Close linkage between the C blood group locus and the locus controlling amino acid transport in sheep erythrocytes

Data from 838 Finnish Landrace or Finnish Landrace crossbred sheep showed a highly significant correlation between phenotypes of the C blood group system and erythrocyte amino acid transport variants. Erythrocytes with normal amino acid transport properties (GSH high, Ly- type) were Cb-positive or Cb-negative. Erythrocytes with the amino acid transport lesion (GSH low, Ly +) were never Cb-negative. Sheep erythrocytes homozygous for C^bshowed stronger lysis reactions with anti-Cb than heterozygous cells. Ly + sheep were nearly always homozygous for C^b, whereas most Ly- sheep were heterozygous or Cb-negative. Inheritance studies provided strong evidence that this association is due to close genetic linkage.     

Sheep twin chimaeras with admixtures of red cell amino acid and potassium transport phenotypes

A pair of sheep twins each had two populations of red cells. Population 1 was positive for antigens Aa, Ma and Mb, was low-potassium type, possessed an amino acid transport system and was lysine-negative phenotype. Population 2 was negative for antigens Aa, and Mb, was high-potassium type, lacked the amino acid transport system and was lysine-positive phenotype. Population 2 disappeared from both sheep over a period of 8 years.     

Genetic control of red-cell nucleoside transport and its association with the B blood-group locus and nucleoside phosphorylase activity in sheep

Nucleoside transport in sheep red cells is controlled by two allelomorphic genes, the gene for nucleoside transport deficiency (Nu ^I) being dominant to that for the functional presence of carrier-mediated nucleoside transport activity (Nu ⁱ). Sheep are also polymorphic with respect to their red-cell nucleoside phosphorylase (NP) activity, some having high activities and others low activities of this enzyme. The gene for high activity (NP ^H) is incompletely dominant to that for low activity (NP ^L). Inheritance data indicate that theNu locus is genetically linked to that for the B blood-group system and, in addition, exerts a pleiotropic effect on NP activity, Nu permeability stabilizing the heat-labileNP ^L gene product. Nu-permeable cells have a higher ATP content than Nu-impermeable red cells, and within the Nu-impermeable subgroup, NP deficiency causes a further reduction in red cell ATP concentration. It is concluded that the nucleoside inosine supplements glucose as a physiological energy substrate in sheep red cells.     

Neutral amino acid transport in Leishmania promastigotes

Neutral amino acid transport was investigated in Leishmania promastigotes. Proline and alanine transport occur against their concentration gradient although there is a very rapid (40% at 30 min) conversion of proline to alanine. Uptake of these amino acids occurs by a sodium-independent route which is completely eliminated by addition of CCCP or KCN. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for proline and alanine are 80 μM and 63 μM with $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ values of 6.4 and 7.2 nmol/min per mg dry weight, respectively. Countertransport of proline, alanine and phenylalanine was measured by loading the cells with a variety of neutral amino acids and proline analogs, followed by CCCP addition. The effect of aminooxyacetic acid, an inhibitor of alanine aminotransferase (EC 2.6.1.2), on proline and alanine countertransport was also examined. The results obtained are consistent with the presence of at least two systems for neutral amino acid transport in Leishmania promastigotes.     

Regulation of amino acid transport in growing cells of Streptomyces hydrogenans

(1) The active uptake of different amino acids by growing cells of Streptomyces hydrogenans was shown to be correlated with the physiological age of the cells. During the lag phase of growth the transport capacity increased and attained its highest level when the growth rate was maximum. During further growth the transport capacity declined progressively. The lowest transport activity was observed when the culture shifted into the stationary growth phase. (2) Such modulation of transport capacity was independent on the presence or absence of amino acids in the growth medium of the cells. (3) The size and the composition of the pool of free intracellular amino acids was also undergoing substantial variations during the growth cycle of the culture. In the lag phase, the levels of all amino acids decreased markedly and attained their lowest values at the end of this phase. During further growth the pool size was slowly replenished. (4) Removal of the pool resulted in a considerable gain of transport capacity. Therefore, it was concluded that active amino acid transport in growing Streptomyces hydrogenans is under feedback control by intracellular amino acids. (5) Quantitatively, the modulation of the pool size could not fully account for the variation of the transport capacity. Since a pool-independent stimulation of transport was found to be correlated with the increase of the growth rate of the cells, the possibility is discussed that the stimulation of transport is either due to increased levels of distinct RNA species, which might provide positive feedback signals for transport, or by increased rates of de novo synthesis of transport limiting proteins.List of Abbreviations AIB 2-aminoisobutyric acid - CM complete medium - MM mineral medium     

