Article

THE PARTICIPATION OF PHOSPHATE IN THE FORMATION OF A "CARRIER" FOR THE TRANSPORT OF MG++ AND MN++ IONS INTO YEAST CELLS

Authors:
To read the full-text of this research, you can request a copy directly from the author.

Abstract

During the absorption of phosphate by yeast, the cells acquire the capacity to absorb Mn++ and Mg++, a capacity which is retained even after phosphate is no longer present in the medium. Cells pretreated with phosphate and then washed, slowly lose their ability to absorb Mn++, the rate of loss depending on the temperature and on the metabolic state. The fermentation of sugars induces a very rapid loss of absorptive capacity, whereas the respiration of ethyl alcohol, lactate, or pyruvate has little effect. Inhibitor studies with sodium acetate, redox dyes, and arsenate, reveal parallel effects on Mn++ absorption, and on phosphate absorption. It is concluded that the synthesis of a carrier for the transport of Mg++ and Mn++ involves a phosphorylation step closely coupled with reactions involved in the absorption of phosphate.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the author.

Article
Saccharomyces cerevisiae and other yeasts are used widely in traditional industries and are subject to increasing frequency of use in the new biotechnology industries. With this in mind, the mechanisms regulating ion uptake and nutrition in yeast are discussed. A lack of industrially relevant studies is painfully evident, and this review has endeavored to interpret the results of the more fundamental studies that have been conducted over the years. The cyclical nature of ion transport is raised as a major factor regulating the growth of yeast, as is the role of cell vacuolar compartments in the storage of excess cationic and anionic species. The role of pH in the regulation of ion uptake is also discussed and possible mechanisms for overcoming some forms of ionic inhibition are raised. The need for further studies of specific interest to the industrial cultivation of yeast is stressed.
Article
Biological barriers are studded with a variety of reactive sites that favor the passage of suitable molecules; in some cases, mechanisms that seem inherently improbable deliver the molecules from these sites against electrochemical gradients, either normally or when a gradient of an analog is artifically produced. In their aggregate, these reactive sites give cytological barriers, their segregating action, and permit physiological and pharmacological control through the increase and decrease of such segregating effects. The coincident nature of such modifications of transport has suggested that the activity is inherent in a common matrix rather than a consequence of the shuttling of dissimilar carriers. Attempts should be intensified to identify these reactive sites on organelles, membranes, and intact cells by the chemical procedures now available for specific sites on macromolecules. Site subject to significant modification in conformation or charge distribution by hormone action should be investigated. Structures having the same binding characteristics as a given transport site should also be looked for in broken-cell preparations. This general search can take advantage of a critical property of the site on the intact cell and need not be deterred by immediate concern for the accessory equipment or mechanism of the transport.
Article
This review highlights the important roles played by magnesium in the growth and metabolic functions of microbial and animal cells, and therefore assigns a key role for magnesium ions in biotechnology. The fundamental biochemical and physiological actions of magnesium as a regulatory cation are outlined. Such actions are deemed to be relevant in an applied sense, because Mg2+ availability in cell culture and fermentation media can dramatically influence growth and metabolism of cells. Manipulation of extracellular and intracellular magnesium ions can thus be envisaged as a relatively simplistic, but nevertheless versatile, means of physiological cell engineering. In addition, biological antagonism between calcium and magnesium at the molecular level may have profound consequences for the optimization of biotechnological processes that exploit cells. In fermentation, for example, it is argued that the efficiency of microbial conversion of substrate to product may be improved by altering Mg:Ca concentration ratios in industrial feedstocks in a way that makes more magnesium available to the cells. With particular respect to yeast-based biotechnologies, magnesium availability is seen as being crucially important in governing central pathways of carbohydrate catabolism, especially ethanolic fermentation. It is proposed that such influences of magnesium ions are expressed at the combined levels of key enzyme activation and cell membrane stabilization. The former ensures optimum flow of substrate to ethanol and the latter acts to protect yeasts from physical and chemical stress.
