POLY(A)-DEPENDENT AND NA+-INDEPENDENT LEUCINE TRANSPORT ACTIVITIES INOOCYTES OF XENOPUS-LAEVIS( RNA FROM THE MUCOSA OF RAT JEJUNUM INDUCESNOVEL NA+)

Complementary DNA clones have been isolated recently from rat (D2) and rabbit kidney (rBAT) which induce increased Na+-independent Leu and L ys transport activities (System b0,+) when expressed in oocytes of Xen opus laevis. These cDNAs encode type II membrane glycoproteins which s how significant homology to the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Injection of human 4F2hc cRNA into oocytes a lso results in induction of Leu/Lys transport activity, but with diffe ring cation requirements for the two amino acids (Na+-dependent for Le u, Na+-independent for Lys: system y+L). System y+L is a newly discove red zwitterionic/cationic amino acid transporter first described in hu man erythrocytes. Here we have examined the characteristics of Leu tra nsport in Xenopus oocytes microinjected with mRNA from the mucosa of r at jejunum. L-Leu uptake during 10 min (0.2 mm, 20-degrees-C) reached 20 pmol/oocyte compared with endogenous uptake by water-injected oocyt es of typically 3-4 pmol/oocyte. The expressed transport activity was 80% Na+-dependent. The Na+-dependent component of the expressed flux w as saturable (K(m app) 0.20 mm) and inhibited by Lys, but not by Ala o r Phe. The minor Na+-independent component of expressed Leu transport activity was also saturable (K(m app) 0.10 mM). Amino acid inhibition studies resolved this flux into two main components, one of which was inhibited by Lys, Ala and Phe and another which was only inhibited by Lys. There was a small residual component of Na+-independent Leu trans port which was insensitive to inhibition by Lys. Experiments utilizing polymerase chain reaction (PCR) demonstrated the presence of both D2 and 4F2hc message in rat jejunum. Hybrid-depletion of jejunal mRNA wit h an antisense oligonucleotide complementary to D2 had no effect on th e expression of Na+-linked Leu transport activity, but reduced the sma ller Na+-independent component of Leu transport by 40%, suggesting onl y a minor role of D2 in the expression of rat intestinal Leu transport activity. Although the properties of Na+-dependent Leu transport were , with the exception of a lack of inhibition by Ala and Phe, consisten t with erythrocyte y+L, hybrid-depletion of jejunal mRNA with an antis ense oligonucleotide complementary to 4F2hc had no detectable effect o n the expressed transport activity. We conclude, therefore, that mRNA from rat jejunum encodes novel Na+-dependent and Na+-independent trans port activities unrelated to the D2/4F2hc glycoproteins. In preparatio n for the expressing cloning of these proteins, we have determined tha t maximal Leu transport activity is encoded by an mRNA size fraction o f 1.5-2.25 kb (peak 2.0 kb).