Dibasic amino acid interactions with Na +-independent transport system asc in horse erythrocytes. Kinetic evidence of functional and structural homology with Na +-dependent system ASC

Abstract

Amino acid transport in horse erythrocytes is regulated by three co-dominant allelomorphic genes coding for high-affinity transport activity (system asc 1), low-affinity transport activity (system asc 2) 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 asc 1 and asc 2 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 asc 1 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 asc 2 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 asc 2 were 14 mM and 6.90 mmol/l cells per h. In contrast, l-arginine was transported by system asc 2 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 asc 2 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 asc 2 activity.