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Oxygen radical generating systems, namely, Cu(II)/ H2O2, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H2O2/catecholamine and Cu(II)/H2O2/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H2O2 enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H2O2 dependent on Cu(II) and H2O2 concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO. scavengers prevented GR inactivation by Cu(II)/H2O2 and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropionylglycine and penicillamine enhanced the effect of Cu(II)/H2O2 in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H2O2 effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and trypanothione disulfide effectively protected GR against Cu(II)/H2O2 inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS.+. GR inactivation by the systems assayed correlated with their capability for HO. radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.

(1) N-Ethylmaleimide (a penetrating SH- reagent) inactivated L-[14C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with N-ethylmaleimide, N-ethylmaleimide concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With D-glucose-energized yeast, N-ethylmaleimide inhibited L-[14C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit L-[14C]leucine binding, except when the cells were preincubated with L-leucine. With the rho- respiratory-deficient mutant (energized cells), N-ethylmaleimide inhibited L-[14C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the N-ethylmaleimide effect as a function of L-[14C]leucine concentration showed a significant decrease of Jmax values of the high- (S1) and low- (S2) affinity amino acid transport systems, but KT values were not significantly modified. (3) When assayed in the presence of D-glucose, N-ethylmaleimide inhibition of D-glucose uptake and respiration contributed significantly to inactivation of L-[14C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of L-[14C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate L-[14C]leucine entrance, while p-chloromercuribenzoate, a slowly penetrating SH-reagent, inactivated it to a limited extent. When compared with the effect of N-ethylmaleimide, these negative results indicate that thiol groups of the L-[14C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.

1. L-[14C]Leucine transport into Saccharomyces cerevisiae involves a high-affinity, low-velocity system (system 1) and a low-affinity, high-velocity system (system 2). These systems are characterized by the different values of the kinetic parameters KT and Jmax, and are both capable of concentrative transport. The general amino acid permease is assumed to be a part of the high-affinity system. 2. The kinetics of L-[14C]leucine entrance show and initial rapid phase (the 'very early uptake') before reaching the steady-state rate. The contribution of the very early uptake to total entrance values affects the values of KT and Jmax, especially when the steady-state rate is relatively slow, as with starved yeast, and then negative KT and Jmax values may result. The very early uptake is increased by pretreatment of starved yeast and D-glucose, this latter effect being counteracted by iodoacetate. 3. After energization of starved yeast by pretreatment with D-glucose or propionaldehyde, the apparent KT,2 value greatly decreases whilst the KT,1 value decreases to a much more limited extent, or does not vary. With the energized yeast, KT,2 decreases throughout incubation whilst KT,1 variation is insignificant. Energization increases Jmax,1 and Jmax,2 several-fold and with the energized yeast at the steady-state phase, Jmax,2 greater than or equal to 4Jmax,1. Variation of KT and Jmax values as a function of the metabolic state of yeast cells may be explained in terms of variation of rate constants k-1, k+1 and k+2 for each transport system. 4. Dicyclohexylcarbodiimide, quercetin and diethylstilbestrol inhibit tranport at 0.05 mM L-[14C]leucine, in good agreement with a function of the plasmalemma ATPase for the operation of system 1. Dio-9, propionic and isobutyric acids, pentachlorophenol, carbonylcyanide 3-chlorophenylhydrazone and carbonylcyanide 4-trifluoromethoxyphenylhydrazone, which affect the proton gradient and/or the membrane potential inhibit L-[14C]leucine uptake at all the assayed amino acid concentrations. 5. The polyene antibiotic, nystatin, which forms channels in membranes permeable to K+ and H+, inhibits systems 1 and 2 activity but enniatin (also a K+ ionophore) does not.

(1) Substrates capable of activating mitochondrial electron transfer and oxidative phosphorylation, namely, pyruvate, acetate, propionaldehyde and butanol, stimulated the concentrative uptake (transport and accumulation) of L-[14-C]leucine by Saccharomyces cerevisiae (wild type strain 207, starved cells). Under adequate experimental conditions, the L-[14-C]leucine uptake versus the oxygen uptake ratio was almost the same with either pyruvate, acetate or D-glucose as energy sources. Substrate oxidation also increased L-[14-C]leucine incorporation into the cell protein. (2) With S. cerevisiae D261 and D247-2 and propionaldehyde as an energy source, or with strain 207 and glucose as energy source, 2,4-dinitrophenol (50 muM) inhibited L-[14-C]leucine uptake, the inhibition being accompanied by stimulation of respiration. With S. cerevisiae 207 and propionaldehyde as energy source, 2,4-dinitrophenol inhibited both respiration and L-[14-C]leucine uptake, but with respiration being less affected than uptake. Displacement of accumulated L-[14-C]leucine was also inhibited by 2,4-dinitrophenol. (3) In the presence of glucose, and for relatively brief incubation periods, anaerobically grown cells of S. cerevisiae 207 and of a p-minus "petite" mutant of this strain incorporated L-[14-C]leucine with less efficiency than the original wild type strain 207, grown aerobically. With D-glucose as energy source, 2,4-dinitrophenol and iodoacetate inhibited alike L-[14-C]leucine uptake by the respiration competent cells. (4) It is postulated that in respiration-competent yeasts, the mitochondrion contributes to 6-[14-C]leucine uptake by supplying high-energy compounds required for amino acid transport and accumulation. Conversely, the promitochondrion in the anaerobically grown yeast, or the modified mitochondrion in the respiratory deficient mutant, competes for high energy compounds generated by glycolysis in the cytosol.