Transport of folinate and related compounds in Pediococcus cerevisiae

The properties of folinate and 5-methyltetrahydrofolate (5-CH(3)-H(4)PteGlu) transport mechanism of Pediococcus cerevisiae were studied. The uptake was dependent on temperature, pH (optimum for both compounds at pH 6.0), and glucose. Iodoacetate, potassium fluoride, and sodium azide inhibited the uptake. 5-CH(3)-H(4)-PteGlu was apparently not metabolized but folinate was metabolized. Metabolism of folinate was reduced by preincubation of cells with fluorodeoxyuridine. The transport system for folinate and 5-CH(3)-H(4)PteGlu were specific for the l-isomers. Pteroylglutamate, aminopterin, and amethopterin did not interfere with the uptake. Tetrahydrofolate competed with the uptake of folinate. The transport of folinate and 5-CH(3)-H(4)PteGlu at 37 C conformed to Michaelis-Menten kinetics; apparent K(m) for both compounds was 4.0 x 10(-7)m, and the V(max) for folinate was 1.0 x 10(-10) moles per min per mg (dry weight) and for 5-CH(3)-H(4)PteGlu it was 1.6 x 10(-10) moles per min per mg (dry weight). Both compounds accumulated in the intracellular pool at a concentration about 80- to 140-fold higher than that in the external medium. Folinate inhibited competitively the uptake of 5-CH(3)-H(4)PteGlu with a K(i) of 0.4 x 10(-7)m. Unlike 5-CH(3)-H(4)PteGlu, which accumulated only at 37 C, folinate was also taken up at 0 C by a glucose- and temperature-independent process, which was not affected by the metabolic inhibitors mentioned above. Since at 0 C the intracellular concentration of folinate was also considerably higher than the external, binding of the substrate to some cellular component is assumed. The finding of an efficient transport system for l-5-CH(3)-H(4)PteGlu is of special interest, since this compound has no growth-promoting activity for P. cerevisiae.     

Carrier-Mediated Transport Phenomena in Enzyme-Loaded Liposomes

Abstract

A mathematical model which describes the kinetic behaviour of enzyme-loaded liposomes containing a substrate transporter in the lipid bilayer is presented. The model accounts for the facilitated diffusion across the membrane and the chemical reaction in the aqueous core. Both steady-state and transient kinetics are analysed. The model allows to quantify the influence of transport phenomena on the catalytic properties of the microencapsulated enzyme and provides some directions for the design of an artificial vesicle in which a selective substrate carrier has been included.     

Plasmid DNA in Strains of Pediococcus cerevisiae and Pediococcus pentosaceus

Five parental strains of Pediococcus were examined for plasmid content. Each strain contained three to six resident plasmids, ranging in size from 4.5 to 39.5 megadaltons. A bacteriocin-like substance produced by Pediococcus cerevisiae FBB63 was tentatively linked to a 10.5-megadalton plasmid after being cured with novobiocin.     

Mathematical Modeling of a Carrier-Mediated Transport Process in a Liquid Membrane

An analysis of the reaction diffusion in a carrier-mediated transport process through a membrane is presented. A simple approximate analytical expression of concentration profiles is derived in terms of all dimensionless parameters. Furthermore, in this work we employ the homotopy perturbation method to solve the nonlinear reaction–diffusion equations. Moreover, the analytical results have been compared to the numerical simulation using the Matlab program. The simulated results are comparable with the appropriate theories. The results obtained in this work are valid for the entire solution domain.     

