitions in the yeast cell membrane the influence of temperature on the reconstitution of active dry yeast

Active dry yeast, with a water content of about 7%, can be reconstituted in water at 38–42°C. When the dried cells are rehydrated in cold water, the cells lose their viability. The influence of temperature on the reconstitution process shows that a phase transition is involved in this cold-shock effect. The experimental results indicate that, in addition to membrane lipids, vicinal water may be involved in this phase transition.     

Fixation of dissolved silicate and sedimentation of biogenic silicate in the lower river Rhine during diatom blooms

Concentrations of dissolved silicate and particulate biogenic silicate were measured in three branches of the lower river Rhine in The Netherlands in order to analyse the role of this element in the eutrophication of the river basin. Particulate silicate followed the seasonal development of the phytoplankton, which was dominated by diatoms. The concentration of dissolved silicate fell during blooms (< 0.1 mg.l^–1 ), but the amounts of biogenic silicate measured ( 1 mg.l^–1) were insufficient to explain the seasonal decrease in the dissolved fraction; this indicates retention of silicate upstream. Some particulate biogenic silicate in river water settled in man-made sedimentation areas in the Rhine delta. The observations suggest that changes in silicate fixation in the Rhine may have contributed to the incidence of non-diatom phytoplankton blooms in receiving waters.