Peter J.A. Van den Broek

Ascorbate Stimulates Ferricyanide Reduction in HL-60 Cells through a Mechanism Distinct from the NADH-dependent Plasma Membrane Reductase

The impermeable oxidant ferricyanide is reduced by the plasma membrane redox system of HL-60 cells. The rate of reduction is strongly enhanced by ascorbate or dehydroascorbate. The aim of this study was to determine the mechanism by which ascorbate and dehydroascorbate accelerate ferricyanide reduction in HL-60 cells. Addition of ascorbate or dehydroascorbate to cells in the presence of ferricyanide led to the intracellular accumulation of ascorbate. Control experiments showed that extracellular ascorbate was rapidly converted to dehydroascorbate in the presence of ferricyanide. These data suggest that intracellular ascorbate originates from extracellular dehydroascorbate. Accumulation of ascorbate was prevented by inhibitors of dehydroascorbate transport into the cell. These compounds also strongly inhibited ascorbate-stimulated ferricyanide reduction in HL-60 cells. Thus, it is concluded that the stimulation of ferricyanide reduction is dependent on intracellular accumulation of ascorbate. Changing the α-tocopherol content of the cells had no effect on the ascorbate-stimulated ferricyanide reduction, showing that a nonenzymatic redox system utilizing α-tocopherol was not involved.p-Chloromercuribenzenesulfonic acid strongly affected ferricyanide reduction in the absence of ascorbate, whereas the stimulated reaction was much less responsive to this compound. Thus, it appears that at least two different membrane redox systems are operative in HL-60 cells, both capable of reducing ferricyanide, but through different mechanisms. The first system is the ferricyanide reductase, which uses NADH as its source for electrons, whereas the novel system proposed in this paper relies on ascorbate.

Photodynamic treatment of yeast cells with the dye Toluidine blue : all-or-none loss of plasma membrane barrier properties

Photodynamic treatment of Kluyveromyces marxianus with the sensitizer Toluidine blue leads to the loss of colony forming capacity. In this paper, the influence of this treatment on the barrier properties of the plasma membrane has been studied. Photodynamic treatment with the dye Toluidine blue resulted in efflux of potassium ions and E260-absorbing material. Moreover, cells became stainable with erythrosine. It is concluded that the permeability change induced by photodynamic treatment proceeds in an all-or-none fashion. Treatment of this yeast strain, with the dye and light, also induced a diminution of the cell volume. This process is most likely not coupled to the cellular potassium content, but rather to the integrity of the vacuole. These data suggest that the vacuole has an important function in the maintenance of cell volume. Finally, it was observed that the loss of cell viability was not induced by the all-or-none loss of barrier properties.

INTRACELLULAR DAMAGE IN YEAST CELLS CAUSED BY PHOTODYNAMIC TREATMENT WITH TOLUIDINE BLUE

Abstract— The positively charged photosensitizer toluidine blue (TB) can induce loss of clonogenicity in Kluyveromyces marxianus. Previous studies have revealed that, as a consequence of the localization of this dye at the cell surface, photodynamic action results in extensive damage at the level of the plasma membrane. In this paper, a study is reported on the effect of photodynamic treatment with TB on intracellular enzymes. It is shown that treatment with TB and light resulted in the inhibition of alcohol dehydrogenase, cytochrome c oxidase, glyceraldehyde‐3‐phosphate dehydrogenase and hexokinase. Photodynamic treatment also lowered the ATP levels. The ATP levels could be partially restored in the presence of glucose but not with ethanol. Toluidine blue binding experiments revealed that photodynamic treatment caused a rapid increase in the amount of cell‐associated dye. Moreover, it also appeared that this treatment decreased the binding of TB to the cell surface. It is concluded that TB enters the cell during the first minutes of illumination, whereafter intracellular enzymes are inactivated. The data indicate that photodynamic damage of intracellular sites contributes to the loss of viability.

