Archives of Insect Biochemistry and Physiology 48:51–62 (2001) © 2001 Wiley-Liss, Inc. Leucine Transport in Membrane Vesicles From Chironomus riparius Larvae Displays a Mélange of Crown-Group Features Paolo Parenti, 1 * Matilde Forcella, 1 Anna Pugliese, 1 Roberto Giacchini, 1 Bruno Rossaro, 2 and Giorgio M. Hanozet 1 1 Department of Environmental Sciences, University of Milan-Bicocca, Milano, Italy 2 Department of Biology, University of Milano, Milano, Italy Leucine uptake into membrane vesicles from larvae of the midge Chironomus riparius was studied. The membrane prepa- ration was highly enriched in typical brush border membrane enzymes and depleted of other membrane contaminants. In the absence of cations, there was a stereospecific uptake of L-leu- cine, which exhibited saturation kinetics. Parameters were de- termined both at neutral (K m 33 ± 5 mM and V max 22.6 ± 6.8 pmol/ 7s/mg protein) and alkaline (K m 46 ± 5 mM 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 17 ± 1 mM) and the maxi- mal uptake rate (V max 74.0 ± 12.5 pmol/7s/mg protein). Stimula- tion of leucine uptake by external alkaline pH agreed with lumen pH measurements in vivo. Competition experiments in- dicated that at alkaline pH, the transport system readily ac- cepts most L-amino acids, including branched, unbranched, and a-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, b-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased af- finity for lysine, glycine, and a-methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transport systems, which combine characters of the lepidopteran amino acid transport system and of the sodium-dependent system from lower neopterans. Arch. Insect Biochem. Physiol. 48:51–62, 2001. © 2001 Wiley-Liss, Inc. Key words: amino acid transport; membrane vesicles; midge larvae; Chir- onomus riparius Abbreviations used: CAPS = 3-cyclohexylamino-1-propan- sulfonic acid; HEPES = 4-(2-hydroxyethyl)-1-piperazine- ethansulfonic acid; MES = 4-morpholineethansulfonic acid; TEP = transepithelial electrical potential; TMA = tetram- ethylammonium. Contract grant sponsor: Italian MURST. *Correspondence to: Dr. Paolo Parenti, Dipartimento di Scienze dell’Ambiente e del Territorio, P.zza della Scienza 1, 20126 Milano, Italy. E-mail: paolo.parenti@unimib.it Received 30 October 2000; Accepted 25 March 2001 INTRODUCTION The insect midgut is the site of nutrients up- take and the cell membrane of the enterocytes contains either passive diffusion systems or cat- ion-driven symporter (for a recent review, see Wolfersberger, 2000). Most of what we know of the molecular physiology of nutrient absorption in insect midgut comes from studies on brush bor- der membrane vesicles from lepidopteran larvae,