Genetic control of nucleoside transport in sheep erythrocytes

Nucleoside transport in sheep erythrocytes is under the genetic control of two allelomorphic genes (Nu ^I and Nu ⁱ ), where Nu ^I codes for the functional absence of a high-affinity nucleoside transport system and is dominant to the gene (Nu ⁱ ) coding for the presence of the transport system. Kinetic and inhibitor experiments show that the high-affinity transport system is not present in heterozygous erythrocytes, demonstrating that the Nu ^I gene is completely dominant over the Nu ⁱ gene. It is suggested that the Nu locus may not represent the structural gene locus of the nucleoside transport system. Instead, it may be a regulator gene locus.     

'Malic enzyme' polymorphism in sheep erythrocytes

Malic enzyme or malate dehydrogenase (NADP +). E.C.I.I.I.40. catalyses the reaction:
L-malate + NADP⇄2 pyruvate + CO₂+ NADPH
Baker & Manwell (1977), in a survey of a number of different enzymes reported that 'malic enzyme' was polymorphic in the erythrocytes and certain other tissues of sheep, and indicated that it would be a potentially useful new genetic marker for this species. This paper confirms the existence of the polymorphism in sheep erythrocytes and presents inheritance and breed data.     

Cationic and anionic amino acid transport studies in rat red blood cells

The transport of L-proline, L-lysine and L-glutamate in rat red blood cells has been studied. L-proline and L-lysine uptake were Na⁺-independent. When the concentration dependence was studied both showed a non-saturable uptake assimilable to a difussion-like process, with high Kd values (0.718 and 0.191 min^–1 for L-proline and L-lysine respectively). Rat red blood cells showed high impermeability to L-glutamate. No sodium dependence was observed and the Kd value was low (0.067 min^–1). Our results show firstly, that rat red blood cells do not have amino acid transport systems for anionic and cationic amino acids and secondly that erythrocytes show no sodium-dependent L-proline transport, and that these cells are very permeable to this amino acid.Abbreviations MeAIB methyl aminoisobutyric acid     

Purine nucleoside phosphorylase polymorphism in sheep erythrocytes

A polymorphism of purine nucleoside phosphorylase is described in sheep erythrocytes. Two isozymes were distinguished electrophoretically, one with high activity (NP-1) and one with low activity (NP-2). Breeding data suggest that the two isozymes are the product of two codominant alleles, NP ¹and NP ². The K _m 's for inosine did not differ between NP-1 and NP-2; however, NP-2 had a lower pH optimum and was relatively unstable when incubated at 48 C.Contribution No. 421-J, from the Department of Pathology, Kansas Agricultural Experiment Station, Manhattan, Kansas. Supported in part by USPHS Grants HL-70119 and HL 12072.     

Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles

Summary We have confirmed previous demonstrations of sodium gradient-stimulated transport ofl-alanine, phenylalanine, proline, and -alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these¹⁴C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability ofd-glucose to inhibit tracer uptake.l-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the¹⁴C-l-alanine uptake could be inhibited by proline and -alanine, while lysine and MeAIB were no more effective thand-glucose. Roughly 50% of the¹⁴C-phenylalanine uptake could be inhibited by proline and -alanine; lysine was as effective as proline and -alanine, and the effects of pairs of these amino acids at 50mm each were not cumulative. MeAIB was no more effective thand-glucose. We conclude that three pathways mediate the uptake of neutral,l, -amino acids. One system is inaccessible to lysine, proline, and -alanine. The second system carries a major fraction of thel-alanine flux; it is sensitive to proline and -alanine, but not to lysine. The third system carries half the¹⁴C-phenylalanine flux, and it is sensitive to proline, lysine, and -alanine. Since the neutral,l, -amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classicalA system, and since all of thel-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classicalASC system.l-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective thand-glucose in inhibiting lysine uptake, while proline and -alanine appear to inhibit a component of the lysine flux. We conclude that the¹⁴C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, -alanine, andl-alanine, and one which is inhibited byl-alanine and phenylalanine but inaccessible to proline, -alanine, and MeAIB. Fluxes of¹⁴C-proline and¹⁴C-MeAIB are completely inhibited byl-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit¹⁴C--alanine uptake. However, -alanine inhibits only 38% of the¹⁴C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports -alanine.     