Carrier-Mediated Transport of Chloride Across the Blood-Brain Barrier

36Cl concentrations in each of eight brain regions and in cisternal cerebrospinal fluid (CSF) were determined 30 min after the intravenous injection of 36Cl in dialyzed-nephrectomized rats with plasma Cl concentrations between 14 and 120 mumol X ml-1. CSF 36Cl exceeded 36Cl concentrations in brain extracellular fluid. The calculated blood-to-brain transfer constants for Cl, kCl, ranged from 1.8 X 10(-5) S-1 at the parietal cortex to 3.8 X 10(-5) S-1 at the thalamus-hypothalamus. kCl fell by 42-62% when mean plasma [Cl] was elevated from 16 to 114 mumol X ml-1. Brain uptake of [14C]mannitol or of 22Na was independent of plasma [Cl], but 22Na influx into CSF fell when plasma [Cl] was reduced. Cl flux into brain and CSF could be represented by Michaelis-Menten saturation kinetics, where, for the parietal cortex, Km = 43 mumol X ml-1 and Vmax = 2.5 X 10(-3) mumol X S-1 X g-1, and for CSF Km = 68 mumol X ml-1. At least 80% of 36Cl influx into the parietal cortex was calculated to occur at the cerebrovascular endothelium, whereas the remainder was derived from tracer that first entered CSF. The CSF contribution was greater at brain regions adjacent to cerebral ventricles. The results show that Cl transport at the cerebrovascular endothelium as well as at the choroid plexus epithelium is a saturable concentration-dependent process, and that the CSF is a significant intermediate pathway for Cl passage from blood to brain.     

Aspartate aminotransferase of Pediococcus cerevisiae.

A five-step procedure is described for preparing highly purified aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC.2.6.1.1) from cell-freee enzyme extracts of Pediococcus cerevisiae. An overall purification of 130-fold was achieved. Some of P. cerevisiae aspartate aminotransferase properties were studied, i.s. pH optimum (7.8--8.0), optimum of temperature (37 degrees), Michaelis constans for 4 enzyme substrates and substrate specificity of enzyme. The enzyme is very thermolabile. During purification the enzyme was stabilizated by 2-oxoglutarate. The highly purified preparation was stored in the solution containing ammonium sulphate. The obtained aspartate aminotransferase preparation was free of alanine and aromatic amino acids aminotransferase activites and did not reveal malate dehydrogenase activity.     

Laser-Activated Electron Transport in a Chlamydomonas Mutant

Following laser activation of electron transport in the pale green mutant of Chlamydomonas reinhardii, the following kinetics are observed: 1) A rapid absorption decrease at 421 mmu (half-time < 2 x 10(-6) sec) recovering with a half-time of approximately 7 x 10(-3) sec. 2) Oxidation of cytochrome f at 554 mmu with a half-time of 1 x 10(-4) sec. 3) Oxidation of cytochrome of type b, at 432 and 564 mmu, with a half-time of approximately 6 x 10(-3) sec, following a 2 x 10(-3) sec lag.THE RESULTS ARE INTERPRETED ACCORDING TO A LINEAR ELECTRON TRANSPORT SEQUENCE: system I trap <-- cytochrome f <-- <-- cytochrome b with an additional molecule of cytochrome b in the cyclic photophosphorylation pathway. Experiments with uncouplers provide evidence for a site of photophosphorylation between cytochrome f and cytochrome b.Additional studies involve inhibitors of electron transport, the temperature dependence and quantum efficiency of cytochrome oxidation, and the effect of oxygen and pre-illumination on the laser-induced absorption changes.     

Physiological and enzymatic properties of a thymidine-requiring Pediococcus cerevisiae mutant.

We describe the isolation and characterization of a Pediococcus cerevisiae thymidine-requiring mutant and its thymidine-independent revertant. The mutant strain lacked thymidylate synthetase activity and had an absolute requirement for low concentrations (2 micrograms/ml) of thymidine in addition to a requirement for N-5-formyl tetrahydrofolic acid (folinate). Even at high concentrations (up to 500 micrograms/ml), thymine could not replace thymidine. In contrast to its wild-type parent, which grows only on folinate, the thymidine-requiring mutant (Thy- Fol+) was able to take up and grow on picogram quantities of unreduced folic acid. When both strains were grown on folinate, the Thy- Fol+ strain was at least 10(3)-fold more resistant to the folic acid analogs aminopterin and methotrexate than the wild-type strain. On the other hand, when grown on folic acid, the Thy- Fol+ strain was as sensitive to the folic acid analogs as the Thy+ Fol+ strain and was 10(2)-fold more sensitive than the wild-type strain grown on folinate. The thymidine-independent revertant (Thy+ Fol+) regained the wild-type level of thymidylate synthetase activity, but maintained the ability to take up and grow on unreduced folic acid like its Thy- Fol+ parent.     

