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A Kotyk

Publications and source records attributed to A Kotyk.

At least 91 records · Page 5Linked to original sources
PubMed1982

Fine cytochemical localization of polyphosphates in the yeast Saccharomyces cerevisiae.

A late exponential culture, cultivated in the absence of phosphates, and a similar culture supplied with phosphate (phosphate overcompensation conditions) were prepared from an industrial strain of Saccharomyces cerevisiae. For the cytochemical staining, the cellular phosphates were transformed into polymeric metal-phosphate complexes by Ca2+ and Mg2+ ions, added to the fixative. The fixative contained 3% glutaraldehyde, buffered by 100 mM Tris-HCl to pH 6.0, plus 100 mM MgCl2 and 100 mM CaCl2. Staining with lead acetate was followed by OsO4 post-fixation. In cells cultivated in the absence of phosphates lead deposits were found in vacuoles only. In the late exponential culture the staining was observed on the surface of the plasmalemma, on the membranes of the endoplasmic reticulum, in mitochondria, in the cell nucleus, and in vacuoles. As a rule, extensive polyphosphate deposits (metachromatic granules) were found in vacuoles. Two hours after phosphate overcompensation, a high quantity of polyphosphate as found also in the cell wall, e.g., in the isthmus of budding cells (scar ring), in the secondary septa of mother and daughter cells, and in the growth apex. When divalent cations were omitted from the fixative, the staining of polyphosphates was limited to the cell wall and large vacuolar granules. The results of cytochemical staining were compared with the biochemical analysis of polyphosphate content in the cultures under study.

Cell Membrane↗
PubMed1981

Factors governing substrate-induced generation and extrusion of protons in the yeast Saccharomyces cerevisiae.

Experiments with respiration deficient (rho-), ADP/ATP transport deficient (op1) and double (op1 rho-) mutants, with glycolytic and tricarboxylic acid cycle substrates showed that the substrate-induced acidification of yeast suspensions is closely associated with glycolysis. The glucose/proton stoichiometry is 2.5 : 1 to 4 : 1 depending on glucose concentration. The kinetics of the process are complex, the acidification curve having a very fast initial component and two slower exponential components. The first component suggests an initial proton efflux from endogenous sources, triggered by exogenous substrates. The acidification process exhibits two Km values at about 1 and 15 mM D-glucose, indicating two distinct saturable pathways of proton extrusion. The total extent of acidification and thus the final pHout reaches a saturation value with increasing glucose concentration and suspension density. Both the total extent and the rate of acidification are subject to control by extracellular pH which reflects the tendency of the cells to build a fixed [H+]out/[H+]in ratio. When the control is lifted, both quantities are considerably increased. A crucial role in the substrate-induced acidification is thus played by active membrane processes and their control mechanisms.

Acid-Base Equilibrium↗
PubMed1981

Processes involved in the creation of buffering capacity and in substrate-induced proton extrusion in the yeast Saccharomyces cerevisiae.

The high pH-maintaining capacity of yeast suspension after glucose-induced acidification, measured as its ability to neutralize added alkali, was found to be due mainly to actively extruded acidity (H+). The buffering action of passively excreted metabolites (CO2, organic acids) and cell surface polyelectrolytes contributed only 15--40% to the overall pH-maintaining capacity which was 10 mmol NaOH/l per pH unit between pH 3 and 4 and 3.5 nmol NaOH/l per pH unit between pH 4 and 7. The buffering capacity of yeast cell-free extract was still higher (up to 4.5-times) than that of glucose-supplied cell suspension; addition of glucose to the extract thus produced considerable titratable acidity but negligible net acidity. The glucose-induced acidification of yeast suspension was stimulated by univalent cations in the sequence K+ greater than Rb+ much greater than Li+ congruent to Cs+ congruent to Na+. The processes participating in the acidification and probably also in the creation of extracellular buffering capacity include excretion of CO2 and organic acids, net extrusion of H+ and K+ (in K+-free media; in K+-containing media this is preceded by an initial rapid K+ uptake), and movements of some anions (phosphate, chlorides). The overall process appears to be electrically silent.

Acid-Base Equilibrium↗
PubMed1981

Membrane potentials in yeast cells measured by direct and indirect methods.

The membrane potential, delta psi, of various yeasts estimated from the distribution of tetraphenylphosphonium cations ranged from -50 to -120 mV, depending on species, incubation conditions and technique of measurement. Values obtained directly with a microelectrode in Endomyces magnusii were consistently lower than those determined indirectly.

Ascomycota↗
PubMed1981

The mechanism of resistance to streptomycin in Escherichia coli. Functional analysis of the permeability barrier of cells harbouring the R1 drd-19Km- plasmid.