Studies on glutathione transport utilizing inside-out vesicle prepared from human erythrocytes

Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Line-weaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity. Most active at acidic pH and at 25°C, this transport activity was easily lost during the storage of vesicles at 4°C. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 0.63 mM; guanosine triphosphate (GTP) substituted for ATP gave a 340% stimulation of transport activity. Neither dithiothreitol nor thiol reagents affected this transport process. The other phase had a low affinity for GSSG and a high transport velocity. Most active at pH 7.2 and 37°C, this transport activity was stable during storage of vesicles at 4°C for several days. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 1.25 mM; GTP substituted with no change in activity. Dithiothreitol increased the V but did not alter the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, and thiol reagents inhibited the transport. These findings suggest that there are two independent transfer processes for GSSG in human erythrocytes.     

Control of amino acid transport in the mammary gland of the pregnant mouse

The regulation of the uptake of the amino acid analog α-aminoisobutyric acid was studied in diced mammary glands from pregnant mice. Stimulation of uptake by insulin was not prevented by inhibitors of protein synthesis; protein synthesis inhibitors decreased uptake by 20%; this response occurred more promptly in insulintreated tissues. Elimination of extracellular amino acids led to a substantial increase in transport which was not abolished by inhibitors of protein synthesis. These results indicate that insulin does not increase amino acid transport in this system by altering synthesis and degradation of transport protein. They are consistent with a model in which the activity of the existing amino acid transport protein is subject to negative feedback regulation from the intracellular amino acid pool.     

Measurements of amino acid transport in internally dialyzed giant axons

Summary It is shown that the axoplasmic composition of acidic and neutral amino acids can be controlled effectively by the method of internal dialysis. Direct assay for specific binding and measurement of diffusion coefficients in axoplasm show that there is no significant binding or compartmentalization of amino acids. The dependence of amino acid efflux on substrate concentration can be measured under well-defined, true steady-state conditions. The taurine efflux-concentration relation in theMyxicola giant axon conforms to a second-order Hill equation. This fact is consistent with either a cooperative process or a mechanism in which membrane translocation is not the rate-controlling step. The effluxes of taurine and glycine from squid axon are an order of magnitude smaller than inMyxicola. The efflux-concentration relations are essentially linear up to 200mm substrate concentration. This result may be produced by specific transporters which have very high asymmetry, or by simple diffusive leak in the absence of specific transporters.     

Transport in halobacterium halobium: Light-induced cation-gradients,amino acid transport kinetics,and properties of transport carriers

Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na⁺. Measurements of ²²Na flux, exterior pH change, and membrane potential, ΔΨ (with the dye 3,3′-dipentyloxadicarbocyanine) indicate that the means of Na⁺ transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H⁺/Na⁺ > 1). The resulting large chemical gradient for Na⁺ (outside > inside), as well as the membrane potential, will drive the transport of 18 amino acids. The 19th, glutamate, is unique in that its accumulation is indifferent to ΔΨ: this amino acid is transported only when a chemical gradient for Na⁺ is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H⁺ collapses within 1 min, while the large Na⁺ gradient and glutamate transporting activity persists for 10–15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na⁺, arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V_max and K_m comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na⁺, in an electrically neutral fashion, so that only the chemical component of the Na⁺ gradient is a driving force. The transport of all amino acids but glutamate is bidirectional. Actively driven efflux can be obtained with reversed Na⁺ gradients (inside > outside), and passive efflux is considerably enhanced by intravesicle Na⁺. These results suggest that the transport carriers are functionally symmetrical. On the other hand, noncompetitive inhibition of transport by cysteine (a specific inhibitor of several of the carriers) is only obtained from the vesicle exterior and only for influx: these results suggest that in some respects the carriers are asymmetrical. A protein fraction which binds glutamate has been found in cholate-solubilized H. halobium membranes, with an apparent molecular weight of 50,000. When this fraction (but not the others eluted from an Agarose column) is reconstituted with soybean lipids to yield lipoprotein vesicles, facilitated transport activity is regained. Neither binding nor reconstituted transport depend on the presence of Na⁺. The kinetics of the transport and of the competitive inhibition by glutamate analogs suggest that the protein fraction responsible is derived from the intact transport system.     

Mechanisms and specificity of amino acid transport in Taenia crassiceps larvae (Cestoda)

The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l-amino acids than d-amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l-methionine uptake by larvae is inhibited more by d-alanine and d-valine than by their respective l-isomers, while d- and l-methionine inhibit l-methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na⁺-insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na⁺. Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.     

