Uptake of l-Phenylalanine in Synchronous Chlorella fusca. Characterization of the Uptake System

Phenylalanine uptake in Chlorella fusca was measured, using the membrane filter technique. The cells were synchronized, and harvested at specific points of the life cycle. Experiments with autospores showed that the uptake followed saturation kinetics, with a K_m= 5 μM. V_max, was 0.1 nmol/min × 10⁷ cells. The optimum temperature for the uptake was 40°C, and the activation energy was 1700 J/mol. The uptake showed a high specificity towards l -phenylalanine; presence of the unlabelled stereoisomer did not inhibit the uptake. Uptake of l -phenylalanine was inhibited in the presence of other analogues or other amino acids, but only if they were present in concentrations considerably higher than that of L-phenylalanine. Variations in the ratio of Na4⁺ to K⁺ in the external solution during uptake experiments did not have any influence upon the uptake rate of l -phenylalanine. The cells were able to take up the amino acid against a concentration gradient. At pool maximum the ratio between internal and external amino acid concentration was 1000/1. 2,4-Dinitro-phenol inhibited the uptake completely. Exchange between internal and external l -phenylalanine could not be demonstrated. The K_m value did not change during the life cycle of the cells. The uptake rate reached a maximum at the end of the light period, and fell to a minimum just before sporulation started. It is concluded that Chlorella fusca cells have a highly specific, active uptake system for l -phenylalanine. The system is constitutive, independent on the K or Na concentration, and the mechanism of uptake does not change during the life cycle of the cells.     

The Influence of Phosphate Uptake on Cation Uptake in Fusarium oxysporum f. sp. vasinfectum

Fusarium oxysporum grown in a low phosphate medium was found to take up several times as much K from KH₂PO₄ as from KCI solutions. Large amounts of phosphate also were taken up from KH₂PO₄. Similar large uptakes of Na and phosphate took place from solutions of NaH₂PO₄. Substantial quanties of phosphate were taken up from solutions of Ca(H₂PO₄)₂ in the absence of any appreciable Ca uptake. When the fungus was grown in a medium containing high phosphate, little or no uptake of phosphate from KH₂PO₄ solutions occured and the K Uptake was at the same level as from KCI solutions. During large phosphate uptake sizable reductions in the organic acid content of the fungal cells were observed. Much, but not all, of the data could be explained on the basis of maintenance of charge balance within the cells. – The respiratory rate of fungus, grown in a low P medium, was markedly increased in KH₂PO₄ solution. Fungus, grown in a medium with high phosphate, had a higher respiratory rate which showed only a slight response to KH₂PO₄ solution. Fungus, grown in a medium with high phosphate, had a higher respiratory rate which showed only a slight response to KH₂PO₄.     

Uptake of dolichol by Vero cells.

Characterization and kinetics of dolichol uptake by a Vero cell line are reported. Vero cells incorporate dolichol in a time- and dose-dependent manner. Optimal uptake is found at 37 degrees C and at a pH of 7.4. In contrast to cholesterol, an inhibitory effect on the dolichol incorporation is found for farnesol, geraniol, and retinol. Long chain polyprenols were slightly stimulatory. The translocation seems not to be highly energy dependent. The lack of substantial inhibition by chloroquine does not plead for a receptor-mediated endocytosis. Incorporated dolichol was distributed over both membranes and supernatant fractions, paralleling the distribution of the lysosomal marker beta-N-acetylhexosaminidase. The incorporated dolichol is subject to a fast efflux process, which is potentiated by the presence of lipid acceptors in the extracellular medium.     

Uptake of liposome-entrapped mannitol by diaphragm.

Tissue uptake of liposome-entrapped radioactive mannitol was examined in rats and mice after both intravenous and intraperitoneal injection. In accord with results from other laboratories, liver and spleen effectively accumulated liposomes. Diaphragm also took up significant amounts of label. In nearly all cases the radioactive content of perfused tissues was less than tissues which were not perfused but this was statistically significant in only a few comparisons.     

Uptake of proteinase-alpha-macroglobulin complexes by macrophages.