The mechanism of phenotypically altered SM resistance in mutants of Escherichia coli JC5455 (Rldrd-19Km-) lrs and JC5455 (pON5300) was compared with that of the standard strain JC5455 (Rldrd-19Km-). On analyzing the membrane polypeptides in polyacrylamide gel both mutants were found to possess a protein spectrum different from that of ths standard strain. Transport of D-xylose and L-arginine was the same in all strains, transport of L-proline was decreased in JC5455 (pON5300) which may indicate a mutational interference with energy metabolism. The basic uptake of dihydrostreptomycin was the same in all strains but there were differences after preincubation of cells with streptomycin or glucose. The increased resistance of JC5455 (Rldrd-19Km-) lrs may be due to observed quantitative differences in membrane polypeptides that might play a role in the binding and functional expression of aminoglycoside-3'adenylyl transferase which modifies streptomycin. The increased sensitivity toward streptomycin in JC5455 (pON5300) can be explained by a mutation due to N-methyl-N'-nitro-N-nitrosoguanidine in the host cell since this change of sensitivity to streptomycin could not be transferred by transformation into a nonmutagenized strain. The coincidence of inducibility of increased transport of streptomycin by this antibiotic and the altered frequency of reversion to high levels of streptomycin resistance in JC5455 (pON5300) and in the transformant JC5455 (pON5302) may indicate that the altered reversibility toward phenotypically high resistance to streptomycin is a property of pON5300 and is transferred by transformation.

Bacterial Proteins↗
PubMed1981

Dependence of phosphate transport in yeast of glycolytic substrates.

Preincubation of baker's yeast (wild strain, respiration-deficient mutant and a low-phosphorus culture) with glucose, trehalose, and other metabolic sugars increases the subsequent uptake of inorganic phosphate 3-5 times. The Kt is reduced by the preincubation from 3.5 to 1.6 mM. The process involves primarily the production of glycolytic energy sources (suppression by iodoacetamide, no effect of antimycin or dicyclohexylcarbodiimide, negligible effect of ethanol, or respiratory mutation). The low-phosphorus yeast takes up phosphate anions about 1-20 times faster than the high-phosphorus (normal) culture. The stimulation is also accompanied by some (apparently nonessential) protein synthesis and has a halftime of 35 min; its decay has a t0.5 of 12 min but affects only less than one-half of the stimulated capacity.

Glucose↗
PubMed1981

Transport properties of two extremely thermophilic species of Thermus.

Thermus flavus and T. ruber grew optimally at 75 and 60 degrees C, respectively, but transport of monosaccharides (D-quinovose) and amino acids (2-aminoisobutyric acid) had optima about 20 degree C lower. Both transports were active, inhibited by 2,4-dinitrophenol but hardly at all by uranyl(2+) ions. Several transport systems are apparently involved with each class of compounds. Preincubation with glucose curtailed subsequent transport severely. Practical cessation of transport below 35 degrees C may be associated with the rather uniform composition of membrane lipids where iso- and anteiso-C15 and C17 acids are practically the only components.

Amino Acids↗
PubMed1979

Uptake of trehalose by Saccharomyces cerevisiae.

Trehalose, a storage sugar of baker's yeast, is known not to be metabolized when added to a cell suspension in water or a growth medium and to support growth only after a lag of about 10 h. However, it was transported into cells by at least two transport systems, the uptake being active, with a pH optimum at 5.5. There was no stoicheiometry with the shift of protons into cells observed at high trehalose concentrations. Trehalose remained intact in cells and was not appreciably lost to a trehalose-free medium. The uptake systems were present directly after growth on glucose, then decayed with a half-life of about 25 min but could be reactivated by aerobic incubation with trehalose, maltose, alpha-methyl-D-glucoside, glucose or ethanol. The uptake systems thus induced were different as revealed by competition experiments. At least one of the systems for trehalose uptake showed cooperative kinetics. Comparative anaysis with other disaccharides indicated the existence in Saccharomyces cerevisiae, after induction with trehalose, of at least four systems for the uptake of alpha-methyl-D-glucoside, four systems for maltose, together with the two for trehalose, variously shared by the sugars, the total of alpha-glucoside-transporting systems being five.

Antimetabolites↗
PubMed1978

Transport kinetics of 6-deoxy-D-glucose in Candida parapsilosis.

