Search PubMed⌕ Search

Biomedical subjects

A Kotyk

Publications and source records attributed to A Kotyk.

At least 37 records · Page 2Linked to original sources
PubMed1998

Different sources of acidity in glucose-elicited extracellular acidification in the yeast Saccharomyces cerevisiae.

Three wild-type strains of Saccharomyces cerevisiae, viz. K, Y55 and sigma 1278b, two mutants lacking one or both of the putative K+ transporters, trk1 delta and trk1 delta trk2 delta, and a mutant in the plasma membrane H(+)-ATPase, viz. pma1-105, were compared in their extracellular acidification following addition of glucose and subsequent addition of KCl; in ATPase activity in purified plasma membranes; and in respiration on glucose. The glucose-induced acidification was the greater the higher the respiratory quotient, i.e. the higher the anaerobic metabolism. A markedly lower acidification was found in the ATPase-deficient pma1-105 strain but also in the TRK-deficient double mutant. The acidification pattern after addition of KCl corresponds to expectations in the TRK mutants; however, a similarly decreased acid production was found in the ATPase-deficient mutant pma1-105. The highest rate of ATP hydrolysis in vitro was found with the trk1 delta trk2 delta mutant where glucose-, as well as KCl-induced acidification were lowest. Likewise, the pma1-105 mutant with extremely low acidification showed only a minutely lower ATP hydrolysis than did its parent Y55 strain. Apparently, several different sources of acidity are involved in the glucose-induced acidification (including extrusion of organic acids); in fact, contrary to the general belief, the H(+)-ATPase may play a minor role in this process in some strains.

Acids↗
PubMed1997

Modifications induced by insulin-dependent diabetes mellitus on human placental Na+/K+-adenosine triphosphatase.

The causes of the reduced activity of Na+/K+-adenosine triphosphatase (ATPase) in human diabetes are still the object of controversy. The aim of this work was to investigate the mechanisms of inhibition by means of the study of the Na+/K+-ATPase purified from human placenta. We purified Na+/K+-ATPase from term placentas of six healthy women and six age-matched women with insulin-dependent diabetes mellitus (IDDM) in good metabolic control. The enzymatic activity was reduced in both the microsomal fraction and the purified Na+/K+-ATPase obtained from diabetic women, whereas no difference was found in the number of active molecules determined by anthroyl ouabain binding. The Na+/K+-ATPase purified from women with IDDM did not show any modification in the ouabain affinity or changes in the physicochemical structure of the ouabain binding site investigated by dynamic fluorescence or alterations in lateral diffusion. The activation energy of the enzyme was increased, whereas the tryptophan accessibility of the enzyme was lower in women with IDDM. The fluidity of the lipid anulus of the enzyme was higher in women with IDDM than in control women, as suggested by fluorescence polarization of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene. The adenosine triphosphate-binding site, investigated by anisotropy decay studies of the fluorescent probe pyrene isothiocyanate, was modified in women with IDDM. It appears that the Na+/K+-ATPase of human placenta is altered in its disposition in IDDM.

Acrylamide↗
PubMed1997

Cell volume regulation in Claviceps fusiformis. An animal-type Na,K-ATPase operating in a fungus?

Claviceps fusiformis, an ergot pathogen of pearl millet, was equilibrated in media without and with 0.1 mM ouabain (g-strophantin). Highly significant effects of ouabain on the contents of sodium ions and water were found, suggesting that an active transport of sodium ions, similar to that in animal cells, regulates the cell volume of the fungus. The basic role of the sodium pump appears to be to decrease larger cell volumes and to increase the small ones; for this purpose it can even reverse its direction.

Biological Transport, Active↗
PubMed1997

Dicarbanonaborates in yeast respiration and membrane transport.

Two derivatives of carborates, sodium 5,6-dichloro-7,8-dicarbanonaborate (CB-Cl) and sodium 5-mercapto-7,8-dicarbanonaborate (CB-SH) were found to inhibit endogenous as well as glucose-induced respiration of the yeast Saccharomyces cerevisiae. Both substances slightly increased endogenous acid production, were neutral toward H(+)-ATPase-associated acidification but pronouncedly inhibited the K(+)-stimulated acidification. The same effects were observed also with an ATPase-deficient mutant of the yeast. The ATP-hydrolyzing activity of yeast plasma membranes in vitro was severely reduced. The membrane potential was substantially increased toward more negative values. The H(+)-symporting uptake of glutamic acid was considerably decreased, that of adenine was diminished much less. The effects of the dicarbanonaborates are obviously pleiotropic but their inhibition of ATP hydrolysis and of uptake of H(+)-symported substances, on the one hand, and absolute lack of effect on ATPase-catalyzed acidification, on the other, pose an unresolved problem.