Article
Full-text available
Lactobacillus plantarum has an unusually high Mn(II) requirement for growth and accumulated over 30 mM intracellular Mn(II). The acquisition of Mn(II) by L. plantarum occurred via a specific active transport system powered by the transmembrane proton gradient. The Mn(II) uptake system has a Km of 0.2 microM and a Vmax of 24 nmol mg-1 of protein min-1. Above a medium Mn(II) concentration of 200 microM, the intracellular Mn(II) level was independent of the medium Mn(II) and unresponsive to oxygen stresses but was reduced by phosphate limitation. At a pH of 5.5, citrate, isocitrate, and cis-aconitate effectively promoted MN(II) uptake, although measurable levels of 1,5-[14C]citrate were not accumulated. When cells were presented with equimolar Mn(II) and Cd(II), Cd(II) was preferentially taken up by the Mn(II) transport system. Both Mn(II) and Cd(II) uptake were greatly increased by Mn(II) starvation. Mn(II) uptake by Mn(II)-starved cells was subject to a negative feedback regulatory mechanism functioning less than 1 min after exposure of the cells to Mn(II) and independent of protein synthesis. When presented with a relatively large amount of exogenous Mn(II), Mn(II)-starved cells exhibited a measurable efflux of their internal Mn(II), but the rate was only a small fraction of the maximal Mn(II) uptake rate.
Article
The concentration dependence of the uptake rate of divalent cations in yeast can be described by a simple diffusion process after accounting for the effect of the surface potential upon the divalent cation concentration near the membrane. It is also necessary to correct for the effect of the cell pH upon the rate of translocation. The apparent saturation kinetics is ascribed to the fact that the quotient of the concentration of the divalent cations near the cell membrane and the bulk aqueous phase concentration is reduced on increasing the divalent cation concentration in the medium. The diffusion process regulated by the surface potential even mimics the saturation kinetics of a two-carrier transport system. The selectivity found between Ca2+ and Sr2+ uptake can probably be traced to differences in their affinity for the negative groups on the cell membrane determining the surface potential rather than to differences in their affinity for a transport system. The enhancement of divalent cation uptake by loading the cells with phosphate is probably due to the concomitant increase in the net negative charge of the cell membrane.
Article
Experiments were performed to obtain information on: (i) the specific properties of Ca2+ binding and transport in yeast; (ii) the relationship between both parameters; (iii) similarities to or differences from other biological systems as measured by the effects of inhibitors; and (iv) the effects of mono and divalent cations, in order to get some insight on the specificity and some characteristics of the mechanism of the transport system for divalent cations in yeast. The results obtained gave some kinetic parameters for a high affinity system involved in the transport of Ca2+ in yeast. These were obtained mainly by considering actual concentrations of Ca2+ in the medium after substracting the amounts bound to the cell. A km of 1.9 microM and a Vmax of 1.2 nmol (100 mg.3 min)-1 were calculated. The effects of some inhibitors and other cations on Ca2+ uptake allow one to postulate some independence between binding and transport for this divalent cation. Of the inhibitors tested, only lanthanum seems to be a potent inhibitor of Ca2+ uptake in yeast. The effects of Mg2+ on the uptake of Ca2+ agree with the existence of a single transport system for both divalent cations. The actions of Na+ and K+ on the transport of Ca2+ offer interesting possibilities to study further some of the mechanistic properties of this transport system for divalent cations.
Article
The amount of arsenate in yeast cells exposed to arsenate plus fermentable sugar for varying periods of time is determined by the kinetics of influx and efflux. Influx occurs only in the presence of fermentable substrate via a transport mechanism that can be divided by kinetic properties into two components with Michaelis constants of 4 × 10−6 and 4 × 10−4. During exposure to arsenate the transport system is gradually reduced in capacity and, with sufficient concentrations of arsenate, may be almost completely inactivated. At the same time, previously absorbed arsenate leaks out of the cell with first-order kinetics. The balance of inward transport, inactivation of the transport system, and outward leakage results in the arsenate content of the cell reaching a maximal value and then decreasing. Arsenate also inhibits the fermentation of the intact cell, but only to a maximal extent of 60 per cent, compared to 100 per cent in broken cells. The data indicate that only a small fraction of the cellular arsenate is involved in the inhibition of metabolism and of transport and that its distribution and metabolic turnover must be complicated. Simple fractionations of cellular arsenate confirm this supposition.
Article
Ni2+, Co2+, Zn2+ can be taken up into a non-exchangeable pool by yeast cells, by a system that also transports Mg2+ and Mn2+. The affinity series is Mg2+, Co2+, Zn2+ > Mn2+ > Ni2+ > Ca2+ > Sr2+. The uptake is small in starved cells, but is enhanced in the presence of glucose. It is remarkably stimulated (5–20 fold) if cells are pretreated with phosphate and glucose. The uptakes are the same under aerobic or anaerobic conditions suggesting that fermentative reactions can supply the energy for transport. Uptake is reduced at low pH (below 5.0), but a H+ exchange system is not involved. Instead, 2 K+ (or 2 Na+ in Na+-loaded cells) are secreted for each divalent cation absorbed.