Complexes of labelled proteinases (subtilopeptidase A, trypsin) with serum alpha 1-macroglobulin or alpha 2-macroglobulin are rapidly taken up in vitro by rabbit alveolar macrophages and peritoneal macrophages but not by mixed rabbit peripheral blood leukocytes. Enzyme, not bound to alpha 1- or alpha 2-macroglobulin, does not become associated with alveolar macrophages. Chemically inactivated subtilopeptidase A does not bind to alpha 1- or alpha 2-macroglobulin; chemically inactivated subtilopeptidase A in mixtures with alpha 1 - or alpha 2-microglobulin, does not interact with alveolar macrophages. Blocking experiments confirmed that the interaction of proteinase with alveolar macrophages is complex specific; uptake of labelled complex was prevented by the simultaneous addition of macroglobulin complexes formed with non-labelled subtilopeptidase A, subtilopeptidase B, trypsin or chymotrypsin but not by macroglobulin alone. The findings demonstrate a complex-specific interaction between proteinase-alpha-macroglobulin complexes and macrophages.     

Uptake of phenol by Trichosporon cutaneum.

The soil yeast Trichosporon cutaneum, which is distinguished by having a strictly oxidative metabolism, can be induced to utilize phenol as a sole carbon source. The present paper shows that such phenol-induced cells contain a specific, energy-dependent uptake system for phenol. Phenol uptake is not directly linked to its o-hydroxylation inside the cell, the first step of phenol metabolism. The Km for uptake is 235 +/- 30 microM, that for hydroxylation only 4.5 +/- 0.5 microM. Further, the phenol analog 2,6-dimethylphenol, which can not be hydroxylated, competes with phenol for the uptake system. The pH dependence of uptake indicates that phenolate is an essential form during the uptake process. The energy requirement for phenol uptake is indicated by effects of various inhibitors of energy generation, including proton-conducting uncouplers. Direct monitoring of proton movements in a pH-stat during phenol uptake indicates a phenol-proton symport. One proton is cotransported with every phenol molecule. Phenol competes with the uptake of sucrose and glycerol by cells grown on these substrates. Under such conditions the uptake of phenol seems to proceed through a different system, with lower affinity for phenol than in phenol-grown cells.     

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.     

Uptake of L-proline by Histoplasma capsulatum.

The uptake and incorporation of L-proline by yeast cells of the dimorphic zoopathogen Histoplasma capsulatum were studied. The amino acid was assimilated in at least two ways: by an active transport system with a Km of 1.7 X 10(-5) M and by simple diffusion. The active transport system was sterospecific and severely restricted to neutral aliphatic side-chain amino acids. Certain analogues inhibited L-proline uptake and prevented incorporation of the amino acid into cellular constituents. The inhibition of L-proline uptake by L-leucine was competitive. Since L-leucine and L-proline are seemingly transported by a system with similar characteristics, must be concluded, as originally postulated, that the buckled ring of L-proline, in solution, acts as an aliphatic side chain and that this cyclic amino acid is transported by a system more or less specific for amino acids with neutral aliphatic side chains.     

Dual Capacity for Nutrient Uptake in Tetrahymena. II. Role of the Two Systems in Vitamin Uptake

SYNOPSIS Previously, we found that a mutant strain of Tetrahymena pyriformis without food vacuoles failed to grow unless the nutrient media were richly supplemented with vitamins and trace metals. Here we show that calcium folinate alone can replace the extra vitamin supplementation. The mutant requires ∼ 90-fold higher concentrations of folinate than the wild-type cells to give similar growth responses in a chemically defined medium. We infer that the food vacuole is an important route of uptake for this vitamin in the wild-type cells. We found no difference between mutant and wild-type cells in their requirements for nicotinic acid, pantothenic acid, riboflavin-monophosphate, and pyridoxal. We infer that an extravacuolar route contributes importantly to uptake of these 4 compounds.     

Uptake of heterologous DNA by Haemophilus influenzae.

