Functional split and crosslinking of the membrane domain of the β subunit of proton-translocating transhydrogenase from Escherichia coli

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α subunit with the NAD(H)-binding domain I and a β subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the α and β subunits. The interface in domain II between the α and the β subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the α subunit and loops connecting the nine transmembrane helices in the β subunit. However, to investigate the organization of the nine transmembrane helices in the β subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type α subunit and the two new peptides β1 and β2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD + by NADPH, the cyclic reduction of 3-acetylpyridine-NAD + by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the α subunit was normally folded, followed by a concerted folding of β1 + β2. Cross-linking of a βS105C-βS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same β subunit has been demonstrated.

The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles in Paramecium

The vacuolar proton-ATPase (V-ATPase) is a multisubunit enzyme complex that is able to transfer protons over membranes against an electrochemical potential under ATP hydrolysis. The enzyme consists of two subcomplexes: V0, which is membrane embedded; and V1, which is cytosolic. V0 was also reported to be involved in fusion of vacuoles in yeast. We identified six genes encoding c-subunits (proteolipids) of V0 and two genes encoding F-subunits of V1 and studied the role of the V-ATPase in trafficking in Paramecium. Green fluorescent protein (GFP) fusion proteins allowed a clear subcellular localization of c- and F-subunits in the contractile vacuole complex of the osmoregulatory system and in food vacuoles. Several other organelles were also detected, in particular dense core secretory granules (trichocysts). The functional significance of the V-ATPase in Paramecium was investigated by RNA interference (RNAi), using a recently developed feeding method. A novel strategy was used to block the expression of all six c- or both F-subunits simultaneously. The V-ATPase was found to be crucial for osmoregulation, the phagocytotic pathway and the biogenesis of dense core secretory granules. No evidence was found supporting participation of V0 in membrane fusion.

Proton pump inhibitor prescribing following Endoscopic diagnosis in a district general hospital

Focal points: A systematic review of the use of proton pump inhibitors was conducted among patients undergoing diagnostic fibreoptic endoscopic examination of the upper gastrointestinal tract during the period July 2001 to February 2002 inclusive A total of 2,557 patients received a PPI following endoscopy and healing doses were prescribed to 75.3 per cent of these patients An “unknown indication” was stated as a diagnosis in 958 patients (37.5 per cent) of patients studied Although endoscopic diagnosis does not appear possible in all cases, the present study demonstrates that NICE guidance to employ the lowest appropriate dose of PPI is followed