Article
The transition metal manganese is considered to be a minor micronutrient in both pro- and eukaryotes, usually being required from the environment at subnanomolar levels. Until recently, Mn was only known to function in cells as a cofactor for a few enzymatic reactions. A notable exception has been reported in many lactic acid bacterial species which require micromolar medium Mn levels for growth and contain up to 35 mM Mn. These high Mn concentrations are accompanied by the near or complete absence of intracellular iron and superoxide dismutase (SOD). Lacking hemes, Lactobacillus plantarum and related species contain a unique Mn-cofactored catalase as well as millimolar Mn(II) in a nonenzymic complex performing the function of the micromolar superoxide dismutase found in most other aerotolerant cells. The high Mn(II) levels are accumulated via an efficient active transport system and are stored intracellularly in a high molecular weight complex. Study of Lactobacillus plantarum has provided an interesting example of the substitution of Mn for Fe in several of the biological roles of Fe, an alternative mechanism of aerotolerance, and a better understanding of the unique biochemistry of the lactic acid bacteria.
Article
1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.
Article
Retention of radioactive chromium chloride by Brewer's yeast, Saccharomyces carlsbergensis, was stimulated by glucose and phosphate. Glucose levels to 25%, increased the amount of chromium retained per gram of cells but high glucose also inhibited growth. Addition of phosphate enhanced Cr retention more than 2.5-fold over the unsupplemented controls. The rate and maximal incorporation of radioactively labeled inorganic chromium salts, which yield little biological activity, was similar to that of organic chromium complexes that display in vitro insulin potentiating activity. The release of the labeled Cr from the yeast cells was pH dependent and more than 85% of the labeled Cr could be extracted with dilute ammonium hydroxide. Disruption of cells with teichozyme-Y released a similar amount of labeled chromium but much lower amounts were extracted with ethanol. Insulin potentiating activity was greatest in the ammonia extract and was lower in ethanol and teichozyme extracts. Essentially all of the radioactive chromium incorporated into the yeast was present in the soluble portion of the cell. These data define conditions for the growth of Brewer's yeast for optimal incorporation of labeled Cr and the conditions for extraction of a biologically active γ-labeled product.
Article
The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.
Chapter
Der Grund, weswegen im Folgenden nur von den Möglichkeiten des Zustandekommens aktiver Transportvorgänge gesprochen werden kann, ist einfach anzugeben: ihr Mechanismus ist noch unbekannt. Ihn kennen zu lernen, ist ein dringendes Anliegen der kausalen Biologie, da die Existenz und die Leistungen der Zellen und Gewebe von seinem Funktionieren abhängen. Der aktive Transport selbst aber ist ein biochemischer Vorgang, der allerdings zunächst als physikalisch-chemische Größe, nämlich als Konzentrationsänderung meßbar wird. Eine als aktiver Transport zu bezeichnende Veränderung von Konzentrationen gegenüber den bei passiver Verteilung zu erwartenden ist wohl eine der einfachsten Äußerungen einer Wechselwirkung zwischen der statischen Feinstruktur der Zelle und den sich an ihr abspielenden chemischen Vorgängen.
Article
It was found that yeast requires a supply of nitrogen and a source of energy in order to take up inorganic sulphate from the medium. Inorganic sulphate enters into cells at a higher rate in normal or S-deficient cells than in N-deficient cells. Michaelis constants of the process amount to 2×10−4 for N-deficient cells and to 7.7×10−5 for S-deficient cells (22°C). Sulphite and thiosulphate act as competitive inhibitors. The uptake of sulphate proceeds at the same rate aerobically and anaerobically. Dinitrophenol, sodium azide and iodoacetic acid inhibit the sulphate uptake; the effect is more marked in N-deficient than in S-deficient yeast. Urethane inhibits in both types to the same degree. Mercurie chloride acts in two different ways, inhibiting enzymatic processes, on the one hand, and increasing the cell membrane permeability, on the other hand. Dinitrophenol inhibits the uptake of sulphate even anaerobically, which points to an effect distinct from the uncoupling of oxidative phosphorylation. During incubation, radioactive sulphate appears first in nucleotide-like compound(s), then in free amino acids (cysteine) and only later in proteins, protein methionine being the last to be labelled. In the discussion the question of differences between the two types of yeast and the effect of inhibitors are taken up.