THE EFFECT OF PHOTODYNAMIC TREATMENT OF YEAST WITH THE SENSITIZER CHLOROALUMINUM PHTHALOCYANINE ON VARIOUS CELLULAR PARAMETERS

Photodynamic treatment of Kluyveromyces marxianus with chloroaluminum‐phthalocyanine resulted in loss of clonogenicity. Several parameters were studied to identify targets that could be related to loss of colony‐forming capacity. Inhibition of various plasma membrane‐bound processes was observed, such as substrate transport and plasma membrane ATPase activity. Moreover, K+ loss from the cells was observed. Photodynamic treatment also reduced the activity of various enzymes involved in energy metabolism, thereby decreasing the cellular ATP level. It will be discussed however that none of these processes is likely to be related directly to loss of clonogenicity. Treatment with phthalocyanine and light resulted in a strong inhibition of the incorporation of 14C‐phenylalanine in trichloracetic acid‐precipitable material. The induction of the β‐galactoside utilization system was also strongly inhibited. The latter two processes did not recover during incubation, subsequent to photodynamic treatment. It is concluded that photodynamically induced inhibition of protein synthesis is a critical factor contributing to the loss of clonogenicity.

Determination of the Ascorbate Free Radical Concentration in Mixtures of Ascorbate and Dehydroascorbate

Ascorbate free radical is considered to be a substrate for a plasma membrane redox system of eukaryotic cells, and might be involved in stimulation of cell proliferation. It can be generated by transition metal-dependent oxidation of ascorbate or by an equilibrium reaction of ascorbate with dehydroascorbic acid. Using ESR spectroscopic measurements at pH 7.4, we show that when ascorbate and dehydroascorbic acid are mixed at concentrations lower than 2.5 mM, the ascorbate free radical concentration was determined by metal-dependent reactions and not by the equilibrium reaction. We conclude that, for studies under physiological conditions, the ascorbate free radical concentration cannot simply be calculated from the equilibrium constant and the ascorbate and dehydroascorbic acid concentration, but has to be determined experimentally.

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.

ELECTRON SPIN RESONANCE STUDY ON THE FORMATION OF ASCORBATE FREE RADICAL FROM ASCORBATE : THE EFFECT OF DEHYDROASCORBIC ACID AND FERRICYANIDE

SummaryAscorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aKequil of 6 × 10−17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.

Single strand breaks and mutagenesis in yeast induced by photodynamic treatment with chloroaluminum phthalocyanine

Photodynamic treatment of the yeast Kluyveromyces marxianus with the sensitizer aluminum phthalocyanine results in loss of clonogenicity. In this paper the effect of this treatment on DNA of this yeast was investigated by searching for single strand breaks and forward mutations. Using the alkaline step elution technique it was found that illumination of the yeast in the presence of aluminum phthalocyanine resulted in an increase in single strand breaks. These could, partially, be repaired by post-incubating illuminated cells in growth medium. At comparable survival levels, photodynamic treatment with aluminum phthalocyanine induced fewer single strand breaks than X-ray treatment. By using a medium containing 5-fluoroorotic acid, mutants in the uracil biosynthetic pathway were selected. Photodynamic treatment resulted in a light dose dependent increase of the mutation frequency. The observed mutagenicity of photodynamic treatment of the yeast with phthalocyanine was lower than the mutagenicity of UVC and X-ray treatment at equal colony forming capacity, indicating that photodynamic treatment is the least mutagenic of those treatments. It is concluded that photodynamic treatment of K. marxianus results in DNA damage. Saccharomyces cerevisiae rad14 and rad52 mutants were used to determine the effect of the nucleotide excision repair and recombinational repair pathways, respectively, on survival after photodynamic treatment. Our data indicate that DNA damage is not the main determinant for cell killing by photodynamic treatment and that the type of damage induced is apparently not subject to RAD14- or RAD52 controlled repair.