|Budget Amount *help
¥6,800,000 (Direct Cost : ¥6,800,000)
Fiscal Year 1991 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1990 : ¥4,900,000 (Direct Cost : ¥4,900,000)
1)The proton and sodium ion dependences of the proline binding and transport activities of the proline carrier in Escherichia coli were investigated in detail. The binding activity in cytoplasmic membrane vesicles from a carrier over-producing strain was absolutely dependent on the presence of Na^+, but did not necessarily require protonation of the carrier. The apparent Michaelis constant of proline (Kt) of the transport, activity in the cytoplasmic membrane vesicles showed dependence on a low concentration of Na^+ and the K_<Na> value was estimated to be 25 uM. The proline transport activities in membrane vesicles and intact cells were modulated by H^+ concentration, showing the inhibitory effect of protons, pKa = 6. Based on these observations a model of the proline/Na^+ symport mechanism is proposed, inwhich a proton is postulated to be a regulatory factor of the transport activity.
2)Proline binding activity of the Escherichia coli proline/Na^+ symport carrier is inhibited by a sul
fhydryl reagent NEM. Proline and its analogs protected the carrier against the NEM-inhibition in a Na^+-dependent manner. Mutant proline carrier, CS281, CS344 and CS349, which have a serine residue in place of Cys-281, Cys-344 and Cys-349, respectively, were analyzed for cation dependent proline binding and NEM-sensitivity. Proline binding activities of CS281 and CS344 were almost completely resistant to NEM, whereas that of CS349 was not. The proline binding activity of CS344 was remarkably lower than those of the wild-type, CS281 and CS349 carriers. We propose that Cys-344 is a cysteine residue functionally involved in the high affinity binding for Na^+ and proline.
3)Proline carrier mutants with altered cation specificity were obtained by mutagenesis. Two mutants strains harboring plasmid pMOP4135 and pMOP4141 could transport proline efficiently only in the presence of an increased concentration of Na^+. The pH dependence of proline binding was also changed in these mutant carriers. DNA sequencing revealed one base alteration of G to A at nucleotides 299 and 656 in pMOP4141 and pMOP4135, respectively, which corresponded to amino acid changes from Gly-22 to glutamic acid and Cys-141 to tyrosine, respectively.
4)The nucleotide sequence of the gltS gene coding for an glutamate/Na^+ symport carrier of Escherichia coli was determined and the amino acid sequence of the carrier was deduced. The predicred glutamate carrier consists of 401 amino acids with a molecular weight of 42, 455. The predicted protein is very hydrophobic, and judging from its hydropathy profile, the protein is composed of 12 membrane-spanning segments. A conserved alignment of 5 amino acid residues Gly-42---Ala-82---Leu-87---Gly-91-Arg-92 was uncovered by sequence homology search, which commonly exists in four Na^+-symport carrier proteins, the glutamate carrier and the proline carrier of E. coli, and the glucose transporters of rabbit and human intestine. Less