Blood-Brain Barrier Carrier-Mediated Transport and Brain Metabolism of Amino Acids

The transport of neutral amino acids through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, is an important control point for the overall regulation of cerebral metabolism, including protein synthesis and neurotransmitter production. The Michaelis-Menten kinetics of BBB amino acid transport have been investigated in vivo with the brain uptake index (BUI) technique, and in vitro with the isolated human brain capillary preparation. The only amino acid that is albumin-bound is tryptophan, and the majority of albumin-bound tryptophan in the plasma is available for transport through the BBB via an enhanced dissociation mechanism that operates at the surface of the brain capillary endothelium. The availability in brain of amino acids is predicted from the BBB Km values to be sharply influenced by supra-physiological concentrations of phenyalanine in the 200–500 M range. Moreover, the measurement of cerebral protein synthesis with an internal carotid artery perfusion technique and HPLC-based measurements of aminoacyl-transfer RNA specific activities shows an inverse relationship between cerebral protein synthesis and plasma phenyalanine concentrations in the 200–500 M range. These findings indicate the neurotoxicity of hyperphenylalninemia is not restricted to the phenylketonuria range of approximately 2000 M, but is exerted in the supra-physiological range of 200–500 M.     

Lipids of Pediococcus cerevisiae and some methicillin-resistant substrains

The phospholipids of Pediococcus cerevisiae were identified as phosphatidyl glycerol, lysylphosphatidyl glycerol, cardiolipin, phosphatidic acid and an unknown. Evidence was obtained for the presence of mono- and diglucosyl diglycerides. The major fatty acids were C18:1, C16:0, and C16:1, with smaller amounts of C14:0, C14:1, and C18:0. The methicillin-resistant strains did not contain more lipid or lipid phosphate than the parent strain when they were grown in the presence of methicillin. The percentages of fatty acids in the organisms were not markedly different. Some variation in the proportions of the phospholipids was noted.     

Properties of a Trifluoroleucine-Resistant Mutant of Saccharomyces cerevisiae

We characterized a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae, TFL20, that has a mutation in the LEU4 gene. We monitored the concentration of extracellular i-AmOH and intracellular amino acids, and compared the ratios of gene expression in TFL20 with the wild-type strain, K30. We found that the LEU1, LEU2, and BAT1 genes were up-regulated in TFL20 for metabolism, and that TFL20 simultaneously produced as much i-AmOH and leucine as K30 does.     

Properties of a sucrose-tolerant Mutant of Saccharomyces cerevisiae

We characterized a sucrose-tolerant mutant of Saccharomyces cerevisiae, S22, that produces about four times as much acetate as the wild-type strain K9. We monitored the concentration of extracellular acetate during cultivation, and compared the gene expression ratios of S22 with those of K9 using DNA microarray. We propose that the sucrose tolerance of S22 may be related to the overexpression of the ENA1, ENA2, and ENA5 genes and some cell wall mannoprotein genes, and that the high acetate productivity of S22 is related to the overexpression of the ALD4 gene and oxidative phosphorylation genes.     

A Unified Kinetic Hypothesis of Carrier-Mediated Transport: Its Applications

A model analysis of the process of carrier mediated membrane transport is presented, wherein the carrier is present in two forms of differing affinity for substrate. The two forms of carrier undergo interconversion by asymmetric metabolic reactions on each side of the membrane. From this model system expressions are derived for the steady-state distribution ratio for substrate, for the unidirectional fluxes of substrate and hence for the initial velocity of uptake of substrate, and for the effect of preloading cells upon the initial velocity of uptake of labeled substrate. These expressions are applied to published data for glycine transport in Ehrlich ascites tumor cells to obtain numerical values for the parameters of a concentrative membrane carrier system. Concentrative uptake is shown to be consequent to the differing affinities of the two forms of carrier. When the affinities of the two forms are equal, equilibrative uptake occurs. The model analysis is applied to the phenomena of metabolic and competitive inhibition.     

