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Biomedical subjects

A Kotyk

Publications and source records attributed to A Kotyk.

At least 55 records · Page 3Linked to original sources
PubMed1992

Are proton symports in yeast directly linked to H(+)-ATPase acidification?

Transport of amino acids in Saccharomyces cerevisiae is an H(+)-driven secondary active transport. Inhibitors of the plasma membrane H(+)-ATPase, particularly heavy water, diethylstilbestrol and suloctidil, were shown to affect the H(+)-extruding ATPase activity as well as the ATP-hydrolyzing activity, to a similar degree as they inhibited the transport of amino acids. The inhibitors had virtually no effect on the membrane electric potential or on the delta pH which constitute the thermodynamically relevant source of energy for these transports. Transport of acidic amino acids was affected much more than that of the neutral and especially of the basic ones. The effects were greater with higher amino acid concentrations. All this is taken as evidence that the amino acid carriers respond kinetically to the presence of protons directly at the membrane site where they are extruded by the H(+)-ATPase, rather than to the overall protonmotive force.

Deuterium↗
PubMed1992

Transport of L-glutamic acid in the fission yeast Schizosaccharomyces pombe.

Transport of L-glutamic acid into the fission yeast Schizosaccharomyces pombe grown to the early stationary phase and preincubated for 60 min with 1% D-glucose is practically unidirectional and is mediated by a single uphill transport system with a KT of 170 microM and Jmax of 4.8 nmol min-1 (mg dry wt.)-1. The system proved to be rather non-specific since all the amino acids transported into the cells acted as potent competitive inhibitors. It has a pH optimum at 3.0-4.0, the accumulation ratio of L-glutamic acid is highest at a suspension density of 0.6-1.0 mg dry wt. per ml and decreases with increasing L-glutamic acid concentrations in the external medium. The system present in the cells after preincubation with D-glucose is unstable and its activity decays after washing the cells with water or after stopping the cytosolic proteinsynthesis with cycloheximide, with a half-time of 24 min in a reaction significantly retarded by phenylmethylsulfonyl fluoride, a serine proteinase inhibitor. The synthesis of the transport protein appears to be repressible by ammonium ions.

Biological Transport↗
PubMed1992

Thialysine-resistant mutants and uptake of lysine in Schizosaccharomyces pombe.

Mutants defective in lysine transport were isolated and characterized. After UV-mutagenesis colonies resistant to thialysine, a toxic analogue of lysine, were isolated and L-lysine uptake into the mutant strains was analyzed. Among the thialysine-resistant strains a group of mutants was found, where the half-saturation constant, KT, of the high-affinity transport system for lysine was higher than in the wild-type, the high-affinity transport system for basic amino acids being specifically affected. This was confirmed by a complementation test in which all the thialysine-resistant strains with a higher KT for lysine uptake belonged to one complementation group. Kinetic and genetic analysis showed that our mutants were identical with can1-1 mutants, showing that a single high-affinity system for the transport of basic amino acids exists in S. pombe.

Arginine↗
PubMed1992

Interaction of 2-deoxy-D-glucose and adenine with phosphate anion uptake in yeast.

The transport of inorganic phosphate anions into yeast cells (after preincubation with glucose, fructose or another metabolizable sugar, and in the presence of glucose) shows two kinetic components with half-saturation constants of 40 mumol/L and 2.4 mmol/L. The uptake was strikingly stimulated by 2-deoxy-D-glucose (2-dGlc) at lower concentrations but inhibited above 100 mmol/L. A similar stimulation was caused by adenine (0.01-1 mmol/L) and a very small one by uracil and inorganic sulfate. It is suggested that either a phosphorylation reaction accompanies the transport (2-dGlc) or that some compounds stimulate the H(+)-ATPase more than inorganic phosphate itself and thus increase its rate of transport.

Adenine↗
PubMed1992

Role of alkaline metal ions in the H(+)-ATPase activity of various yeast species.

Saccharomyces cerevisiae, Schizosaccharomyces pombe, Endomyces magnussi, Lodderomyces elongisporus and Rhodotorula gracilis, yeast species ranging from a glycolytic type to a strictly aerobic one, were tested for the activity of their plasma membrane H(+)-ATPase and the effect of alkaline metal cations thereon. The ATP-hydrolyzing activity of membranes from glucose-activated cells ranged from 456 to 932 mumol inorganic phosphate released per min per 1 g membrane protein. The effect of 0.2 M Li+, Na+, K+, Rb+ and Cs+ never exceeded the statistical range of error. In contrast, acidification after glucose addition ranged from 0.15 (for R. gracilis) to 14.8 nmol H+ per min per mg dry weight (for S. cerevisiae) and it was markedly influenced by the presence of alkaline metal chlorides, the highest effect observed being a seven-fold increase by K+ in a S. cerevisiae suspension. The effects were additive to those observed without ions in solution and are ascribed to the operation of independent channels and/or exchange systems for H+ with a clear selectivity toward K+. The separate nature of the ion-triggered extracellular acidification is supported by a different ratio of titration to pH-derived acidity with and without K+.