With the use of highly competent Haemophilus influenzae cells, it was possible to demonstrate the uptake of heterologous DNAs. However, these DNAs, as expected, were only 1% or less as effective when competing for uptake with Haemophilus DNA. Escherichia coli DNA was removed from solution by competent cells to the extent expected if all the E. coli DNA particles contained at least one uptake recognition signal. The data were consistent with a model in which there was one uptake signal per 20 X 10(6) to 30 X 10(6) daltons of E. coli DNA. Since H. influenzae DNA has many more recognition signals, approximately one per 2 X 10(6) daltons (Danner et al., Gene 77:311-318, 1980; K. Vogt and S. H. Goodgal, submitted for publication), it has been suggested that the slower rate of E. coli DNA binding and the so-called specificity of Haemophilus DNA binding are due to the number of recognition signals per molecule of DNA as well as the nature of the DNA receptor (Vogt and Goodgal, submitted for publication). The specificity of native H. influenzae DNA binding does not apply to the uptake of denatured DNA in the transforming system (low pH) for denatured DNA.     

Uptake of glycine by non-mycorrhizal Lolium perenne.

Plants of Lolium perenne were grown in sterile solution culture. 15N-labelled glycine (Gly) coupled with gas chromatograph mass spectrometry was used to prove that non-mycorrhizal plants of L. perenne are capable of acquiring N in the form of intact Gly. It was estimated that a minimum of 80% of Gly-N uptake, over a 3 h period, was as intact Gly, though possible processes resulting in deviation from this estimate are discussed. The relative incorporation of 15N derived from Gly uptake into serine (Ser) compared with other amino acids in the root amino acid pool suggested the enzyme serine:glyoxylate aminotransferase was at least partly responsible for the synthesis of Ser from Gly. Defoliation was shown to reduce Gly uptake by L. perenne. The addition of either 25 mol x m(-3) sucrose or 50 mol x m(-3) glucose to the uptake solution of defoliated plants increased Gly-N uptake compared with both defoliated plants without sugars and with undefoliated plants. Addition of a glucose analogue, 3-O-methyl-D-glucopyranose, that is absorbed but not metabolized by plants, did not affect Gly uptake by defoliated plants. Increasing pH from 3.5 to 9.2 caused a reduction in Gly uptake. Results of the effects of defoliation and pH are consistent with Gly uptake by L. perenne being by an energy-dependent proton symport. When either or Gly were supplied to plants at equimolar concentrations, uptake was five times greater than that of Gly at pH 6 and 13 times greater at pH 9.     

Uptake of fatty acids by Mycoplasma capricolum.

The energy requirements for fatty acid uptake by Mycoplasma capricolum were studied. Fatty acid transport and esterification to phospholipid appeared to be tightly coupled, since there was little intracellular accumulation of free fatty acid. Uptake was blocked by iodoacetate, n-ethylmaleimide, and p-chloromercuribenzoate. Glucose, glycerol, and potassium ions were necessary for fatty acid uptake by whole cells. A reduction in uptake was observed in cells treated with valinomycin or dicyclohexylcarbodiimide. The effect of temperature on the rate of oleate uptake showed a discontinuity at 24 degrees C. Above 24 degrees C an energy of activation of 4.6 kcal (ca. 19.2 kJ)/mol was obtained. The data suggest that uptake of fatty acid by M. capricolum is an energy-linked, protein-mediated process. A membrane-bound enzyme activity that catalyzed the synthesis of fatty acyl-hydroxamate was demonstrated. This activity was virtually independent or only marginally dependent on coenzyme A, depending on the assay system, but was stimulated approximately twofold by ATP.     

Uptake of glutathione by renal cortical mitochondria.

