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Transport systems y + , asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na + -dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na + site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na + -site inhibitor) with the same affinity as asc (apparent K i range 1–4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na + -independent, dibasic amino acid transport system y + accepts neutral amino acids when Na + or another acceptable cation is also present. This latter observation implies that the y + translocation site binds Na + and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y + -mediated lysine uptake (5 μM, 20°C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na + functioning as a competitive inhibitor of transport. Apparent K m and K m values for lysine transport at 20°C were 15.2 μM and 183 μmol/l cells per h, respectively, in sucrose medium and 59.4 μM and 228 μmol/l cells per h in Na + medium. Similar results were obtained with y + in erythrocytes of a primitive vertebrate, the Pacific hagfish ( Eptatretus stouti ), indicating that Na + -inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 μM, the IC 50 value for Na + -inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K + and guanidinium + , also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y + translocation site, the presence of 150 mM Na + increased the harmaline inhibition constant ( K l ) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y + , asc and ASC represent a family of closely related transporters with a common evolutionary origin.

Red blood cells from the Pacific hagfish (Eptatretus stouti) were found to possess a facilitated diffusion nucleoside transport system insensitive to inhibition by the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). Uridine uptake by this route was saturable (apparent Km 0.14 mM; Vmax 2 mmol/l cells per h at 10°C), inhibited by inosine and adenosine, and blocked both by the vasodilator dipyridamole and by the thiol-reactive agent p-chloromercuriphenylsulphonate. The properties of this carrier resemble closely those of NBMPR-insensitive nucleoside transport systems in some mammalian neoplastic cell lines and in rat red cells. The presence of this type of carrier in a primitive vertebrate suggests that such transporters have a broad biological distribution and that they pre-date or arose at an early stage of vertebrate evolution.

1. (1) Sheep erythrocytes possess a facilitated diffusion transport system selective for small neutral amino acids of intermediate size (C-system). The effects of seven thiol-reactive agents on this system were investigated. 2. (2) L -Alanine influx by this route was inhibited by HgCl2, p- chloromercuriphenylsulphonate (PCMBS), azodicarboxylic acid bisdimethylamide (diamide), N- ethylmaleimide and t- butylhydroperoxide . Iodoacetamide and 5,5′-dithiobis(2-nitrobenzoate) had no effect. 3. (3) Detailed analysis of these inhibitor effects suggested the presence of three distinct classes of cellular thiol groups essential for normal transport function. 4. (4) Class 1 thiols react with PCMBS and are located on the outer surface of the cell membrane in the region of the transport site. Class 2 thiols react with N- ethylmaleimide and diamide but are not affected by t- butylhydroperoxide . Class 3 thiols are oxidized during t- butylhydroperoxide treatment and are presumably also attacked by diamide and N- ethylmaleimide . 5. (5) Class 3 thiols are either intracellular GSH or reactive thiols which readily form mixed disulphides with GSSG. Any direct involvement of GSH in amino acid transport is not mediated by the γ-glutamyl cycle.

1. 1. Two automated colorimetric methods have been developed for assaying the GSH and total thiol in protein-free extracts of erythrocytes. They employ as chromogens 5,5′-dithiobis-(2-nitorbenzoate) (DTNB) and alloxan. 2. 2. The concentrations of GSH, GSSG and total non-protein thiol have been estimated in high and low GSH erythrocytes from Finnish Landrace and Tasmanian Merino sheep. 3. 3. In both breeds of sheep low GSH cells were found to have low concentrations of total non-protein thiol and GSSG as well as of GSH. 4. 4. Nevertheless high and low GSH cells have similar values for the oxidation-reduction potential of the GSH : GSSG couple.

Erythrocytes from the Yucatan miniature pig, like those from the normal domestic pig, lack functional glucose transporters and were unable to utilize plasma glucose as an energy source. In contrast, inosine and adenosine entered the cells rapidly. The nucleoside transporter responsible for this uptake was identified as a band 4.5 polypeptide (5000 copies per cell; apparent Mr 45 000-66 000). Inosine concentrations in the physiological plasma range (1.6-2.5 microM) were found to maintain normal erythrocyte ATP levels and ATP/ADP ratios during prolonged in vitro incubation of cells at 37 degrees C, an effect that was blocked by the specific nucleoside transport inhibitor, nitrobenzylthioguanosine. In the absence of extracellular nucleoside, cells 'protected' themselves against some of the consequences of deprivation of energy substrate by glycolyzing the ribose moiety of inosine produced during ATP catabolism. Although erythrocytes from the miniature pig were capable of utilizing extracellular adenosine as an energy substrate, plasma samples from these animals contained less than 0.4 microM adenosine. It is concluded that inosine is a major physiological energy source of pig erythrocytes.