Article
This review details the major interactions of fungi with toxic metal species. Physico-chemical and biological aspects are discussed including definitions and classification of metals in biological contexts, mechanisms of metal fungitoxicity, resistance and tolerance, environmental influences and the occurrence of fungi in metal-polluted habitats. Cellular interactions include extracellular precipitation and complexation, binding to cell walls, transport of monovalent and divalent metal cations, intracellular fates of toxic metal species (including metal-binding proteins and peptides, and vacuolar compartmentation), and metal transformations including organometal(loid) synthesis. Significant interactions between metals and mycorrhizal fungi and macrofungi are summarized with reference to the amelioration of plant phytotoxicity and metal accumulation respectively. Biotechnological aspects of metal-fungal interactions relate to the biological treatment of metal-contaminated effluents and waste streams for metal removal/recovery and environmental protection.
Article
The uptake of arsenate by Candida humicola requires an active transport system and may operate through low- and high-affinity sites. Arsenite, methylarsonate and dimethylarsinate are deduced to enter the cell by slow passive diffusion.
Article
The effect of monovalent cations on derepression of phosphate transport was studied. It was found that ammonium, K+ and Rb+ accelerate the derepression of phosphate transport produced by glucose in yeast (Saccharomyces cerevisiae). Na+ and Li+ were ineffective in accelerating derepression; Cs+ produced only a minor stimulation. The concentration range of both K+ and NH4+ that accelerated derepression was similar to that required for transport to occur. In the case of ammonium, the effects seem to depend exclusively on the so-called low-affinity transport system. The effect was strongly dependent on pH, with an optimum around 6; however, the increase in the pH of the medium did not produce in itself a high increase of the depression. Derepression was dependent on the presence of glucose, and it was very low with ethanol as substrate. The mechanism seems to depend on the ability that both K+ and NH4+ have to decrease the membrane potential of the cell while transported, and not on the capacity to produce the alkalinization of the cell interior. In addition, the phenomenon depends on the presence of glucose as substrate, which indicates the involvement of some product of glucose metabolism in the mechanism, and possibly some relation to catabolic repression.
Article
Uptake of ⁵⁴Mn by excised oat roots from dilute manganese chloride solutions has been investigated. The time-course of uptake has been analysed into the customary but somewhat arbitrary fast and slow phases. Uptake is not metabolic in either of these. The fast phase (‘exchangeable’ manganese) is essentially complete in about 30 minutes and represents the attainment of equilibrium in a process of ion-exchange. It is shown that analyses appropriate for enzyme kinetics cannot be applied in this situation, and an alternative formulation is based on Donnan equilibration, taking account of the selectivity of the ion exchanger towards different counter-ions; the predictions of this latter theory are compared with the experimentally determined uptake. The slow phase (‘absorbed’ manganese) may also involve exchange sites, either chemically different from, or more difficult of access than, those involved in the fast phase, or both. Equilibrium was certainly not reached in three hours in this slow-phase process. Release of manganese, taken up by the roots from manganese chloride solutions, into calcium chloride solutions does not seem to be simply the reversal of uptake, particularly with very dilute solutions. This is particularly shown by the kinetics of uptake and release, uptake being a much faster process than release. Manganese may transfer from the first phase to the second phase, but there is no evidence that uptake by the roots proceeds in series from first to second phase. It is considered more likely that the two phases function independently, linked by the surrounding solution.
Article
Using a commercial lager brewing yeast, the immediate release of magnesium, potassium and phosphate ions by cells when inoculated into wort was evaluated to be directly related to its subsequent fermentation performance. Yeast which released appreciable amounts of these ions immediately after inoculation mediated improved fermentations as evidenced by better growth, higher ethanol concentrations and lower diacetyl levels at the end of fermentation. Conversely, yeast that absorbed these ions or released them at very low concentrations performed poorly throughout fermentation producing beers with lower ethanol concentrations and higher diacetyl levels. These observations led to the identification and development of a rapid, practical and highly sensitive method to measure Mg++ released or absorbed by yeast as an indicator of its vitality and a predictor of its subsequent fermentative performance. Full method details of the Magnesium Release Test (MRT) are given.
Article
Copper uptake by and toxicity to Saccharomyces cerevisiae Hansen is greatly enhanced by glucose. Uptake and toxicity are markedly reduced at low temperature (1–2 °C) and the enhancement by glucose abolished. Energy dependent transport of copper is indicated but further investigation was precluded by high toxicity of very low copper concentrations.
