Johnny Van Steveninck

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-link mechanisms exist.

Kinetic analysis of simultaneously occurring proton-sorbose symport and passive sorbose transport in Saccharomyces fragilis

Abstract Sorbose transport in Saccharomyces fragilis takes place both via an active sugar-H + symport system and via facilitated diffusion. To establish whether the two modes of transport proceed via the same transporter or via two different carriers, the kinetic consequences of both models were investigated. The kinetic equations for initial transport were derived for three possible reaction sequences with respect to sugar and H + binding to the symport carrier: random binding and obligatory ordered binding with either sugar or H + binding first, yielding six sets of kinetic parameters. Analysis of experimental data of sorbose transport in S. fragilis showed the existence of separate carriers for active, sorbose-H + symport and facilitated diffusion. Furthermore, it could be concluded that the symport carrier shows random binding of sugar and H + . In recent literature, a similar combination of active and passive sugar transport in Rhodotorula gracilis and Chlorella vulgaris was interpreted as two modes of action of the same carrier, viz., active symport via the protonated, and facilitated diffusion via the unprotonated carrier. Analysis of the experimental data according to the criteria presented in this paper showed, however, that this supposition is untenable and that two different carriers must also be involved in these micro-organisms.

Kinetic analysis of H+/methyl β-d-thiogalactoside symport in Saccharomyces fragilis

Abstract A theoretical description of initial uptake kinetics of H+/sugar symport is given, with emphasis on the differences between carrier and non-carrier systems. Transport of methyl β- d -thiogalactoside in Saccharomyces fragilis is shown to proceed via the inducible lactose transporter. Uptake of this sugar stimulates electrogenic H+ influx. Together with the correlation between methyl β- d -thiogalactoside accumulation and the proton-motive force this shows that transport proceeds via H+ symport. Kinetic analysis of initial influx revealed that transport proceeds via a single transport system, sensitive to changes in membrane potential. The pH dependence of the kinetic parameters showed that Kapp is almost pH insensitive, whereas Vapp decreases strongly at increasing extracellular pH. It is shown that transport proceeds, most likely, via a non-carrier system, with random binding of H+ and sugar, in a system where binding of the first ligand does not influence binding of the second.

Regulation of sugar transport systems of Kluyveromyces marxianus: the role of carbohydrates and their catabolism

In Kluyveromyces marxianus grown on a glucose-containing synthetic medium four different sugar transporters have been identified. In cells, harvested during the exponential phase, only the constitutive glucose/fructose carrier, probed with 6-deoxy-D-glucose or sorbose, appeared to be active. In cells from the stationary phase three proton symporters can be active, recognizing 6-deoxyglucose (a glucose/galactose carrier), sorbose (a fructose carrier) and galactosides (lactose carrier), respectively. These symporters appeared to be sensitive to catabolite inactivation. This process is induced by incubating cells in the presence of glucose, fructose or mannose. Catabolite inactivation was not influenced by the inhibitor of protein synthesis, anisomycin. Derepression of the proton/sorbose and the proton/galactoside symporters proceeded readily when cells were incubated in a medium without glucose. Activation of the proton/galactose symporter needed, in addition, the presence of specific molecules (inducers) in the medium. The activation of each of these active transport systems was inhibited by anisomycin, showing the involvement of protein synthesis.

The influence of ozone on human red blood cells. Comparison with other mechanisms of oxidative stress

Exposure of red blood cells to ozone resulted in K+ leakage, lipid peroxidation and inhibition of some membrane-associated enzymes. On the other hand, carrier-mediated transport of glucose, leucine, sulfate and glycerol and the nonspecific permeation of glycerol, L-glucose and erythritol were not affected by ozone. The cellular level of reduced glutathione declined, whereas the ATP content of the cells was quite insensitive to ozone exposure. It was shown that, most probably, lipid peroxidation and K+ leakage are not causally related. Further, K+ leakage did not reflect gradual, progressive loss of K+ from all cells simultaneously, but occurred in an all-or-none fashion. Finally, ozone-induced damage was compared to damage induced by H2O2, t-butyl hydroperoxide and photosensitizers plus light. It appeared that the pathways leading to membrane deterioration are quite dissimilar in these various forms of oxidative stress.

