Proton extrusion and attendant transport phenomena in Candida utilis induced by ethanol

Summary Ion fluxes after ethanol addition to Candida utilis depend crucially on aeration (air versus oxygen). In O₂-aerated non-growing cells ethanol causes an H ⁺ / K ⁺ exchange and an extrusion of acetate and lactate accompanied mostly by K ⁺, and their subsequent reimportation together with H ⁺. Cells from continuous culture display generally stronger acidification and more marked K ⁺ movements than non-growing ones. Offprint requests to: A. Prell     

Coupling of energy-dependent H+ and K+ ion fluxes in Streptococcus faecalis

Grampositive bacteria S. faecalis are capable of uptaking potassium ions during many hours in the media containing glucose. Such behaviour of K+-uptake indicates that this system is not regulated as it takes place in gramnegative bacteria E. coli (1,3). The stoichimetry of DCCD-sensitive exchange between H+ and K+ ions equals 2:1. It is possible that S. faecalis possesses an electrogenic proton-potassium pump which can exchange 2H+ from the cell for external K+.     

Behaviour of Candida utilis during growth in relation to Mg2+ and K+ concentration in sugar cane molasses

Magnesium and potassium ions are present in sugar cane molasses in a concentration of about 0.3% and 2.5%, respectively, which is high enough to support biomass production from Candida utilis. Culture broth with 40 g/l of total reducing substances supplemented by the addition of 1 ppm of Mg²⁺ leads to a higher yield (Y x/s) in batch fermentation experiments. The subsequent addition of Mg²⁺ up to 10 ppm decreases the yield coefficient from 0.53 to 0.42 without affecting the growth rate. Fermentation media supplemented with 1 to 10 ppm of K⁺ decreased both the yield coefficient and the specific growth rate. A Mg/K ratio of about 0.1 seems to be optimal for yeast biomass propagation.     

Interdependence of H+ and K+ fluxes during the Ca2+-pumping activity of sarcoplasmic reticulum vesicles

The release of H⁺ during the oxalate-supported Ca²⁺ uptake in sarcoplasmic reticulum vesicles is kinetically coincident with the initial phase of Ca²⁺ accumulation. The Ca²⁺ uptake is increased and the H⁺ release is decreased in the presence of KCl and other monovalent chloride salts as expected for a H⁺-monovalent cation exchange. The functioning of the Ca²⁺-pump is disturbed by the presence of potassium gluconate and, to a lesser extent, of choline chloride. These salts do not inhibit the ATPase activity of Ca²⁺-permeable vesicles, suggesting a charge imbalance inhibition which is specially relevant in the case of gluconate. Therefore, K⁺, and also Cl^–, appear to be involved in secondary fluxes during the active accumulation of Ca²⁺. The microsomal preparation seems homogeneous with respect to the K⁺-channel, showing an apparent rate constant for K⁺ release of approximately 25 s^–1 measured with the aid of⁸⁶Rb⁺ tracer under equilibrium conditions. A Rb⁺ efflux, sensitive to Ca²⁺-ionophore, can be also detected during the active accumulation of Ca²⁺. The experimental data suggest that both monovalent cations and anions are involved in a charge compensation during the Ca²⁺ uptake and H⁺ release. Fluxes of these highly permeable ions would contribute to cancel the formation of a resting membrane potential through the sarcoplasmic reticulum membrane.     

Effect of external pH on the respiration activity of Candida utilis induced by ethanol

Summary The kinetics of ethanol oxidation by non-growing cells of Candida utilis in different media at various external pH values was determined experimentally. The statistical discrimination between two rival mathematical models has shown that the mechanism of non-specific substrate inhibition of respiration kinetics fits better the experimental data. It has been found that the maximum respiration activity is controlled by the buffering properties of organic polycarbonic acids in the medium. The pH values at which the maximum respiration rate was observed were close to the pK values of the organic acids in the buffer solution, independently of whether the acids were metabolized or not. Offprint requests to: Jan Paca     

H+ transport by reconstituted gastric (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+-H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212).     

Effects of A23187 and Ca2+ on volume- and thiol-stimulated, ouabain-resistant K+C1- fluxes in low K+ fluxes in low K+ sheep erythrocytes

Ouabain-resistant (OR), C1- -dependent K+ (K+C1-) transport measured by Rb+ influx in isosmotic and anisosmotic media was stimulated by the Ca2+ ionophore A23187 and EGTA (ethylene-glycol-tetracetic acid) in low K+ (LK) but not in high K+ (HK) sheep red cells. Increasing external Ca2+ concentrations, [Ca2+]o, from about 10(-7) to 10(-3)M in presence of A23187 and in absence of EGTA inhibited OR Rb+ influx, in LK red cells osmotically shrunken or swollen as well as treated with the thiol reagent N-ethylmaleimide (NEM). Hence the volume- and the NEM-stimulated K+C1- transport system in LK cells can be experimentally modulated by cellular Ca2+ or other Me2+, which may interact with sites on the K+C1- transporter under the control of membrane sulfhydryl (SH) groups.     

