Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes.

Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.     

Amino acid transport in pig lymphocytes Enhanced activity of transport system asc following mitogenic stimulation

Changes in neutral amino acid transport activity caused by addition of phytohaemagglutinin-P to quiescent peripheral pig lymphocytes have been evaluated by measurements of ¹⁴C-labelled neutral and analogue amino acids under conditions approaching initial entry rates. Utilizing methylaminoisobutyric acid, the best model substrate of System A, we confirmed our previous report (Borghetti, A.F., Kay, J.E. and Wheeler, K.P. (1979) Biochem. J. 182, 27–32) on the absence of this transport system in quiescent cells and its emergence following stimulation. Furthermore, we demonstrated the presence in quiescent cells of an Na⁺-dependent transport system for neutral amino acids that has been characterized as System ASC by several criteria including intolerance to methylaminoisobutyric acid, strict Na⁺-dependence, the property of transtimulation and specificity for pertinent substrates such as alanine, serine, cysteine and threonine. Analysis of the relationship between influx and substrate concentration revealed that two independent saturable components contribute to entry of alanine in quiescent cells: a low affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= ≈4}\text{mM}$) and a high affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= ≈0.2}\text{mM}$) component. The high affinity component could be inhibited in a competitive way by serine, cysteine and threonine, but methylaminoisobutyric acid did not change appreciably its constants. The enhanced activity of alanine transport through the ASC system observed in activated cells resulted from a large increase in the capacity ($\text{V}$) of the high affinity component without any substantial change in the apparent affinity constant ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$).     

Enhanced stability of the Na+-dependent amino acid-transport system after lymphocyte activation.

Two cytochromes b with absorption maxima at 555 and 562 nm and differing in their mid-point redox potentials are synthesized in Pseudomonas AM1 during growth on methanol or succinate in batch culture, or in NH4+-limited or carbon-limited continuous culture. Both cytochromes b were also present in a cytochrome c-deficient mutant in all growth conditions.     

Reversed transport of amino acids in Ehrlich cells.

Gramicidin induces a marked Na+-dependent efflux of amino acids from Ehrlich cells. In absence of Na+, gramicidin does not alter the efflux. In presence gramicidin, glycine efflux is inhibited by methionine and less so by leucine. Glycine efflux caused by HgCl2 is neither Na+ dependent nor inhibitable by amino acids. Neither efflux of inositol which is transported by an Na+-dependent route, nor efflux of several other solutes which are transported by Na+-independent routes, is affected by gramicidin. The antibiotic appears to permit a reversal in the direction of of the operation of the Na+-dependent amino acid transport system. The increased efflux is partly, but not entirely, due to an increase in the cellular Na+ concentration and a reduction of the electrochemical potential difference for Na+.     

Trypanosoma gambiense: membrane transport of amino acids.

Trypanosoma gambiense absorbed ¹⁴C-labeled lysine, arginine, glutamate, phenylalanine, methionine, threonine, glycine, and alanine by mediated transport systems. The interactions of these compounds as inhibitors or stimulators formed complex patterns of uptake which suggested the presence of five binding and/or transport loci: Locus A bound glutamate, arginine, and lysine, and the binding of glutamate or arginine stimulated the transport of lysine. Locus B transported threonine, glycine, and alanine and appeared to be partially sensitive to ouabain and Na⁺. Locus C transported glutamate, locus D transported phenylalanine and methionine, and locus E transported lysine and arginine.     

Kinetic analysis of blood-brain barrier transport of amino acids.

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of 14C-labelled amino acids and 3H2O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringer's solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15s following injection. A brain uptake index (Ib) was determined from the ratio of 14C dpm in the brain tissue and the injection mixture divided by the same ratio for the 3H2O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded Km (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship P = V/Km where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the Ib for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the 3H2O reference 15s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min-1.     

Inhibition of phorbol ester-accelerated amino acid transport in bovine lymphocytes.

