Effects of salts on aerobic metabolism of Debaryomyces hansenii.

Debaryomyces hansenii was grown in YPD medium without or with 1.0 M NaCl or KCl. Respiration was higher with salt, but decreased if it was present during incubation. However, carbonylcyanide-3-chlorophenylhydrazone (CCCP) markedly increased respiration when salt was present during incubation. Salt also stimulated proton pumping that was partially inhibited by CCCP; this uncoupling of proton pumping may contribute to the increased respiratory rate. The ADP increase produced by CCCP in cells grown in NaCl was similar to that observed in cells incubated with or without salts. The alternative oxidase is not involved. Cells grown with salts showed increased levels of succinate and fumarate, and a decrease in isocitrate and malate. Undetectable levels of citrate and low-glutamate dehydrogenase activity were present only in NaCl cells. Both isocitrate dehydrogenase decreased, and isocitrate lyase and malate synthase increased. Glyoxylate did not increase, indicating an active metabolism of this intermediary. Higher phosphate levels were also found in the cells grown in salt. An activation of the glyoxylate cycle results from the salt stress, as well as an increased respiratory capacity, when cells are grown with salt, and a 'coupling' effect on respiration when incubated in the presence of salt.

Glycolytic sequence and respiration of Debaryomyces hansenii as compared to Saccharomyces cerevisiae.

The fermentation and respiration activities of Debaryomyces hansenii were compared with those of Saccharomyces cerevisiae grown to stationary phase with high respiratory activity. It was found that: (a) glucose consumption, fermentation and respiration were lower than for S. cerevisiae; (b) fasting produced a much smaller decrease of respiration; (c) glucose consumed and not transformed to ethanol was higher; (d) in S. cerevisiae, full oxygenation prevented ethanol production but this effect was reversed by CCCP, whereas D. hansenii still showed some ethanol production under aerobiosis, which was moderately increased by CCCP. ATP levels were similar in the two yeasts. Levels of glycolytic intermediaries after glucose addition, and enzyme activities, indicated that the main difference and limiting step to explain the lower fermentation of D. hansenii is phosphofructokinase activity. Respiration and fermentation, which are lower in D. hansenii, compete for the re-oxidation of reduced nicotinamide adenine nucleotides; this competition, in turn, seems to play a role in defining the fermentation rates of the two yeasts. The effect of CCCP on glucose consumption and ethanol production also indicates a role of ADP in both the Pasteur and Crabtree effects in S. cerevisiae but not in D. hansenii. D. hansenii shows an alternative oxidase, which in our experiments did not appear to be coupled to the production of ATP.

Electrical properties of the plasma membrane of microplasmodia of Physarum polycephalum.

Microplasmodia of Physarum polycephalum have been investigated by conventional electrophysiological techniques. In standard medium (30 mM K+, 4 mM Ca++, 3 mM Mg++, 18 mM citrate buffer, pH 4.7, 22 degrees C), the transmembrane potential difference Vm is around -100 mV and the membrane resistance about 0.25 omega m2. Vm is insensitive to light and changes of the Na+/K+ ratio in the medium. Without bivalent cations in the medium and/or in presence of metabolic inhibitors (CCCP, CN-, N3-), Vm drops to about 0 mV. Under normal conditions, Vm is very sensitive to external pH (pH0), displaying an almost Nernstian slope at pH0 = 3. However, when measured during metabolic inhibition, Vm shows no sensitivity to pH0 over the range 3 to 6, only rising (about 50 mV/pH) at pH0 = 6. Addition of glucose or sucrose (but not mannitol or sorbitol) causes rapid depolarization, which partially recovers over the next few minutes. Half-maximal peak depolarization (25 mV with glucose) was achieved with 1 mM of the sugar. Sugar-induced depolarization was insensitive to pH0. The results are discussed on the basis of Class-I models of charge transport across biomembranes (Hansen, Gradmann, Sanders and Slayman, 1981, J. Membrane Biol. 63:165-190). Three transport systems are characterized: 1) An electrogenic H+ extrusion pump with a stoichiometry of 2 H+ per metabolic energy equivalent. The deprotonated form of the pump seems to be negatively charged. 2) In addition to the passive K+ pathways, there is a passive H+ transport system; here the protonated form seems to be positively charged. 3) A tentative H+-sugar cotransport system operates far from thermodynamic equilibrium, carrying negative charge in its deprotonated states.