Article
Magnesium and manganese have proved physically and functionally interchangeable in many isolated biological systems investigated in vitro. This lack of discrimination contrasts sharply with the high biological specificity exhibited by intact mammals under a large variety of conditions. The dichotomy between intact animals and their isolated systems might be due at least partially to presence vs. absence of an intact circulation. Hence the capability of mammalian plasma to discriminate between the alkaline earth and the transition metal was investigated by means of equilibrium dialysis, exchange, ultrafiltration, ultracentrifugation, and zone electrophoresis. The states of the respective elements are thus contrasted as follows: (a) Magnesium is partially bound, manganese totally. (b) Magnesium is nonselectively bound by serum proteins, manganese selectively by a ß1-globulin. (c) Under conditions approaching physiological, the two metals do not interchange. This is interpreted as indicating that the plasma proteins contribute to biological specificity by discriminating between a trace metal and a macronutrient.
Chapter
This Chapter is concerned with those systems which are responsible for the transport of inorganic ions, amino acids and sugars into vegetative fungal cells. One of the virtues of fungi is the ease with which mutants can be obtained. Mutants have been used both to identify transport systems and to provide information about the way transport is regulated. With respect to this latter topic, a recurring theme in much of the information is feedback inhibition of transport either by the solute transported or by some compound closely related metabolically. The extensive use of mutants has of course meant that transport has been thought of in terms of transport proteins, with a consequent emphasis on transport kinetics.
Article
Manganese was accumulated by cells of Escherichia coli by means of an active transport system quite independent of the magnesium transport system. When the radioisotope (54)Mn was used, manganese transport showed saturation kinetics with a K(m) of 2 x 10(-7)m and a V(max) of 1 to 4 nmoles/min per 10(12) cells at 25 C. The manganese transport system is highly specific; magnesium and calcium did not stimulate, inhibit, or compete with manganese for cellular uptake. Cobalt and iron specifically interfered with (54)Mn uptake, but only when added at concentrations 100 times higher than the K(m) for manganese. Active transport of manganese is temperature- and energy-dependent: uptake of (54)Mn was inhibited by cyanide, dinitrophenol, and m-chlorophenyl carbonylcyanide hydrazone (CCCP). Furthermore, the turnover or exit of manganese from intact cells was inhibited by energy poisons such as dinitrophenol and CCCP.
Nerve Impulse, Tr. 2nd Josiah Macy Conf
  • H B Steinbach
Steinbach, H. B., Nerve Impulse, Tr. 2nd Josiah Macy Conf., 1951, 15.
  • D Needham
Needham, D., and PiUai, J., Biochem. J., 1937, 81, 1937.
  • J M Wiame
Wiame, J. M., d. Bid. Chem., 1949, 178, 919.
  • G Schmidt
  • L Hecht
  • S J Thanhanser
Schmidt, G., Hecht, L., and Thanhanser, S. J., J. Biol. Chem., 1949, 178, 733.
  • F E Samson
  • A M Katz
Samson, F. E., Katz, A. M., and Harris, D. L., Arch. Biochem. and Biophysics, 1955, M, 406.
  • O Warburg
Warburg, O., and Christian, W., Biochem., Z., 1939, 868, 40.
  • A Rothstein
  • A D Hayes
Rothstein, A., and Hayes, A. D., Arch. Biochem. and Biophysics, 1956, 68, 87.
  • A Rothstein
  • M A Bruce
Rothstein, A., and Bruce, M. A., J. Cell. and Comp. Physiol., 1958, in press.
  • J Goodman
  • A Rothstein
Goodman, J., and Rothstein, A., Y. Gen. Physiol., 1957, 40, 915.
  • A Rothstein
  • A D Hayes
  • D H Jennings
  • D C Hooper
Rothstein, A., Hayes, A. D., Jennings, D. H., and Hooper, D. C., Y. Gen. Physiol., 1958, 41, 585.
  • M Sussman
  • S Spiegelman
Sussman, M., and Spiegelman, S., Arch. Biochem. and Biophysics, 1950, 29, 85.
  • J V Maizel
  • A A Benson
  • N E Tolbert
Maizel, J. V., Benson, A. A., and Tolbert, N. E., Plant Physiol., 1956, 81, 407.
  • E Juni
  • M D Kamen
  • J M Reiner
  • S Spiegelman
Juni, E., Kamen, M. D., Reiner, J. M., and Spiegelman, S., Arch. Biochme. and Biophysics, 1948, 18, 387.
  • F E Samson
  • A M Katz
  • D L Harris
Samson, F. E., Katz, A. M., and Harris, D. L., Arch. Biochem. and Biophysics, 1955, M, 406.