12-O-Tetradecanoylphorbol-13-acetate, a highly active tumor-promoting agent and lymphocyte comitogen, rapidly accelerates the transport of alpha-aminoisobutyric acid in cultured bovine lymphocytes. Structure-activity studies show that the ability of phorbol diesters to accelerate alpha-aminoisobutyric acid uptake runs parallel to their potency as lymphocyte comitogens and as tumor promoters in mouse skin. This phorbol ester-accelerated, amino acid transport is largely insensitive to the inhibition of RNA and protein synthesis by actinomycin D and cycloheximide, respectively, and is insensitive to the inhibition of membrane movement by cytochalasin B and colchicine. Retinoic acid, an antagonist of the tumor-promoting and comitogenic actions of phorbol esters also inhibits the acceleration of amino acid uptake by 12-O-tetradecanoylphorbol-13-acetate; however, the epoxy derivatives of retinoic acid and structurally related analogs, which are potent antagonists of the other aspects of phorbol ester activation of lymphocytes, are inactive in blocking amino acid uptake. Comparative studies in lymphocytes show that this phorbol ester elicits a number of metabolic responses which appear to originate at the cell membrane and that these are differentially antagonized by retinoic acid, the 5,6-epoxide of retinoic acid, and related retinoid analogs.     

Deoxycytidine transport and pyrimidine deoxynucleotide metabolism in phytohaemagglutinin-stimulated pig lymphocytes.

Increased entry of deoxy[3H]cytidine begins at about 12h after addition of phytohaemagglutinin to peripheral pig lymphocyte cultures, and is accompanied by a parallel stimulation of deoxycytidine kinase up to the beginning of DNA synthesis at 24h. The increased deoxycytidine uptake is characterized by an increase in Vmax. without alteration of the apparent Km (0.7 +/- 0.11 muM). Although the entries of both nucleosides are promoted at the same time, the stimulation of deoxycytidine uptake is less than that of thymidine, and the two nucleosides are transported by separate systems. In addition to deoxycytidien kinase, the synthesis of deoxycytidylate deaminase and thymidylate synthetase are stimulated after addition of phytohaemagglutinin, but to a lesser extent than that of thymidine kinase. The importance of the latter enzyme in forming dTMP, and of thymidylate kinase in providing dTTP, is discussed.     

Organic ion transport during seven decades. The amino acids

The amino acids are ions of various charge combinations, and one can argue that historically they were the first ions for which the ongoing problem of membrane transport was presented; also that among transported ions these may undergo a highly detailed molecular recognition. Furthermore, the distribution of charge on the amino acid molecule determines by what route or routes it is conducted across the biological membrane, with what directional and structural specificity, and therefore what regulation is imposed, and where. Cases where a presumably charged chemical group behaves as if it were somehow absent from the amino acid have been observed to fall into several categories: Straightforward cases where the pH has been low enough or high enough to remove the charge by protonation or deprotonation, even in free solution. Cases where that protonation or deprotonation is facilitated at the binding site, and perhaps by the total transport process. The cystine molecule can apparently thus be rendered either a tripolar anion or a tripolar cation for transport. Cases where an otherwise co-transported Na+ is omitted to redress charge, or where a Na+ serves as a surrogate for a missing charged group on the amino acid molecule. A case where the protonation occurs reversibly at the receptor site rather than on the amino acid molecule.     

Mechanism of activation of pyruvate dehydrogenase by mitogens in pig lymphocytes.

The activity of pyruvate dehydrogenase in extracts of pig mesenteric lymphocytes was measured under different preincubation conditions. The mitogens concanavalin A and ionophore A23187 both increased pyruvate dehydrogenase activity. In both cases activation required extracellular Ca2+. Digitonin-permeabilized cells required 0.5 microM free Ca2+ for half-maximal activation of pyruvate dehydrogenase. The stimulation by concanavalin A in intact cells was probably not due to changes in effectors of pyruvate dehydrogenase kinase. This evidence suggests that activation of pyruvate dehydrogenase is by Ca2+ activation of pyruvate dehydrogenase phosphatase and supports the view that the cytoplasmic free [Ca2+] rises to something less than 1 microM on stimulation with mitogens.     

Membrane transport in Schistosoma mansoni: transport of amino acids by adult males.

