Tsunao Tetsuka

Nucleotide sequence of a complementary DNA for human ST2.

Human ST2 cDNA, a homologue of murine ST2 that is only expressed in growth-stimulated BALB/c-3T3 cells and a member of the primary response gene family induced by growth factors, was isolated from the cDNA library of an activated human helper T cell line, 5C10. Human ST2 has 67.6% identity in a 327 amino acid overlap to murine ST2. Furthermore, as in the case of murine ST2, human ST2 encodes a protein remarkably similar in sequence to the extracellular portion of human interleukin 1 receptor, both types 1 and 2. The expression of ST2 in human lymphocytes could trigger further investigations into its physiological role in humans.

Identification of the product of the murine ST2 gene

The murine ST2 gene is expressed in growth-stimulated BALB/c-3T3 cells. This gene encodes a protein that is similar to the extracellular portions of the interleukin-1 receptors (types 1 and 2). In this study, we prepared a polyclonal antibody against the recombinant ST2 protein produced in Escherichia coli. This antibody detected recombinant ST2 protein in the culture fluid of COS7 cells transfected with a mammalian expression vector (pEF-BOS) carrying ST2 cDNA. Using this antibody, we could detect the ST2 protein in the culture fluid of growth-stimulated BALB/c-3T3 cells, and in the medium of continuously growing cells, but not in that of growth-arrested cells. ST2 proteins produced in COS7 cells and BALB/c-3T3 cells were N-glycosylated as predicted from nine putative N-glycosylation sites in its deduced amino-acid sequence.

The existence of a growth-specific DNA binding factor for the promoter region of mouse ST2 gene

A comparison of the 5′‐flanking regions of human and mouse ST2 genes revealed the presence of two highly conserved DNA sequences. The promoter activity assay with a luciferase gene as a reporter showed that the deletion of the upstream conserved region diminished the transcriptional activity in growing BALB/c‐3T3 cells. By electrophoretic mobility‐shift analysis, the presence of a factor that binds to the positive regulatory region of the mouse ST2 gene was found in growing but not in quiescent BALB/c‐3T3 cells. These results suggest the functional importance of this conserved region and the requirement of a binding factor for the expression of the ST2 gene.

Solubilization and reconstitution of high-and low-affinity Na+-dependent neutral l-α-amino acid transporters from rabbit small intestine

High- and low-affinity Na(+)-dependent neutral L-alpha-amino acid transporters were solubilized with 0.25% octaethylene glycol dodecyl ether (C12E8) after removal of the proteins from the brush-border membrane vesicles with 2% CHAPS and 4 M urea. When the CHAPS-insoluble protein was treated with papain before its solubilization with C12E8, a substantial amount of protein was removed without any decrease of the transport activities. The solubilized transporters were reconstituted into proteoliposomes after removal of C12E8 with Bio-Beads SM2. Several parameters proved to be important for optimal reconstitution efficiency: (a) the type of detergent, and (b) the phospholipid/protein and detergent/protein ratio during reconstitution, and (c) the salt concentration during reconstitution. Reconstituted proteoliposomes showed rapid uptake of neutral L-alpha-amino acids but not imino acid, basic or acidic amino acids driven by an electrochemical potential of Na+ (out > in). The uptakes under low- and high-substrate condition were further augmented by an artificial membrane potential introduced by K+ diffusion via valinomycin (negative interior). Kinetic analysis revealed that both the brush-border membranes and the solubilized fraction involved two carrier-mediated pathways for alanine transport. The kinetic parameters were determined by curve fitting with a computer to be Kt1 = 0.28 mM (0.21 mM) and Kt2 = 43.2 mM (28.4 mM), respectively (those with brush-border membrane vesicles in parentheses). Studies on the specific activities for transport of individual amino acids under low or high substrate concentration and the cross-inhibitory effects of various amino acids on alanine uptake (low concentration) revealed that these transporters possess broad specificity for neutral L-alpha-amino acids.