Transport of GSH into renal cortical mitochondria was studied. Mitochondria were highly enriched with little contamination from other subcellular organelles (as assessed by marker enzymes), they exhibited coupled respiration (respiratory control ratio greater than 3.0), and they had initial GSH concentrations of 5.71 +/- 0.65 nmol/mg protein (n = 47). Incubation of mitochondria with GSH in a triethanolamine, pH 7.4, buffer containing sucrose, potassium phosphate, MgCl2, and KCl, produced time- and concentration-dependent increases in intramitochondrial GSH content. Uptake was linear versus time for at least 2 min and exhibited kinetics consistent with one low-affinity, high-capacity process (Km = 1.3 mM, Vmax = 5.59 nmol/min per mg protein), although the results cannot exclude the presence of other, less quantitatively significant pathways. The initial rate of uptake of 5 mM GSH was not significantly altered by uncouplers (0.1 mM 2,4-dinitrophenol and 25 microM carbonyl cyanide m-chlorophenylhydrazone) or by 1 mM ADP. In contrast, incubation with 1 mM ATP, 1 mM KCN, 0.1 mM or 1 mM CaCl2 inhibited uptake by 41, 39, 43, or 55%, respectively. GSH uptake was markedly inhibited by gamma-glutamylglutamate and by a series of S-alkyl GSH derivatives. Strong interactions (i.e., both cis and trans effects) were observed with other dicarboxylates (i.e., succinate, malate, glutamate) but not with monocarboxylates (i.e., lactate, pyruvate). Preincubation of mitochondria with GSH protected against tert-butyl hydroperoxide- or methyl vinyl ketone-induced inhibition of state 3 respiration. These results demonstrate uptake of GSH into renal cortical mitochondria that appears to involve electroneutral countertransport (exchange) with other dicarboxylates. Functionally, GSH uptake into mitochondria can protect these organelles from various forms of injury, such as oxidative stress.     

Uptake of transferrin-bound zinc by human lymphocytes.

Uptake of transferrin-bound zinc was stimulated in phytohemagglutinin-treated human lymphocytes as compared to untreated lymphocytes. Stimulation of zinc uptake depended upon the concentration of phytohemagglutinin and was maximal for the first hour after addition of phytohemagglutinin to lymphocyte cultures. Thereafter, increased zinc uptake declined until approximately basal levels were reached 5 hr after addition of phytohemagglutinin. Stimulation of zinc uptake was insensitive to sulfhydryl reagents, but was decreased by KCN, actinomycin D, aurin tricarboxylic acid, and by lowering the incubation temperature. Two compounds, NaF and poly-l-ornithine, were found to markedly increase zinc uptake over that seen with only phytohemagglutinin. Additionally, compounds known to increase cellular levels of cyclic AMP, such as epinephrine, histamine, serotonin, glucagon, and prostaglandin E₁, as well as 8-bromo-cyclic AMP and dibutyryl-cyclic AMP, also increased uptake of transferrin-bound zinc by phytohemagglutinin-stimulated lymphocytes.     

Uptake of tyramine cellobiose by rat liver.

The uptake of 125I-tyramine cellobiose (TC) by isolated rat hepatocytes and by total rat liver is markedly higher than that of 14C-sucrose and 125I-PVP, suggesting that TC does not enter the cells by fluid phase endocytosis. The distribution of radioactivity after differential centrifugation shows that the compound is shared out amongst sedimentable structures and unsedimentable fraction. Analysis by isopycnic centrifugation indicates that quickly after its penetration into the cells, most of sedimentable 125I-TC is associated with lysosomes. Such an intracellular localization is confirmed by the distributions observed after free flow electrophoresis and by the fact that radioactivity and cathepsin C, a lysosomal hydrolase, are simultaneously released from a mitochondrial fraction treated with glycyl-L-phenylalanine-2-naphthylamide. Pretreatment of the rats with chloroquine, an acidotropic drug that accumulates in lysosomes, prevents to some extent the entry of 125I-TC into these organelles. Experiments performed with purified lysosomes show that 14C-sucrose does not cross the lysosomal membrane when 125I-TC accumulates linearly with time in the fractions. These results are explained by supposing that the linkage of tyramine to cellobiose allow the disaccharide to diffuse through the plasma and the lysosome membranes, and that the accumulation of the molecule in these organelles results from its weak basic properties. 125I-TC could be an interesting molecule with which to study acidotropism in the whole animal and in isolated and cultured cells.