Amino acid transport in horse erythrocytes is regulated by three co-dominant allelomorphic genes coding for high-affinity transport activity (system asc1), low-affinity transport activity (system asc2) and transport-deficiency, respectively. The asc systems are selective for neutral amino acids of intermediate size, but unlike conventional system ASC, do not require Na+ for activity. In the present series of experiments we have used a combined kinetic and genetic approach to establish that dibasic amino acids are also asc substrates, systems asc1 and asc2 representing the only mediated routes of cationic amino acid transport in horse erythrocytes. Both transporters were found to exhibit a strong preference for dibasic amino acids compared with neutral amino acids of similar size. Apparent K m values (mM) for influx via system asc1 were l -lysine (9), l -ornithine (27), l -arginine (27), l -alanine (0.35). Corresponding V max estimates (mmol/l cells per h, 37°C) were l -lysine (1.65), l -ornithine (2.15), l -arginine (0.54), l -alanine (1.69). Apparent K m values for l -lysine and l -ornithine influx via system asc2 were ≈ 90 and > 100 mM , respectively, with V max values > 2 and > 1 mmol/l cells per h, respectively. Apparent K m and V max values for l -alanine uptake by system asc2 were 14 mM and 6.90 mmol/l cells per h. In contrast, l -arginine was transported by system asc2 with the same apparent K m as l -alanine (14 mM), but with a 77-fold lower V max . This dibasic amino acid was shown to cause cis- and trans-inhibition of system asc2 in a manner analogous to its interaction with system ASC, where the side-chain guanidinium group is considered to occupy the Na+-binding site on the transporter. Concentrations of extracellular l -arginine causing 50% inhibition of zero-trans l -alanine influx and half-maximum inhibition of l -alanine zero-trans efflux were 14 mM (extracellular l -alanine concentration 15 mM) and 3 mM (intracellular l -alanine concentration 15.5 mM), respectively. We interpret these observations as evidence of structural homology between the horse erythrocyte asc transporters and system ASC. Physiologically, intracellular l -arginine may function as an endogenous inhibitor of system asc2 activity.

The pig erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide ( M r 64 000) on the basis of photoaffinity labelling experiments with the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). This protein was purified 140-fold by treatment of haemoglobin-free erythrocytes ‘ghosts’ with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with n- octyl - glucoside and subsequent gradient-elution ion-exchange chromatography on DEAE-cellulose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified material revealed the presence of only two detectable protein bands, one which co-migrated with the radiolabelled NBMPR-binding protein, and a lower molecular weight species with an M r of 43 000. The latter protein may be a degradation product of the band 3 anion-exchange transporter. The overall purification of the NBMPR-binding protein with respect to the M r 64 000 band was 350-fold. Reversible NBMPR-binding to the partially-purified band 4.5 preparation was saturable (apparent K d 7.2 nM). Adjustment of the chromatography conditions to allow elution of the NBMPR-binding protein along with the majority of solubilised membrane phospholipid reduced the apparent K d value to 3.0 nM. Purification of reversible NBMPR-binding activity during ion-exchange chromatography was paralleled by an increase in the specific activity of nitrobenzylthioguanosine (NBTGR) -sensitive uridine transport as assayed in proteoliposomes reconstituted by a freeze-thaw-sonication procedure.

Guinea pig reticulocytes are shown to possess an Na+-dependent glycine transporter which also requires Cl- for activity. Glycine transport by this route is saturable (apparent Km 98 microM; Vmax 24 mumol/g Hb per h) and inhibited by sarcosine. The properties of this carrier closely resemble those of System Gly previously demonstrated in pigeon and human erythrocytes. In contrast, no System Gly activity was detected in mature guinea pig erythrocytes.

An amino acid transport defect which occurs in the erythrocytes of adult sheep is also present in foetal erythrocytes from newborn lambs which have inherited the lesion. The transport defect in erythrocytes from adult sheep is associated with high intracellular levels of ornithine and lysine and a markedly diminished GSH concentration. Although the lesion in foetal cells also results in the accumulation of ornithine and lysine, the intracellular GSH concentration is only moderately diminished.

L-Phenylalanine benzyl ester (Phe-Bz) and a number of ester analogues prevent sickling of erythrocytes from sickle cell disease patients. The compounds tested exhibit anti-sickling activity in the concentration range 0.5-3.0 mM. A general feature of these compounds is the presence of two aromatic rings in their molecular structure. The anti-sickling agents rapidly enter the erythrocyte and are hydrolysed to their component molecules. Incubation of human erythrocytes with 3.0 mM L-phenylalanine for 30 min at 37 degrees C results in accumulation of 2.0 mmol L-phenylanine/l cells, while incubation of erythrocytes with 3.0 mM Phe-Bz under similar conditions results in the production of 4.0 mmol L-phenylalanine/l cells and an equivalent amount of benzyl alcohol. Both L-phenylalanine and benzyl alcohol are inhibitors of the gelation of deoxyhaemoglobin S (deoxy-HbS) in vitro. Moreover, Phe-Bz and related anti-sickling agents fluidize the lipid bilayer of the erythrocyte membrane, inhibiting several transport systems, including those for L-phenylalanine, uridine and sulphate ions, as well as the Na+ pump and the Na+/K+ cotransporter, but increasing the passive influx and efflux of both cations and anions. The accumulation of Phe-Bz hydrolysis products within the erythrocyte together with the effects of Phe-Bz on cation permeability result in the influx of water causing the cell to swell. Thus, treatment of erythrocytes with 3.0 mM Phe-Bz at 37 degrees C for 30 min causes an increase in mean cell volume of 14.8%, decreasing the mean intracellular haemoglobin concentration from 34 to 29.6 g%. The increase in cell volume caused by Phe-Bz and its analogues together with the direct effects of their hydrolysis products on HbS probably act in concert to bring about the anti-sickling effect.