The Na+/H+ exchanger is an integral membrane protein that is universally distribute in mammalian tissues and is responsible for intracellular pH regulation. Several isoforms of the Na+/H+ exchanger exist (NHE-1-NHE-4). The first that was cloned is the amiloride sensitive isoform (NHE-1). Using a fragment of the rabbit cardiac Na+/H+ exchanger cDNA clone we isolated and sequenced Na+/H+ exchanger cDNA from a human heart coding for the complete human Na+/H+ exchanger (NHE-1 isoform). Two overlapping cDNA clones were obtained, giving a combined sequence that contained both 3' and 5' untranslated regions. The 5' and 3' untranslated regions proved to be highly homologous to human sequences described earlier but contained some variations that could affect the mRNA stability and/or the efficiency of translation of the Na+/H+ exchanger. Northern blot analysis and reverse transcriptase polymerase chain reaction confirmed the presence of the 5 kb NHE-1 message in primary cultures of isolated myocytes.

The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creating a library of variants with a stepwise decrease in the midpoint potential of the heme from the wild-type value of +20 mV down to −80 mV. This difference in midpoint potential is enough to alter the reactivity of the heme towards succinate and thus its redox state under turnover conditions. Our results show both the steady state succinate oxidase and fumarate reductase catalytic activity of the enzyme are not a function of the redox potential of the heme. As well, lower heme potential did not cause an increase in the rate of superoxide production both in vitro and in vivo . The electron paramagnetic resonance (EPR) spectrum of the heme in the wild-type enzyme is a combination of two distinct signals. We link EPR spectra to structure, showing that one of the signals likely arises from an out-of-plane distortion of the heme, a saddled conformation, while the second signal originates from a more planar orientation of the porphyrin ring.

binding channel shows conservation of positively charged residues, which are possibly involved in complex formation with the H-protein. The structure of I c consists of a major -type domain and an Nterminal helical segment. I c has two CPY-binding sites: the N-terminal inhibitory reactive site and the secondary CPY-binding site which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. I c also has the ligand-binding site, the putative binding site of the polar head group of phospholipid, which is conserved among PEBPs and accommodates a sulfate ion in the crystal structure.Along with the complex structure of I c , its mutational analyses for inhibitory activity and binding to CPY demonstrate that the Nterminal inhibitory reactive site is essential for the complex formation with CPY as well as enzyme inhibition and that the I c binding to CPY forms a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of Ic toward CPY gives insights into not only the inhibitory mechanism of PEBPs toward serine proteinases but also the biological functions of I c belonging to the PEBP family.[1] Mima J., Hayashida M., Fujii T., Hata Y., Hayashi R., Ueda M. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine in the biosynthesis of spermidine. PAPT of Helicobacter pylori (HpPAPT) is encoded by the speE gene. HpPAPT has less than 20% of sequence identity with other PAPTs, even containing no signature sequence. The threedimensional structure of HpPAPT has been determined by multiwavelength anomalous dispersion (MAD) in this study. HpPAPT consists of an N-terminal beta-stranded domain and a Cterminal Rossmann-like domain, with a binding pocket between two domains. The oligomerization of HpPAPT is mostly made by the Nterminal domain and sensitive to the pH values of buffer. Our structure illustrates that HpPAPT has a distinctive binding pocket with a bigger space, a unique electrostatic potential surface of less acidity, and numerous unconserved residues. Due to the lack of the gatekeeping loop, HpPAPT may need to perform a significant conformation change to accommodate the ligand binding. When the tryptophan synthase -and 2 -subunits combine to form the 2 2 complex, the enzymatic activity of each subunit is stimulated by one to two orders of magnitude. In order to elucidate the structural basis of this mutual activation, it is necessary to determine the structures of the -and -subunits alone and together with the 2 2 complex. The crystal structures of the tryptophan synthase 2 2 complex from S. typhimurium (St 2 2 ) has been reported. Therefore, we determined the crystal structure of the tryptophan synthasesubunit alone from E. coli (Ec ) at 2.3Å resolution. The biggest difference between the structures of the Ec and the -subunit in the St 2 2 (St ) was as follows. The helix-2' in the St including an ...