Adenosine Triphosphate↗
PubMed1992

Lifetime determination of low sample concentrations: a new cuvette for highly accurate and sensitive fluorescence measurements.

A new cuvette was developed for spectrofluorometric measurements which increases the efficiency of recorded fluorescence emission by at least two-fold, decreasing, at the same time, the noise component of the record. It is suited for both steady-state and dynamic fluorescence measurements. Comparative data for both the classical and the novel cuvettes are presented here.

Data Interpretation, Statistical↗
PubMed1991

Increased endocytosis in the Saccharomyces cerevisiae fragile mutant VY1160.

The VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for which Saccharomyces cerevisiae cells are not permeable. Two mutations, srb1 and ts1, have been identified in VY1160 mutant, and previous studies (Kozhina et al., 1979) have shown srb1 to be responsible for cell lysis. We now present evidence that the ts1 mutation leads to increased endocytosis in VY1160 cells. The internalization of lucifer yellow carbohydrazide in VY1160 cells is time-, temperature- and energy-dependent and consistent with a fluid-phase mechanism of endocytosis. The rate of steady-state accumulation of the dye at 37 degrees C is 145 ng/micrograms DNA per h for VY1160 mutant and 23 ng/micrograms DNA per h for S288C parental strain. Studies with isogenic strains having either the srb1 or the ts1 mutation, or SRB1 TS1 wild-type alleles have shown that only ts1 strains possess increased endocytosis. Quantitation of endocytosis in cells grown at 24 degrees C and shifted at 38 degrees C shows that ts1 strains, but not srb1 and wild-type strains, increase ten-fold the internalization of lucifer yellow 2 h after the shift at 38 degrees C. The analysis of ts1 x wild-type crosses provides evidence that the temperature-sensitive phenotype segregates together with the enhanced endocytosis. It is concluded that the increased endocytosis might explain the generally increased permeability of VY1160 mutant cells.

Adsorption↗
PubMed1991

Protonmotive force in yeasts--pH, buffer and species dependence.

Using yeast species Saccharomyces cerevisiae K, Rhodotorula gracilis, and Lodderomyces elongisporus, their intracellular pH value and their membrane potential were estimated at pH 3.5-7.5 in four different buffers: triethanolamine--phthalic acid (TEPA), citric acid--trisodium citrate (CASC), acetic acid--NaOH (AANA) and MES. The pHin followed the same pattern in all buffers, with rather constant values below pHout = 5 and again above pHout = 7. The membrane potential decreased regularly with decreasing pHout. The apparent protonmotive force increased with decreasing pHout. It is seen that for all the yeast species pHin and pHout are the same between 5.0 and 6.0 which is thus the pH range of choice for various transport measurements. Of the four buffers, TEPA gives smoothest pHin dependences on pHout and, being metabolically inert, is the one to be recommended.

Biological Transport, Active↗
PubMed1990

Deuterons cannot replace protons in active transport processes in yeast.

Replacement of ordinary water with heavy water causes a sharp reduction of the rates of both primary hydrogen ion transport (at the plasma membrane ATPase) and secondary symports (H(+)-associated transports of sugars and amino acids) in several species of yeast. At the same time, the hydrolytic activity of the ATPase is affected only very little. Likewise, the membrane potential, the delta pH and, correspondingly, the accumulation ratios of the various symported solutes are altered much less. This serves as evidence that H+ or H3O+ ions are direct participants in the various active transports of nutrients in yeast.

Adenosine Triphosphatases↗
PubMed1990

Phenylmethylsulfonyl fluoride protects L-lysine transport in Schizosaccharomyces pombe against inactivation by ammonium ions.

Ammonium ions inactivate the basic amino acid transport system in Schizosaccharomyces pombe in an irreversible manner. The inactivation is accompanied by a 4-fold decrease of KT of L-lysine transport, leaving its Jmax unchanged; phenylmethylsulfonyl fluoride protects the system against inactivation. In contrast, two basic amino acid transport systems in a gap1 mutant of Saccharomyces cerevisiae are influenced by NH4+ ions in such a way that only the Jmax decreases while the KT of L-lysine transport is unchanged. Phenylmethylsulfonyl fluoride does not act here as a protective agent.

Ammonia↗
PubMed1990

Transport of L-tryptophan in Saccharomyces cerevisiae.

In addition to the general amino acid transport system (GAP) of S. cerevisiae L-tryptophan is transported by another system with approximately 25% capacity of GAP, with a KT of 0.41 +/- 0.08 mmol/L and with a similar specificity as GAP (lower inhibition by Met, Pro, Ser, Thr and 2-aminoisobutyric acid; greater inhibition by Glu and His). The pH optimum of this system is at 5.0-5.5, activation energy above the transition point (20 degrees C) was 20 kJ/mol, below the transition point 55 kJ/mol. The transport by this system was virtually unidirectional, efflux amounting to at most 10% into a tryptophan-free medium. The transport itself was blocked by 2,4-dinitrophenol, antimycin A and uranyl nitrate. The system was synthesized de novo during preincubation with glucose = fructose greater than trehalose greater than ethanol within 30 min, and was degraded with a half-time of 15 min in the absence of further synthesis. The accumulation ratios of L-tryptophan in gap1 mutants were concentration-dependent (200:1 at 1 mumol L-Trp/L, 4:1 at 2.5 mmol L-Trp/L) and decreased with increasing suspension density from 200:1 to 5:1 (for 10 mumol L-Trp/L). The involvement of hydrogen ions in the uptake was clearly demonstrated by the effect of D2O even if it could not be established by either shifts of pHout or membrane depolarization.