Energy sources for amino acid transport in animal cells

The existence of an electrogenic Na⁺ pump in Ehrlich cells which substantially contributes to the membrane potential, previously derived from the distribution of the lipid soluble cation tetraphenylphosphonium (TPP⁺), could be confirmed by an independent method based on the quenching of fluorescence of a cyanine dye derivative, after the mitochondrial respiration had been suppressed by appropriate inhibitors. The mitochondrial membrane potential, by adding to the overall potential as measured in this way is likely to cause an overestimation of the membrane potential difference (p.d.). But since this error tends to diminish with increasing pump activity, the true p.d. of the plasma membrane should easily account for the driving force to drive the active accumulation of amino acids in the absence of an adequate Na⁺ concentration gradient. Accordingly, the F₂-aminoisobutyric acid (AIB) uptake rises linearly with the distribution of TPP⁺ at constant Na⁺ concentrations, suggesting that each responds directly to membrane potential. There is evidence that the electrogenic (free) movement of Cl^? is slow, at least at normal p.d., whereas a major part of the Cl^? movement across the cellular membrane appears to occur by an electrically silent Cl^?-base exchange mechanism. By such a mode Cl^?, together with an almost stoichiometric amount of K⁺, may under certain conditions move into the cell against a high adverse electrical potential difference. This “paradoxical” movement of K⁺Cl^? contributing to the deviation of the Cl^? distribution from the electrochemical equilibrium distribution, is not completely understood. It is insensitive towards ouabain but can almost specifically be inhibited by furosemide. As a likely explanation a H⁺–K⁺ exchange pump was previously offered, even though unequivocal evidence of such a pump is so far lacking. According to available evidence the electrogenic movement of free Cl^? is too small, at least at normal orientation of the p.d., to significantly shunt the electrogenic pump potential so that the establishment of such a potential is plausible. The evidence presented is considered strong in favor of the gradient hypothesis since even in the absence of an adequate Na⁺ concentration gradient, the electrogenic Na⁺ pump will contribute sufficient extra driving force to actively transport amino acid into the cells.     

Hormonal regulation of hepatic amino acid transport

The transport of 2-aminoisobutyric acid (AIB) into liver tissue was increased by both insulin and glucagon. We have now shown that these hormones do not stimulate the same transport system. Glucagon, possibly via cAMP, increased the hepatic uptake of AIB by a mechanism which resembled system A. This glucagon-sensitive system could be monitored by the use of the model amino acid MeAIB. In contrast, the insulin-stimulated system exhibited little or no affinity for MeAIB and will be referred to as system B. On the basis of other reports that the hepatic transport of AIB is almost entirely Na⁺ dependent and the present finding that the uptake of 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH) was not stimulated by either hormone, we conclude that system B is Na⁺ dependent. Furthermore, insulin added to the perfusate of livers from glucagon-pretreated donors suppressed the increase in AIB or MeAIB uptake. Depending upon the specificities of systems A and B, both of which are unknown for liver tissue, the insulin/glucagon ratio may alter the composition of the intracellular pool of amino acids.     

Transport of neutral amino acids by human erythrocytes

The transport of several neutral amino acids by human erythrocytes in vitro was studied. The measurements made included steady-state distributions, kinetics of initial rates of uptake, effects of monovalent cations and anions, general mutual inhibitory interactions, kinetics of inhibitions, effluxes, ability to produce accelerative exchange diffusion, and the inhibitory action of the thiol reagent N-ethylmaleimide. The results are interpreted as showing that the human erythrocyte membrane possesses several distinct transport systems for these amino acids, including one Na⁺-dependent system and one dependent on both Na⁺ and a suitable anion, that are qualitatively similar to those systems previously described in pigeon erythrocytes and mammalian reticulocytes. Quantitatively, however, the systems differ among the different kinds of red cell and a major difference lies in their abilities to produce accelerative exchange diffusion.     

生产氨基酸的基因工程菌研究进展

随着抗癌药物制剂、氨基酸输液制剂及甜味二肽生产的飞速发展,对原料氨基酸的需求量日益增长。传统的发酵工业越来越不能满足需求,势必被以基因工程为基础的新兴发酵工业所代替。通过建立大肠杆菌及棒状杆菌的高效载体受体系统,运用ＤＮＡ重组、定向突变等手段,对代谢途径及关键酶进行了深入系统的研究,为代谢工程注入了新的活力,为获得高产、优质且易于自动化生产的菌株打下了基础