Mammalian equilibrative nucleoside transporters are typically divided into two classes, es and ei, based on their sensitivity or resistance respectively to inhibition by nitrobenzylthioinosine (NBMPR). Previously, we have reported the isolation of a cDNA clone encoding a prototypic es-type transporter, hENT1 (human equilibrative nucleoside transporter 1), from human placenta. We now report the molecular cloning and functional expression in Xenopus oocytes of a cDNA from the same tissue encoding a homologous ei-type transporter, which we designate hENT2. This 456-residue protein is 46% identical in amino acid sequence with hENT1 and corresponds to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in a form bearing a sequence deletion. In addition to placenta, hENT2 is found in brain, heart and ovarian tissue. Like hENT1, hENT2 mediates saturable transport of the pyrimidine nucleoside uridine (Km 0.2±0.03 mM) and also transports the purine nucleoside adenosine. However, in contrast with hENT1, which is potently inhibited by NBMPR (Ki 2 nM), hENT2 is NBMPR-insensitive (IC50 < 1 μM). It is also much less sensitive to inhibition by the coronary vasoactive drugs dipyridamole and dilazep and to the lidoflazine analogue draflazine, properties that closely resemble those reported for classical ei-type transport in studies with intact cells.

Two different protein families, designated CAT (cationic amino acid transporter) and BAT (broad-specificity amino acid transporter) mediate the plasma membrane transport of cationic amino acids in animal cells. CAT transporters have 12-14 transmembrane domains and are selective for cationic amino acids. BAT proteins, in contrast, have one to four transmembrane domains and induce the transport of both cationic and zwitterionic amino acids when expressed in Xenopus oocytes. Mutations in the human BAT gene cause type I cystinuria, a disease affecting the ability of intestinal and renal brush border membranes to transport cationic amino acids and cystine. We have used functional expression cloning in oocytes to isolate a BAT-related cDNA from rat jejunal epithelium. The cDNA encodes the rat 4F2 heavy chain (4F2hc) cell-surface antigen, a 527-residue (60 kDa) protein that is 26% identical in amino acid sequence with rat renal BAT (also known as NBAT/D2). Expression of rat jejunal 4F2hc in oocytes induced the lysine-inhibitable Na+-dependent influx of leucine and the leucine-inhibitable Na+-independent influx of lysine. Lysine efflux was stimulated by extracellular (Na+ plus leucine). These characteristics identify the expressed amino acid transport activity as system y+L, a transporter that has been implicated in basal membrane transport of cationic amino acids in intestine, kidney and placenta.

A full-length cDNA encoding a putative diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) was obtained from sunfl ower (Helianthus annuus L.) seeds. The 1524-bp open reading frame of this cDNA, designated as HaDGAT1, encodes a protein of 507 amino acids with a molecular mass of 58.5 kDa showing high homology to DGAT1 enzymes of other plants. The protein characters, such as a predicted structure with a long N-terminal hydrophilic domain followed by 9 transmembrane domains, acyl-CoA-binding signature, diacylglycerol (DAG)-binding and putative endoplasmic reticulum retrieval motifs (ER-DIR), also indicated that HaDGAT belongs to the DGAT1 family. HaDGAT1 is expressed in all plant tissues especially in developing seeds. Expression of recombinant HaDGAT1 in yeast showed an 1.76-fold increase of total fatty acids, especially unsaturated fatty acids such as palmitoleic acid (enhanced by 86.6%) and oleic acid (enhanced by 81.6%).