K+,H+ and Cl- fluxes across erythrocyte membrane irradiated with radio frequency electromagnetic fields

The effect of 460 MHz microwave radiation on the ion-transporting properties of the isolated rat erythrocytes was studied with the use of K+, H+ and Cl(-)-selective electrodes. In comparison with the control cells kept at 0 degree C the most significant changes were observed in the K+ transport system. Particularly, microwave radiation (specific absorbed rate 280 W/kg) caused an increased loss of K+ during treatment and 2-fold decrease in the rate of K+ efflux from the irradiated erythrocytes, when the latter were incubated in the isoosmotic, unbuffered sucrose. The same changes were observed when the erythrocytes were conventionally heated up to 39 degrees C for 20 minutes. It is concluded that high levels of microwave radiation cause temperature-induced changes of the membrane structure resulting in alterations in potassium transport across the membrane.     

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells.

The K+ content and the K+ flux were measured in the cell lines ME2 and MF2 isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the seem K+ content and the same K+ steady state flux per unit of surface area. In ME2 cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K+ net movement. The (Na+ plus K+)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K+ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells. The K+ net flux is considerable increased when the intracellular K+ level is reduced after preincubation of the cells in a K+ -free medium. Thus, internal K+ seems to regulate the K+ influx.     

Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.     

Turgor-controlled K+ fluxes and their pathways in Escherichia coli

Escherichia coli like most gram-negative bacteria with walls maintains a cytoplasmic osmolarity exceeding that of the medium; the resulting hydrostatic pressure (turgor pressure) pushes the cytoplasmic membrane against the peptidoglycan and creates a tension in the two envelopes. Potassium is the only cation which takes part in the regulation of cellular osmolarity. The adaptation of intracellular K+ concentration to external osmolarity involves K+ turgor-controlled fluxes. When the medium osmolarity is raised an osmodependent influx of K+ can be observed; this is carried out by the K+ transport system TrkA which can also taken up rubidium. A specific and unidirectional pathway allows K+ ions to flow out of the cell when the medium osmolarity is decreased; this pathway reveals two characteristics: it has no affinity for rubidium and it can be blocked by the blockers of eukaryotic K+ channels. Osmodependent fluxes are turned on immediately after the medium osmolarity is disturbed; in contrast, they are turned off gradually as the rate of K+ fluxes approach zero. The rate of K+ influx seems to depend on the level of internal osmolarity and not on the extent of the increase in medium osmolarity. The rate of the efflux is directly proportional to the decrease in medium osmolarity and is independent on the level of internal osmolarity.     

Inactivation of H+,K+-ATPase by a K+-competitive photoaffinity inhibitor

A light-sensitive derivative, 2,3-dimethyl-8-[(4-azidophenyl)methoxy]imidazo[1,2-a]pyridine (DAZIP), of the drug 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine (SCH 28080) has been synthesized and shown to be a K+-competitive inhibitor of gastric H+,K+-ATPase in the dark. The apparent dissociation constants calculated for DAZIP at pH 6.4 and 7.4 were 1.8 +/- 0.2 and 4.7 +/- 1.2 microM, respectively. Inhibition required binding of DAZIP to a luminal-facing site on the enzyme. Irradiation in the presence of DAZIP and 2 mM Mg2+ resulted in irreversible loss of ATPase activity that was more than 2-fold greater at pH 6.4 than at pH 7.4, showing the enhanced efficiency of covalent incorporation at the lower pH. Further photolyses were conducted at pH 6.4 in the presence of either 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), ATP and CDTA, or MgATP. The specificity of light-dependent, covalent insertion of DAZIP for the site of reversible inhibition was shown both by protection against photoinactivation given by K+ (the competing ligand) and by the observation that the amount of K+-protectable photoinactivation approached a maximum limiting value as a function of DAZIP concentration. The effectiveness of K+ in protecting against photoinactivation was 100-fold greater in the presence of ATP and CDTA than in the presence of either Mg2+ or CDTA and suggests the formation of a ternary complex of the apoenzyme with ATP and tightly bound K+. The dissociation constant for DAZIP (2 microM) calculated from photolyses in the presence of MgATP without added K+ agreed with the kinetic experiments and suggests that DAZIP inhibits turnover by binding to E.MgATP.     