Transport Alterations in a Phosphatidylethanolamine-Deficient Mutant of Bacillus subtilis

A mutant of Bacillus subtilis ATCC 6051, demonstrated to be deficient in phosphatidylethanolamine, was found to differ from the parent strain in the rate of uptake of a variety of metabolites.     

Effect of folinate on thymidine uptake by Pediococcus cerevisiae

Uptake of (3)H-thymidine by resting cells of Pediococcus cerevisiae was found to be energy- and temperature-dependent. The pH optimum was between 6.5 and 8.0, and after 2 min of incubation most of the radioactivity was found in the deoxyribonucleic acid (DNA) fraction. Iodoacetate at a concentration of 10(-2)m caused a 50% inhibition of uptake. Preincubation of resting cells for 10 min with folinate (10(-3)mu mole/ml) diminished the (3)H-thymidine uptake by 75%. In growing cells, the folinate-induced inhibition was still more striking. Deoxyuridine augmented the folinate effect, whereas fluorodeoxyuridine and aminopterin or amethopterin abolished it. Preincubation with folinate did not interfere with the uptake of (3)H-amethopterin, and thus the inhibitor did not compete for uptake sites within the cell. The role of these inhibitors in reversing the folinate effect is discussed. Cells preincubated with folinate showed an increased incorporation of (14)C-uracil into DNA, presumably after prior conversion to thymidylate. We concluded that the folinate effect was due to stimulation of de novo thymidylate synthesis with concomitant inhibition of the uptake of external thymidine.     

Pediococcus cerevisiae mutant with altered transport of folates.

A Pediococcus cerevisiae mutant that actively accumulated folate (PteGlu), in contrast to the wild-type, was also found to exhibit changes in the pattern of uptake of 5-methyl-tetrahydrofolate (5-CH3-H4PteGlu) and amethopterin. Most of the 5-CH3-H4PteGlue accumulated through a glucose- and temperature-dependent process, and a concentrative uptake was also found in gluocse-starved cells and in cells incubated at OC. About 75% of the accumulated 5-CH3-H4PteGlu exchanged with amethopterin. In contrast to the wild type, the mutant accumulated both diastereoisomers of 5-CH3-H4PteGlue by glucose-dependent and glucose-independent processes. Amethopterin and PteGlue competitively inhibited the uptake in both processes, with an apparent lower affinity of the carrier for PteGlu than for the analogue. p-Chloromercuribenzoate strongly inhibited the uptake (75%). The p-chloromercuribenzoate-nonsusceptible and temperature-independent uptake was also competed by amethopterin. Metabolic poisons like sodium azide, potassium fluoride, iodoacetate, and 2,4-dimitrophenol inhibited the glucose-dependent process. Uptake, in the absence of glucose, was enhanced by sodium azide and potassium fluoride.     

Carrier-Mediated Transport of Thyroid Hormones into Rat Glial Cells in Primary Culture

The uptake of 3,3',5-[3'-125I]triiodo-L-thyronine ([125I]L-T3) and of L-[3',5'-125I]thyroxine ([125I]L-T4) by cultured rat glial cells was studied under initial velocity (Vi) conditions. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The following respective values of Km (microM) and Vmax (fmol/min/microgram of DNA) were obtained at 25 degrees C: 0.52 +/- 0.09 and 727 +/- 55 for L-T3 and 1.02 +/- 0.21 and 690 +/- 85 for L-T4. Ki values (microM) for the inhibition of [125I]L-T3 uptake by unlabeled analogues were as follows: L-T4, 0.88; 3,3',5'-triiodo-L-thyronine, 1.4; 3,3'-diiodo-L-thyronine, 2.9; 3,3',5-triiodo-D-thyronine, 4.8; and triiodothyroacetic acid, 5.3. These values indicate that the uptake system is stereospecific. Unlabeled L-T3 was a better competitor than unlabeled L-T4 for the uptake of [125I]L-T4, an observation suggesting that both hormones were taken up by a common carrier system. L-T3, and L-T4 uptake was pH dependent, a finding suggesting that the phenolic unionized form of the hormones was preferentially taken up. L-T3 uptake was studied in the presence of various inhibitors; the results suggest that uptake was independent of the transmembrane Na+ gradient and of the cellular energy. Compounds that inhibited cellular uptake but were without effect on L-T3 binding to isolated nuclei also inhibited L-T3 nuclear binding in intact cells, an observation suggesting that uptake could be rate limiting for the access of L-T3 to nuclear receptors when transport is severely inhibited.     