Adenosine Triphosphate↗
PubMed1995

The transmembrane gradient of the dielectric constant influences the DPH lifetime distribution.

The fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in egg-phosphatidylcholine liposomes was measured in normal and heavy water. The lower dielectric constant (by approximately 12%) of heavy water compared with normal water was employed to provide direct evidence that the drop of the dielectric constant along the membrane normal shifts the centers of the distribution of both DPH and TMA-DPH to higher values and sharpens the widths of the distribution. The profile of the dielectric constant along the membrane normal was not found to be a linear gradient (in contrast to [1]) but a more complex function. Presence of cholesterol in liposomes further shifted the center of the distributions to higher value and sharpened them. In addition, it resulted in a more gradient-like profile of the dielectric constant (i.e. linearization) along the normal of the membrane. The effect of the change of dielectric constant on the membrane proteins is discussed.

Cholesterol↗
PubMed1995

Distribution of individual cytoplasmic pH values in a population of the yeast Saccharomyces cerevisiae.

Fluorescence ratio imaging microscopy using pH-sensitive fluorescent dyes makes it possible to evaluate statistical distribution of intracellular pH in a population of the yeast S. cerevisiae examined in a thin layer of suspension in a Petri dish. The distribution appears to fit a Gaussian curve with a half-width around the 0.4 pH unit. The curve became slightly narrower after resuspension in a strong buffer; the mean values shifted with the pH of the buffer. The shape of the distribution curves of both resting and growing cells in various phases of growth does not change significantly. Likewise, addition of 1% of glucose, 50 microM suloctidil or 100 microM diethylstilbestrol brings about no alteration. The only value which clearly changes is the average cytoplasmic pH.

Cytoplasm↗
PubMed1995

Enzymically generated electron-excited species increase the membrane order parameter without changing the rotational relaxation time of TMA-DPH.

The effect of enzymically generated triplet acetone produced from 60 mmol/1 2-methylpropanal (MPAL) in the presence of 2 mumol/l horseradish peroxidase on the properties of artificial unilamellar liposomes was studied. The concentration of malondialdehyde, membrane lateral pressure (membrane order parameter), lifetime distribution of the excited state of TMA-DPH incorporated into liposomes, and rotational relaxation time of TMA-DPH were determined. The concentration of MDA increased with the MPAL concentration, and the membrane lateral pressure increased linearly with the concentration of MDA. The mean lifetime values changed (p < 0.01) as did the limiting anisotropy (p < 0.005), the widths of the main peak of the lifetime distribution and the rotational relaxation time remaining unchanged. This indicates that there is no significant difference between the widths and rotational relaxation times in the control and triplet-acetone-treated sample; i.e. peroxidation by MPAL increased the lipid order of the bilayer but the effect of neighboring lipid molecules on the dynamics of the probe was negligible.

Acetone↗
PubMed1994

Human hypertensive placenta contains an increased amount of Na,K-ATPase with higher affinity for cardiac glycosides.

Placentas of women suffering from pregnancy-induced hypertension (PIH) were found to contain a greater amount of Na,K-ATPase molecules, estimated from anthroyl ouabain binding, than normotensive individuals. Both the microsomal fraction of placental cells and purified Na,K-ATPase showed an increased affinity for the specific inhibitor ouabain which, in the case of the microsomes, bound with a dissociation constant of 0.9 nM as compared with 3.4 nM in the controls. Likewise, the dissociation constant of the ouabain complex with purified Na,K-ATPase was about 3.5 times lower in the hypertensive patients. The differences are apparently caused by a different microenvironment of the ouabain-binding site, as reflected in the quantum yield of bound anthroyl ouabain. If an endogenous digitalis-like factor is present in the body fluids to regulate Na,K-ATPase activity, the present results render its role quite plausible.

Adult↗
PubMed1994

Both glucose-type monosaccharides and one of their metabolites are required for activation of yeast plasma membrane H(+)-ATPase.