Anaerobiosis↗
PubMed1989

Transport of L-lysine in the fission yeast Schizosaccharomyces pombe.

Systems of L-lysine transport in Schizosaccharomyces pombe are not constitutive, as at no phase of growth in a rich medium is lysine taken up. Transport activity appears only after preincubation of harvested cells with glucose or another suitable source of energy. If cycloheximide is added during this preincubation no transport systems are synthesized. After removal of glucose, the activity of the transport system decays with a half-time of 13 min. The transport of L-lysine into S. pombe cells from the stationary phase of growth preincubated for 60 min with 1% D-glucose is mediated by at least two systems, the high-affinity one with a Kt of 26 mumol/l and Jmax of 4.95 nmol/min per mg dry wt., the low-affinity one with a KT of 1.1 mmol/l and Jmax of 11.8 nmol/min per mg dry wt. The transport of lysine mediated by these two systems proceeds uphill. The high-affinity system has a pH optimum at 4.0-4.2, the accumulation ratio is highest at a cell density 2-5 mg dry wt. per ml and decreases with increasing lysine concentrations. Lysine accumulated by this system does not exit from cells. The only potent competitive inhibitors are L-arginine, L-histidine and D-lysine. The other amino acids tested do not behave as competitive inhibitors. Of the various metabolic inhibitors tested, the most potent were proton conductors and antimycin A.

Biological Transport, Active↗
PubMed1989

Inhibition of hexose transport by glucose in a glucose-6-phosphate isomerase mutant of Saccharomyces cerevisiae.

The rate of hexose transport was approximately 60% lower for both the high- and the low-affinity components of hexose uptake when a glucose-6-phosphate isomerase mutant of Saccharomyces cerevisiae was preincubated with glucose, as compared with preincubation with water. Similarly the Jmax value of the high-affinity system of the mutant was 25-35% of the corresponding Jmax value for normal cells incubated with glucose. Accumulation of glucose 6-phosphate or of some other metabolite, such as fructose 6-phosphate or trehalose, may be responsible for this striking inhibition.

Biological Transport, Active↗
PubMed1989

Peculiarities of amino acid transport in Schizosaccharomyces pombe: effects of growth medium.

Transport systems for amino acids in the wild-type strain of Schizosaccharomyces pombe are not constitutive. During growth on different media no transport of acidic, neutral and basic amino acids is detectable. To acquire the ability to transport amino acids, cells must be preincubated with a metabolic source of energy, such as glucose. The appearance of transport activity is associated with protein synthesis (suppression by cycloheximide) at all phases of culture growth. After such preincubation the initial rate of amino acid uptake depends on the phase of growth of the culture and on the amount of glucose in the growth medium but not on the nitrogen source used. L-Proline and 2-aminoisobutyric acid are practically not transported under any of the conditions tested.

Amino Acids↗
PubMed1988

Exocytosis as a possible mechanism for the secretion of coproporphyrin from a chloroquine-treated yeast.

Chloroquine, which is known to be concentrated in intracellular acid vesicles, stimulates the release of porphyrins from yeast. Experiments with normal baker's yeast Saccharomyces cerevisiae and the sec-1 mutant, with suppressed exocytosis, showed that the release of porphyrins was stimulated more in normal yeast than it was in the mutant. It is suggested that chloroquine stimulates the exocytosis of porphyrins.

Chloroquine↗
PubMed1988

Membrane transport in an osmotically fragile mutant of Saccharomyces cerevisiae.

Transport properties of the osmotically fragile strain VY1160 of Saccharomyces cerevisiae were compared with those of the parent S288c strain. Mediated diffusion of 6-deoxy-D-glucose was practically unaffected; membrane-potential-dependent transport of D-glucosamine was very much depressed in the fragile strain. The H+-driven transport of L-lysine and L-proline, as well as that of the hitherto uninvestigated D-glucose-6-phosphate, were also very depressed. 2-Deoxy-D-glucose transport displayed slightly different kinetic parameters. Primary H+ extrusion by the plasma membrane H-ATPase was not diminished although the ATP-splitting activity was depressed by about 50%. The overall proton-motive force (pmf) of the fragile mutant at pH 5.5 was only 20 mV while in the parent strain it was 108 mV. In parallel with this, spontaneous acidification of the external medium (a CO2-associated event) was only about 2% of that in the parent strain. The defect in this, together with the inability to stimulate transport protein synthesis by glucose, may account for the generally poorer transport performance of the fragile mutant.

Amino Acids↗