Evidence for Carrier-Mediated Transport of Monosaccharides in the Ehrlich Ascites Tumor Cell

Evidence for carrier-mediated transport of monosaccharides in the Ehrlich ascites tumor cells was provided through kinetic analysis of data obtained by: (a) studying sugar uptake by dilute cell suspensions with an optical densimetric apparatus, (b) studying sugar uptake by thicker cell suspensions by means of direct chemical analytical methods using packed cell plugs, (c) observing the effects of a competitive inhibitor upon sugar uptake with the chemical analytical method, and (d) measurement of tracer uptake of a high affinity sugar in thick cell suspensions in the absence of net movement. Quantitative application of the data obtained with the above experimental procedures to theoretical model systems derived for both carrier-mediated transport and simple passive diffusion indicated that the results were consonant with predictions for the carrier-mediated transport model, but could not be explained on the basis of uncomplicated diffusion.     

Osmotic Remedial Response in a Galactose-negative Mutant of Saccharomyces cerevisiae

We investigated an osmotic remedial mutant of Saccharomyces which was deficient in galactose-1-phosphate uridyl transferase. Both the rate of growth and the transferase activity of the mutant culture were dependent on the osmotic activity of the growth medium. Organic and ionic solutes proved to be equally effective in inducing the osmotic remedial response. The galactose pathway enzymes were separable by ultracentrifugation, indicating that a stable interenzyme complex was not formed. The results were consistent with the hypothesis that the corrective effect occurs at the tertiary or quaternary level of organization in an environmentally sensitive protein. The possibility that the osmotic remedial response represents an effect of translation is discussed.     

Coupling of Energy to Folate Transport in Lactobacillus casei

Lactobacillus casei cells can accumulate folate to an intracellular concentration in excess of 500 muM and to concentration gradients (relative to the extracellular compartment) of several thousand-fold. Maximum rates of folate transport are achieved rapidly (t(1/2) < 1 min) after the addition of glucose to energy-depleted cells and occur at intracellular adenosine 5'-triphosphate concentrations above 625 muM. The rate of folate transport and the adenosine 5'-triphosphate content of cells are both extremely sensitive to arsenate and decrease in parallel with increasing concentrations of the inhibitor, indicating a requirement for phosphate-bond energy in the transport process. The energy source is not a membrane potential or a pH gradient generated via the membrane-bound adenosine triphosphatase, since dicyclohexylcarbodiimide (an adenosine triphosphatase inhibitor) and carbonyl cyanide m-chlorophenylhydrazone (a proton conductor) have little effect on the uptake process. The K(+)-ionophore, valinomycin, is an inhibitor of folate transport, but does not act via a mechanism involving dissipation of the membrane potential. This can be deduced from the facts that the inhibition by valinomycin is relatively insensitive to pH, is considerably greater in Na(+)- than in K(+)-containing buffers, and is not enhanced by the addition of proton conductors. Folate efflux is not affected by valinomycin, glucose, or various metabolic inhibitors, although a rapid release of the accumulated vitamin can be achieved by the addition of unlabeled folate together with an energy source (glucose). These results suggest that the active transport of folate into L. casei is energized by adenosine 5'-triphosphate or an equivalent energy-rich compound, and that coupling occurs not via the membrane-bound adenosine triphosphatase but by direct interaction of the energy source with a component of the transport system.