Leucine transport in membrane vesicles from Chironomus riparius larvae displays a melange of crown-group features

Leucine uptake into membrane vesicles from larvae of the midge Chironomus r iparius was studied. The membrane preparation was highly enriched in typica l brush border membrane enzymes and depleted of other membrane contaminants . In the absence of cations, there was a stereospecific uptake Of L-leucine , which exhibited saturation kinetics. Parameters were determined both at n eutral (K-m 33 +/- 5 gM and V-max 22.6 +/- 6.8 pmol/7s/mg protein) and alka line (K-m 46 +/- 5 muM and V-max 15.5 +/- 2.5 pmol/7s/mg protein) pH values . At alkaline pH, external sodium increased the affinity for leucine (K-m 1 7 +/- 1 muM) and the maximal uptake rate (V-max 74.0 +/- 12.5 pmol/7s/mg pr otein). Stimulation of leucine uptake by external alkaline pH agreed with l umen pH measurements in vivo. Competition experiments indicated that at alk aline pH, the transport system readily accepts most L-amino acids, includin g branched, unbranched, and alpha -methylated amino acids, histidine and ly sine, but has a low affinity for phenylalanine, beta -amino acids, and N-me thylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and alpha -methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transpor t systems, which combine characters of the lepidopteran amino acid transpor t system and of the sodium-dependent system from lower neopterans. (C) 2001 Wiley-Liss, Inc.