The mechanisms by which adult male Schistosoma mansoni transport amino acids have been investigated using radioactive amino acids during 2-min incubation times. The transport constants (K_t) for mediated uptake of glycine, proline, methionine, arginine, glutamate, and tryptophan were calculated to be 0.60-1.05, 1.67-1.98, 2.0, 0.10-0.35, 0.30-0.50, and 0.5-1.0 mM, respectively. Maximal velocities (V_max) were 5.5–7.5, 25, 6.4, 1.5-2.0, 2.5, and 3.0–6.0 μmoles absorbed/g worm protein/2 min, respectively. Cysteine is taken up solely by diffusion. Proline uptake is unique in that no significant diffusion component was found. The other amino acids studied were absorbed by diffusion as well as by specific transport systems. In the 2-min incubation periods employed glycine, proline, glutamate, and methionine were not significantly metabolized indicating that the uptake studies using these substrates reflect transport. Metabolism of the other amino acids used in these studies was not examined. The specificity of the transport systems was studied by testing the inhibitory effects of various amino acids on the uptake of each of the amino acids studied. The results suggest the presence of at least five transport systems. There is a highly specific transport locus for proline, and one for acidic amino acids. There are probably at least two transport systems, each of broad and overlapping specificity, for most of the neutral amino acids. Basic amino acids also appear to be taken up by complex transport systems, at least one of which overlaps with the neutral sites. The results are discussed with respect to the nutrition of the parasite and the host-parasite relationship.     

Classification of essential amino acids for the weanling pig

Feeding experiments with mixtures of purified amino acids show that arginine, leucine, phenylalanine, and valine are required for good growth of weanling pigs. The pig resembles the rat in its ability to synthesize part, but not all of the arginine required. It is now possible to tentatively classify the known amino acids with respect to major growth effects in weanling pigs. The amino acids which must be present in the diet for good growth are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. The remaining amino acids may be tentatively classed as dispensable.     

A multicomponent analysis of amino acid transport systems in human lymphocytes. 1. Kinetic parameters of the A and L systems and pathways of uptake of naturally occurring amino acids in blood lymphocytes

We have determined the kinetic parameters of natural and system-specific synthetic amino acid transport by human blood lymphocytes, using a multi-component computer analysis that separates carrier-mediated uptake from diffusion. These studies were initiated in order to provide the basis for studies of human blood T and B lymphocytes and malignant lymphocytes. Methylaminoisobutyric acid (methyl-AIB) and 2-amino-2-carboxy-bicyclo (2,2,1) heptane (BCH) uptakes into lymphocytes were measured as prototypes of A- and L-system amino acid transport. The Michaelis constant for methyl-AIB uptake was 540 microM; the maximal velocity of uptake was 28 mumol/L cell water/min, and the diffusion coefficient was .004 min-1. In contrast, the Michaelis constant for BCH uptake was 63 microM; the maximal velocity was 969 mumol/L cell water/min, and the diffusion coefficient was .141 min-1. The transport of the naturally occurring amino acids, alanine, proline, and leucine was defined by studies of: (1) competitive inhibition with the system-specific synthetic amino acids, methyl-AIB and BCH, (2) the effect of the transcellular sodium gradient on transport, and (3) evaluation of the time-dependent increase of transport in amino acid-deficient medium (adaptation). Alanine was transported principally (approximately 70%) by the ASC-system, and leucine was transported principally (70%) by the L-system in lymphocytes. The analysis of proline transport was more complex because of a large component of uptake by diffusion even at low amino acid concentrations. Taken together, the kinetics of sodium-sensitive uptake and the results of competitive inhibition studies indicated that proline was transported by the A-system (30%), the ASC system (30%), and also by the L-system (15%).     

Reversed transport of amino acids in Ehrlich cells

Gramicidin induces a marked Na⁺-dependent efflux of amino acids from Ehrlich cells. In absence of Na⁺, gramicidin does not alter the efflux. In presence of gramicidin, glycine efflux is inhibited by methionine and less so by leucine. Glycine efflux caused by HgCl₂ is neither Na⁺ dependent nor inhibitable by amino acids. Neither efflux of inositol which is transported by an Na⁺-dependent route, nor efflux of several other solutes which are transported by Na⁺-independent routes, is affected by gramicidin. The antibiotic appears to permit a reversal in the direction of the operation of the Na⁺-dependent amino acid transport system. The increased efflux is partly, but not entirely, due to an increase in the cellular Na⁺ concentration and a reduction of the electrochemical potential difference for Na⁺.