Manganese transport into yeast cells is energy-dependent. It is dependent on endogenous sources of energy and is inhibited by olygomycin (12.5 -25 kg/ml), 2,4-dinitrophenol (1 mM), 2-deoxyglucose (1 -50 mM) and sodium azide (1 -10 mM), but is stimulated by cyanide and glucose. The stimulating effect of glucose is eliminated by N-ethylmaleimide and iodoacetate, which apparently inhibit the transport of glucose itself. About 75% of the manganese accumulated in the presence of glucose is found in yeast protoplasts and nearly 25% in the cell walls. A major portion of the accumulated manganese is found in vacuoles. The concentration of osmotically free manganese in the cytosol did not exceed 2 m M , but the concentration in vacuoles was up to 14mM. The tonoplast is assumed to have a transport system for divalent cations, thereby regulating their concentration in the cytosol.The transport of bivalent manganese ions has long attracted the attention of investigators [l -41.Data have been obtained which support the hypothesis that a M 2 +-dependent plasmolemma ATPase provides energy for this process; this enzyme uses ATP only of glycolytic origin [4-61. The state of the accumulated ions (free and bound manganese) and also their distribution inside the yeast cells have not been studied yet.Recently using a cytochemical method of analysis we found that magnesium ions are localized in yeast vacuoles and that the accumulated manganese is also concentrated in these organelles [7]. An independent biochemical approach supported the idea that a considerable portion of the total magnesium (up to 40%) is localized in yeast vacuoles [8,9]. In this work we have used this approach in order to study the distribution of manganese in the cells of Saccharomyces carhbergensis. In addition, the energy-dependent character of manganese accumulation by yeast was demonstrated. Microorgan ismsAll the experiments were carried out on the yeasts Saccharomyces cereuisiae (strain IBPM-355) and Succharomyces carlsbergensis (strain IBPM-366). Yeasts were grown in flasks in Reader medium at 29 "C. Inoculation was carried out with 24-h innocula; after 5 h the cells were collected by centrifugation, and were washed with distilled water. Then they were incubated for 60 min in a medium containing potassium phosphate (0.33 M) and glucose (0.1 M) at 30 "C or 37 "C under the same shaking conditions as during growth. Then the cells were washed with distilled water and were used for experiments to investigate the accumulation of manganese ions. The accumulation of manganese was carried out for 45 min at 30 "C, pH 5.5 and under continuous shaking. The concentration of manganese sulphate was 3 mM and glucose (if added) 100 mM [3].Usually cells were separated from medium by centrifugation followed by washing with water.In the experiments on the influence of different inhibitors on manganese uptake, this process was stopped at specific times by the addition of La3+ ions to a final concentration 0.3 mM. Then the cells were separated by centrifugation and washed with...
The content of total, bound and osmotically free magnesium was estimated in various fungi and in the yeast Saccharomyces cerevisiae. Total magnesium increases at lower growth rates of Endomyces magnusii and Penicillium chrysogenum 140A as well as during the logarithmic stage of growth of Penicillium chrysogenum Q-176. The binding of magnesium requires orthophosphate, decreasing during lack of external phosphate when the intracellular concentration of free magnesium rises. The fungi were found to contain a novel form of bound magnesium, a polymeric magnesium orthophosphate (PO Mg), which appears to take part in the control of free magnesium level in Penicillium chrysogenum Q-176. The level of free magnesium is proportional to the growth rate of Endomyces magnusii and Penicillium chrysogenum Q-176 and 140A. Total, as well as free, magnesium changes less than three-fold as external Mg concentration is changed 13,000-fold. The magnesium is taken up against concentration gradients of 1 : 25 to 1 : 1300, the metal being distributed non-uniformly in the cells of Saccharomyces cerevisiae.
The uptake of Mn2+ by Saccharomyces cerevisiae at the expense of endogenous sources of energy depends on the stage of culture development and is maximum in the middle of the exponential phase. The ability of cells to take up Mn+ is related to the content of intracellular potassium at all stages of growth, to the content of ATP during the exponential phase and it is not related to the content of inorganic polyphosphates. The uptake is inhibited by oligomycin (25 microgram/ml) by 50-85% and under anaerobic conditions by 10-50%, depending on the stage of growth, indicating the role of aerobic phosphorylation in the process. The uptake of Mn+ is apparently associated with a hydrolysis of low-molecular weight polyphosphates and ATP, as well as with the exit of K+ from cells.
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