Multiple pathways for L-methionine transport in brush-border membrane vesicles from chicken jejunum

Abstract

1. The intestinal transport of L-methionine has been investigated in brush-border membrane vesicles isolated from the jejunum of 6-week-old chickens. L-Methionine influx is mediated by passive diffusion and by Na+-dependent and Na+-independent carrier-mediated mechanisms. 2. In the absence of Na+, cis-inhibition experiments with neutral and cationic amino acids indicate that two transport components are involved in L-methionine influx: one sensitive to L-lysine and the other sensitive to 2-aminobicyclo[2.2. 1]heptane-2-carboxylic acid (BCH). The L-lysine-sensitive flux is strongly inhibited by L-phenylalanine and can be broken down into two pathways, one sensitive to N-ethylmaleimide (NEM) and the other to L-glutamine and L-cystine. 3. The kinetics of L-methionine influx in Na+-free conditions is described by a model involving three transport systems, here called a, b and c: systems a and b are able to interact with cationic amino acids but differ in their kinetic characteristics (system a: Km = 2.2 +/- 0.3 microM and Vmax = 0.13 +/- 0.005 pmol (mg protein)-1 (2 s)-1; system b: Km = 3.0 +/- 0.3 mM and Vmax = 465 +/- 4.3 pmol (mg protein)-1 (2 s)-1); system c is specific for neutral amino acids, has a Km of 1.29 +/- 0.08 mM and a Vmax of 229 +/- 5.0 pmol (mg protein)-1 (2 s)-1 and is sensitive to BCH inhibition. 4. The Na+-dependent component can be inhibited by BCH and L-phenylalanine but cannot interact either with cationic amino acids or with alpha-(methylamino)isobutyrate (MeAIB). 5. The kinetic analysis of L-methionine influx under a Na+ gradient confirms the activity of the above described transport systems a and b. System a is not affected by the presence of Na+ while system b shows a 3-fold decrease in the Michaelis constant and a 1.4-fold increase in Vmax. In the presence of Na+, the BCH-sensitive component can be subdivided into two pathways: one corresponds to system c and the other is Na+ dependent and has a Km of 0.64 +/- 0. 013 mM and a Vmax of 391 +/- 2.3 pmol (mg protein)-1 (2 s)-1. 6. It is concluded that L-methionine is transported in the chicken jejunum by four transport systems, one with functional characteristics similar to those of system bo, + (system a); a second (system b) similar to system y+, which we suggest naming y+m to account for its high Vmax for L-methionine transport in the absence of Na+; a third (system c) which is Na+ independent and has similar properties to system L; and a fourth showing Na+ dependence and tentatively identified with system B.

Figure 1. Time course for uptake of 100 μM L-methionine under zero-trans 100 mM NaSCN (○), KSCN (•) or a KSCN gradient and 50 mM L-methionine (□)

Membranes were prepared in 300 mM mannitol, 0.1 mM MgSO₄·7H₂O, 0.02 % LiN₃ and 20 mM Hepes-Tris, pH 7.4 and incubated (37 °C) in 100 mM NaSCN or KSCN, 100 mM mannitol, 0.2 mM MgSO₄·7H₂O, 0.02 % LiN₃, 20 mM Hepes-Tris, pH 7.4 and 100 μM L-methionine (50 μM L-[¹⁴C]methionine). Osmolarity was maintained by reducing mannitol concentration. Each value represents the mean ±s.e.m. of 4-5 membrane preparations. Only s.e.m. that exceed the size of the symbol are shown.

Figure 2. Effect of an increasing zero-trans Na⁺ gradient on 100 μM L-methionine uptake (50 μM L-[¹⁴C]methionine)

Vesicles were prepared as described in legend of Fig. 1 and incubated in the presence of increasing NaSCN concentrations (1-100 mM). Osmolarity was maintained with mannitol. Each value represents the mean ±s.e.m. of 3 membrane preparations.

Figure 3. Cis-inhibition of L-[¹⁴C]methionine influx (50 μM) by L-amino acids, BCH, MeAIB and D-glucose (all at 10 mM) under a 100 mM zero-trans KSCN gradient

Each value represents the mean ±s.e.m. of 3-5 membrane preparations. The bars labelled with different letters show statistical differences (P < 0.05).

Figure 4. Cis-inhibition of L-[¹⁴C]methionine influx (50 μM) by L-amino acids, BCH and MeAIB (all at 10 mM) under a 100 mM zero-trans NaSCN gradient

The Na⁺-dependent component (dashed bars) was obtained by subtracting KSCN results (Fig. 3) from NaSCN values (open bars). The passive diffusion of L-methionine in the presence of Na⁺ or K⁺ was the same (P≥ 0.05). Each value represents the mean ±s.e.m. of 3-5 membrane preparations. The bars labelled with different letters show statistical differences (P < 0.05).

Figure 5. Relative transport rates of L-[¹⁴C]methionine (50 μM) influx under a 100 mM zero-trans KSCN gradient in the presence of varying concentrations of unlabelled L-methionine incubated with (A) or without (B) 10 mM L-lysine (□) or BCH (▿)

The lines of A represent the best fit obtained in the presence of L-lysine (one transport system) or BCH (two transport systems). B shows the curve drawn by inserting the kinetic parameters of Table 4 in eqn (6) (r= 0.998). Each value represents the mean ±s.e.m. of 3-5 membrane preparations. Only s.e.m. that exceed the size of the symbol are shown.

Figure 6. Relative transport rates of L-[¹⁴C]methionine (50 μM) influx in the presence of varying concentrations of unlabelled L-lysine (A) and BCH (B), under a 100 mM zero-trans KSCN gradient

The lines represent the best fit of the L-lysine-sensitive transport component (two transport systems) and of the BCH-sensitive component (one transport system). Each value represents the mean ±s.e.m. of 3-5 membrane preparations. Only s.e.m. that exceed the size of the symbol are shown.

Figure 7. Relative influx of L-methionine under a 100 mM zero-trans NaSCN gradient

A, relative transport rates of L-[¹⁴C]methionine (50 μM) influx in the presence of varying concentrations of unlabelled L-methionine and 10 mM L-lysine (□) or BCH (▿). The lines represent the best fit obtained in the presence of L-lysine (one transport system) or BCH (two transport systems). B, relative transport rates of Na⁺-dependent, BCH-sensitive influx calculated by subtracting the values obtained in the absence of Na⁺ (Fig. 5A) from those obtained under a Na⁺ gradient (Fig. 7A). The best fit obtained was for a single transport system. Each value represents the mean ±s.e.m. of 3-5 membrane preparations. Only s.e.m. that exceed the size of the symbol are shown.