Intravesicular pH changes in submitochondrial particles induced by monovalent cations: relationship to the Na+/H+ and K+/H+ antiporters

The fluorescence of internalized fluorescein isothiocyanate dextran has been used to monitor the intravesicular pH of submitochondrial particles (SMP). Respiring SMP maintain a steady-state delta pH (interior acid) that results from the inwardly directed H+ flux of respiration and an opposing passive H+ leak. Addition of K+, Na+, or Li+ to SMP results in a shift to a more alkaline interior pH (pHi) in both respiring and nonrespiring SMP. The K+-dependent change in pHi, like the K+/H+ antiport in intact mitochondria, is inhibited by quinine and by dicyclohexylcarbodiimide. The Na+-dependent reaction is only partially inhibited by these reagents. Both the Na+- and the K+-dependent pH changes are sensitive to amiloride derivatives. The Km for both Na+ and K+ is near 20 mM whereas that for Li+ is closer to 10 mM. The K+/H+ exchange reaction is only slightly inhibited by added Mg2+, but abolished when A23187 is added with Mg2+. The passive exchange is optimal at pHi 6.5 with either Na+ or K+, and cannot be detected above pHi of 7.2. Both the Na+/H+ and the K+/H+ exchange reactions are optimal at an external pH of 7.8 in respiring SMP (pHi 7.1). Valinomycin stimulates the K+-dependent pH change in nonrespiring SMP, as does nigericin. It is concluded that SMP show K+/H+ antiport activity with properties distinct from those of Na+/H+ antiport. However, the properties of the K+/H+ exchange do not correspond in all respects to those of the antiport in intact mitochondria. Donnan equilibria and parallel uniport pathways for H+ and cations appear to contribute to cation-dependent pH changes in SMP.     

Cation transport by gastric H+:K+ ATPase.

A vesicular microsomal fraction isolated from hog fundic mucosa demonstrates the capacity to take up equal amounts of RB+ and Cl-. The amount of the Rb+ uptake is sensitive to the extravesicular osmolarity, and rate of uptake is sensitive to temperature. 86Rb+ efflux is dependent upon the cation composition of the diluting solution. ATP, but not beta-gamma methylene ATP, induces a reversible efflux of 86Rb+ from loaded vesicles, and this is dependent upon a functional K+-ATPase. The ATP induced efflux is not affected by CCCP (carbonyl cyanide m-chlorophenylhydrazone) or TCS (tetrachlorosalicylanilide) nor by lipid soluble ions or valinomycin. Nigericin inhibits the efflux by 40%. Uptake of the lipid soluble ion 14C-SCN- has been demonstrated and is enhanced by ATP only in the presence of valinomycin. The results are consistent with a neutral or isopotential exchange of H+ for Rb+ mediated by K+-ATPase.     

The interaction of H+ and K+ with the partial reactions of gastric (H+ + K+)-ATPase

The influence of H+ and K+ on the partial reactions and transport of gastric (H+ + K+)-ATPase was studied. Using transient kinetics, the effects and sidedness of effects of H+ and K+ on formation and breakdown of phosphoenzyme were determined in intact and lyophilized reconstituted vesicles in the absence and presence of gramicidin. Whereas increasing H+ concentrations on the ATP-binding face of the vesicles accelerates phosphorylation, increasing K+ concentrations inhibits phosphorylation. Increasing H+ on this side reduces K+ inhibition of the phosphorylation rate. At low ATP/K+ ratios, the phosphorylation step can become rate-limiting for steady state hydrolysis. Decreasing H+ accelerates dephosphorylation in the absence of K+. K+ on the internal or luminal face of the vesicles accelerates dephosphorylation, and this rate is reduced with increasing H+ concentrations. At low internal pH, K+-dependent dephosphorylation may become rate-limiting. H+ transport measurements using fluorescence quenching of acridine orange show that whereas internal K+ is required for H+ transport, external K+ inhibits the rate of formation of a pH gradient, and the inhibition is reduced by decreasing medium pH. The pH optimum for ATPase activity and transport correlated in the vesicles, and the K0.5 of K+ for transport correlated with data for intact parietal cells.     

Na+ and K+ fluxes stimulated by Na+-coupled glucose transport: Evidence for a Ba2+-insensitive K+ efflux pathway in rabbit proximal tubules

Summary Addition of glucose or the nonmetabolizable analogue -methyl-d-glucoside to rabbit proximal tubules suspended in a glucoseand alanine-free buffer caused a sustained increase in intracellular Na⁺ content (+43±7 nmol · (mg protein)^–1) and a concomitant but larger decrease in K⁺ content (–72±11 nmol· (mg protein)^–1). A component of the net K⁺ efflux was Ba²⁺ insensitive, and was inhibited by high (1mm) but not low (10 m) concentrations of the diuretics, furosemide and bumetanide. The increase in intracellular Na⁺ content is consistent with the view that the increased rates of Na⁺ and water transport seen in the proximal tubule in the presence of glucose can be attributed (at least in part) to a stimulation of basolateral pump activity by an increased [Na⁺] _i .