The strictly aerobic yeast Candida parapsilosis transports the nonmetabolizable monosaccharide 6-deoxy-D-glucose by an active process (inhibition by 2.4-dinitrophenol and other uncouplers but not by iodoacetamide), the accumulation ratio decreasing with increasing substrate concentration. Measured accumulation ratios are in agreement with those predicted from kinetic constants for influx and efflux. Energy for transport is probably required in the translocation step. The maximum rate is temperature-dependent with a transition point at 21 degrees C. the accumulation ratio is not. The uptake is most active at pH 4.5--8.5. It appears not to involve stoichiometric proton symport. The transport system is shared by D-glucose, D-mannose, D-galactose and possibly maltose but not by fructose, sucrose or pentoses. The apparent half-life of the transport system was 3.5--4 h.

Aerobiosis↗
PubMed1978

Stimulation of amino acid transport in Saccharomyces cerevisiae by metabolic inhibitors.

Inhibitors of energy metabolism (3-chlorophenylhydrazonomalononitrile, antimycin A, iodoacetamide, dicyclohexylcarbodiimide) but not of transport (uranyl ions) stimulate at low concentrations the uptake of L-leucine, L-glutamic acid, L-arginine and, to a lesser degree, of 2-aminoisobutyric acid in Saccharomyces cerevisiae. The effect is apparent only after augmenting the energy reserves of cells by preincubation with D-glucose or, more strikingly, with ethanol. It is absent in a mutant (op1) lacking the translocation system for ADP--ATP in mitochondria. The presence of two different energy reserves for amino acid transport is indicated (one in energy-poor, the other in energy-rich cells). The stimulating effect appears to be caused by a retarded degradation of the transport proteins as occurs at a lowered level of mitochondria-produced ATP.

Adenosine Triphosphate↗
PubMed1978

Effect of inhibitors on acid production by baker's yeast.

Glucose-induced acid extrusion, respiration and anaerobic fermentation in baker's yeast was studied with the aid of sixteen inhibitors. Uranyl(2+) nitrate affected the acid extrusion more anaerobically than aerobically; the complexing of Mg2+ and Ca2+ by EDTA at the membrane had no effect. Inhibitors of glycolysis (iodoacetamide, N-ethylmaleimide, fluoride) suppressed acid production markedly, and so did the phosphorylation-blocking arsenate. Fluoroacetate, inhibiting the citric-acid cycle, had no effect. Inhibition by uncouplers depended on their pKa values: 2,4,6-trinitrophenol (pKa 0.4) less than 2,4-dinitrophenol (4.1) less than azide (4.7) less than 3-chlorophenylhydrazonomalononitrile (6.0). Inhibition by trinitrophenol was only slightly increased by its acetylation. Cyanide and nonpermeant oligomycin showed practically no effect; inhibition by dicyclohexylcarbodiimide was delayed but potent. The concentration profiles of inhibition of acid production differed from those of respiration and fermentation. Thus, though the acid production is a metabolically dependent process, it does not reflect the intensity of metabolism, except partly in the first half of glycolysis.

Acids↗
PubMed1978

Apparent half-lives of sugar transport proteins in Saccharomyces cerevisiae.

Using incubation in the presence of 0.4 mM cycloheximide the half-lives of the principal membrane transport proteins in baker's yeast were found to be: more than 24 h for the constitutive glucose carrier, 2.2 h for the inducible galactose carrier, 1.2 h for the inducible maltose carrier and 0.8 h for the inducible alpha-methyl-D-glucoside carrier. The distinct nature of the two last-named carriers was thus supported. De-induction of the galactose carrier was enhanced in the presence of glucose plus cycloheximide but not of either substance alone. Chloramphenicol suppressed all effects of cycloheximide. In contrast to the enzymes of galactose metabolism, the induction of the glactose carrier was not under the control of a mitochondrial factor and took place in a rho-mutant. The system induced by maltose but not the one induced by alpha-methyl-D-glucoside was de-induced rapidly by the intervention of a cytoplasm-synthesized protein.

Carbohydrate Metabolism↗
PubMed1977

Effect of high substrate concentrations on active transport parameters.

A general model is described to account for the observation that steady-state accumulation ratios (mainly of non-electrolytes) decrease with increasing solute concentration, frequently reaching values of less than unity. Three variants of the model are treated, all of them including the assumption that the immediate supply of the source of energy is limited and its local concentration is appreciably reduced by its interaction with the transport system. Consequences of this assumption for the kinetic parameters of the initial rate of transport are analyzed and compared with experimental data.

Biological Transport, Active↗
PubMed1977

Specificity of trans-inhibition of amino acid transport in baker's yeast.

The trans-inhibition potency of intracellular amino acids on the transport of various amino acids follows the same sequence, viz. Pro(Lys), Phe, Glu, Ala, Gly, Leu, and alpha-aminoisobutyric acid. The same sequence was found for the reciprocal of trans-inhibition constants. It appears that the intracellular amino acid itself or a derivative thereof acts on a component that is common to all amino acid transport systems of baker's yeast.

Amino Acids↗