Saccharomyces cerevisiae and Schizosaccharomyces pombe cells were grown on D-glucose, D-galactose, D-fructose, D-mannose, maltose, trehalose and ethanol. All these substrates were separately added to cells thus grown and the onset and rate of acidification mediated by the plasma membrane H(+)-ATPase were determined. Irrespective of the growth substrate, the best triggers of acidification in both species were fructose, mannose and glucose (with average rates of 5.2, 5.0 and 4.8 nmol H+ per min per mg dry weight, respectively, for S. cerevisiae, and 4.5, 6.8 and 5.8 for S. pombe). These were followed in S. cerevisiae by galactose in Gal-, Man- and Tre-grown cells (about 0.40 nmol H+) and by maltose in Mal- and Tre-grown cells (about 0.15 nmol H+). Trehalose elicited some response in only ethanol-grown cells while ethanol itself was completely ineffective in activating the H(+)-ATPase. In S. pombe, however, maltose caused an acidification rate of 3.6 nmol H+ per min per mg dry wt., followed by EtOH (().38), Gal (0.13) and Tre (0.05). 6-Deoxy-D-glucose and 2-deoxy-D-glucose, not metabolized or improperly metabolized analogues of glucose, had no effect whatsoever. It appears that the sensor triggering the ATPase-activating pathway is a complex responding both to a glucose-type sugar (Glc, Man, Fru) and possibly identical with one of the glucose carriers, and to one of its metabolites, most probably fructose-6-phosphate.

Deoxyglucose↗
PubMed1994

Dependence of the kinetics of secondary active transports in yeast on H(+)-ATPase acidification.

Acidification of the external medium of the yeast Saccharomyces cerevisiae, mainly caused by proton extrusion by plasma membrane H(+)-ATPase, was inhibited to different degrees by D2O, diethylstilbestrol, suloctidil, vanadate, erythrosin B, cupric sulfate and dicyclohexylcarbodiimide. The same pattern of inhibition was found with the uptake of amino acids, adenine, uracil, and phosphate and sulfate anions. An increase of the acidification rate by dioctanoylglycerol also increased the rates of uptake of adenine and of glutamic acid. In contrast, a decrease of the membrane potential at pH 4.5 from a mean of -40 to -20 mV caused by 20 mM KCl had no effect on the transport rates. The ATPase-deficient mutant S. cerevisiae pmal-105 showed a markedly lower uptake of all the above solutes as compared with the wild type, while its membrane potential and delta pH were unchanged. Other types of acidification (spontaneous upon suspension; K+ stimulated) did not affect the secondary uptake systems. A partially competitive inhibition between some individual transport systems was observed, most pronouncedly with adenine as the most avidly transported solute. These observations, together with the earlier results that inhibition of H(+)-ATPase activity affects more the acidic than the basic amino acids and that it is more pronounced at higher pH values and at greater solute concentrations, support the view that it is the protons in or at the membrane, as they are extruded by the ATPase, that govern the rates of uptake by secondary active transport systems in yeast.

Biological Transport, Active↗
PubMed1994

Enhancement of synthesis and activity of yeast transport proteins by metabolic substrates.

The transport rates of amino acids, ranging from L-Glu to L-Lys, uracil, adenine and sulfate and phosphate anions by Saccharomyces cerevisiae are greatly increased by preincubation with D-glucose in a nongrowth medium when a de novo synthesis of proteins takes place. In addition, some substrates, especially the inorganic anions, require the presence of glucose during their transport. This requirement has to do both with ongoing protein synthesis and degradation, as well as with providing energy and/or activating the plasma membrane H(+)-ATPase which supplies the protons to the H+ symports studied here.

Adenine↗
PubMed1994

Univalent-cation-elicited acidification by yeasts.

Addition of univalent cations to sugar-metabolizing Saccharomyces cerevisiae, Schizosaccharomyces pombe and Lodderomyces elongisporus brought about a powerful acidification of the external medium with rates up to nearly 20 nmol H+ per min per mg dry wt. in S. cerevisiae, over 15 nmol in S. pombe, and 4.7 nmol in L. elongisporus. These rates were as much as 20 times, 5.5 times and 10.3 times, respectively. higher than in the absence of K+. Use of galactose-induced cells, of H(+)-ATPase-deficient mutants and observations over the entire growth curve indicated that the K+ effect on H+ extrusion is not connected with the H(+)-ATPase function as such but rather depends on metabolic reactions producing ATP. The effect has apparently nothing to do with the electrical potential across the plasma membrane.