Kinetic analysis of 2-deoxy-d-glucose uptake in Saccharomyces fragilis

Abstract During uptake of 2-deoxy- d -glucose by Saccharomyces fragilis an alkalinization of the medium occurs, suggesting the possibility that deoxyglucose may be a substrate of a proton symport system. Therefore the uptake kinetics were analyzed according to the criteria presented in a previous paper. It is shown that transport proceeds via only one uptake system. Both the kinetic parameters and the steady-state accumulation level appeared to be virtually pH-independent. Deoxyglucose uptake stimulates H+-influx and K+-efflux. However, the H+/deoxyglucose stoichiometry appeared to depend on pH. It is discussed that deoxyglucose uptake can not be interpreted in terms of proton symport, but that the experimental data are in accordance with the previously proposed phosphotransferase transport system.

The influence of uncouplers on proton-sugar symport in Saccharomyces fragilis

Abstract Uncouplers of oxidative phosphorylation inhibit proton-sugar symport in Saccharomyces fragilis . However, they do not induce efflux of accumulated sugar. It is shown that the effect cannot be explained by uncoupler-induced alterations in the transmembrane potential or transmembrane pH difference. It is also indicated that a decrease in intracellular pH is not involved in inhibition of sugar transport. It is argued that inhibition of transport by uncouplers is most likely caused by a direct interaction with the translocator.

Effects of semicarbazide on oxidative processes in human red blood cell membranes.

Semicarbazide can interfere with oxidative processes in the red blood cell membrane via different modes of action. Treatment of human red blood cell membranes with O3, results, among other effects, in cross-linking of membrane proteins and inhibition of glyceraldehyde-3-phosphate dehydrogenase activity. Semicarbazide inhibits these effects by acting as an O3 scavenger. The effect of semicarbazide as an O3 scavenger is complicated by the fact that ozonolysis of semicarbazide yields a product that causes inhibition of glyceraldehyde-3-phosphate dehydrogenase. Glyceraldehyde-3-phosphate dehydrogenase inhibition can also be provoked by incubation of membrane suspensions with O3-treated phospholipids. Semicarbazide prevented this effect by interaction with an inhibitory O3-phospholipid reaction product. Protoporphyrin-induced photodynamic cross-linking of membrane proteins is chemically distinct from O3-induced cross-linking. Photodynamic cross-linking is also inhibited by semicarbazide, in this case via reaction with a histidine photooxidation product.

The involvement of high-energy phosphate in 2-deoxy-d-glucose transport in Kluyveromyces marxianus

Abstract Under aerobic conditions 2-deoxy- d -glucose was accumulated in Kluyveromyces marxianus mainly in a phosphorylated form. During sugar uptake both ATP, polyphosphate and orthophosphate levels decreased. Under anaerobic conditions considerably less sugar was taken up. The intracellular free sugar concentration did not exceed the medium concentration, whereas sugar phosphorylation leveled off at about 3 μmol/g yeast. In response to anaerobic 2-deoxy- d -glucose uptake only ATP and polyphosphate appeared to decrease. Within the experimental error sugar phosphorylation was counterbalanced by the polyphosphate decrease. Pulse labeling experiments revealed transport-associated phosphorylation under these anaerobic conditions, Further, kinetic studies on permeabilized cells showed that cytoplasmic ATP could not be the phosphoryl donor in this transport-associated phosphorylation. These results confirm and extend previous observations, indicating that polyphosphate plays a crucial role in 2-deoxy- d -glucose transport in Kluyveromyces marxianus .

The influence of anesthetics on glycerol and K+ translocation across the membrane of red blood cells

A wide variety of chemically unrelated anesthetics protects red blood cells against osmotic hemolysis. The protective effects of most anesthetics are mutally additive, suggesting common rather than separate mechanisms of action. Other properties shared by these anesthetics are their inhibition of facilitated diffusion of glycerol into human erythrocytes and their stimulation of passive transport of this solute in bovine red blood cells. With respect to their influence on passive K+ translocation. anesthetics can be divided into two groups. Neutral and anionic anesthetics cause in isotonic NaCl solutions a large K+ loss and in hypotonic NaCl solutions a K+ leakage which decreases rapidly with increasing cell volume. Cationic anesthetics have the opposite effect: no K+ loss occurs at the optimal protective concentration, whereas at higher concentrations a K+ leakage occurs which increases with increasing cell volume. The possible significance of these differences in action is discussed.