Carbohydrate Metabolism↗
PubMed1994

Na,K-ATPase from placenta of women with pregnancy-induced hypertension exhibits an increased affinity for cardiac glycosides.

Microsomes were prepared from placentas of normotensive women and of patients suffering from pregnancy-induced hypertension (PIH). Activity of Na,K-ATPase (estimated as ATP hydrolysis) from the hypertensive tissue was lower than from tissue of normotensive women, even if the number of Na,K-ATPase molecules (monitored by anthroyl ouabain binding) was actually greater in the hypertensive tissue. The affinity of Na,K-ATPase for anthroyl ouabain was about four times higher in plasma membranes of hypertensives, indicating some structural change in the Na,K-ATPase or in its vicinity. Assuming the presence of an endogenous digitalis-like factor, the results suggest a simple way of explaining not only the lower Na,K-ATPase activity in the placental membranes of hypertensives but also the different extent of enzyme inhibition in different tissues of PIH patients.

Anthracenes↗
PubMed1993

Kinetic properties of yeast lysine permeases coded by genes on multi-copy vectors.

Amplification of the LYP1 transport system in the plasma membrane of Saccharomyces cerevisiae did not change the substrate specificity, the affinity and the pH optimum of the transport system for lysine, but significantly increased the uptake velocity and accumulation ratio. The dependence of active lysine uptake and accumulation on pH is probably given by the properties of the permease itself rather than by the value of available protonmotive force.

Biological Transport↗
PubMed1993

Effects of the physiological state of five yeast species on H(+)-ATPase-related processes.

Effects of starvation and glucose preincubation on membrane potential, ATPase-mediated acidification and glutamic acid transport were studied in yeast species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Dipodascus magnusii, Lodderomyces elongisporus and Rhodotorula gracilis. The membrane potential was highest after preincubation with glucose in all species but L. elongisporus and R. gracilis. In all cases the membranes were depolarized in the presence of 20 mmol/L KCl and hyperpolarized with 50 mumol/L diethylstilbestrol (DES). The extracellular acidification caused by addition of glucose was highest after preincubation with glucose in all cases except in R. gracilis where there was none. In all cases except in R. gracilis addition of KCl caused a marked increase in the acidification rate. Addition of DES with glucose caused a large decrease in rate in S. cerevisiae but had much less effect on the other species. Transport of glutamic acid was clearly increased after pretreatment with glucose in S. cerevisiae, S. pombe and D. magnusii (mainly due to enhanced synthesis of the carrier) but actually decreased in R. gracilis and L. elongisporus. Addition of DES had an inhibitory effect in all species but much more pronounced in S. cerevisiae and S. pombe than in others. In general, both the acidification and the transport of glutamate were enhanced after preincubation with glucose but much more so in the semianaerobic species, such as S. cerevisiae, than in the strict aerobes (R. gracilis) where the effect was occasionally negative. There was no relationship between the ATPase-mediated acidification and the membrane potential.

Biological Transport, Active↗
PubMed1993

Functional analysis of apf1 mutation causing defective amino acid transport in Saccharomyces cerevisiae.

Mutation in the Apf1 locus causes a pleiotropic effect of H(+)-driven active amino acid transport in baker's yeast Saccharomyces cerevisiae. The uptake of other, presumably H(+)-driven, substances, e.g. of purine and pyrimidine bases, maltose and phosphate ions, is not significantly influenced by this mutation. The apf1 mutation decreases not only the initial rates of amino acid uptake but also the accumulation ratios of amino acids taken up but has virtually no effect on the membrane potential or on the delta pH which constitute the thermodynamically relevant source of energy for their transport. Similarly, no changes in intracellular ATP content, in ATP-hydrolyzing and H(+)-extruding H(+)-ATPase activities, in the efflux of intracellularly accumulated amino acids, or in rates of endogenous respiration, were observed in the apf1 mutant phenotype. Hence, all these data are in accordance with the experiments showing that the Apf1 protein, an integral protein of the endoplasmic reticulum, is required exclusively for efficient processing and translocation of transport proteins specific for amino acids from the endoplasmic reticulum to their final destination, the plasma membrane.

Adenosine Triphosphate↗