Role of sugar residues for recombinant gastric H+,K+-ATPase

Contains fulltext : 24837___.PDF (publisher's version ) (Open Access

Ethanol stimulates expression of functional H+,K+-ATPase in SF9 cells

Contains fulltext : 21983___.PDF (publisher's version ) (Open Access

The use of primary care services by drug users attending a HIV prevention unit.

Intravenous drug using clients of a Dublin HIV Prevention Unit were interviewed about their use of general practitioner services. Sixty eight percent of clients had visited a GP within the previous year and 48% were registered with a GP under the General Medical Services (GMS) Scheme. Of 161 interviewees 60 were being treated with a fixed dose regimen of methadone at the Prevention Unit; this group was far less likely to visit a GP with a drug related problem and far less likely to have received methadone from a GP. Members of the GMS were much more likely than non members to have visited a GP and also more likely to have attended a specific GP for all problems besides methadone treatment. Methadone treatment and medical cover within the GMS Scheme emerged as important influences on the behaviour of clients with respect to general practitioners

Role of negatively charged residues in the fifth and sixth transmembrane domains of the catalytic subunit of gastric H+,K+-ATPase

Contains fulltext : 28302.pdf (publisher's version ) (Open Access

Biochemical characterization of sporadic/familial hemiplegic migraine mutations

Contains fulltext : 133904.pdf (publisher's version ) (Closed access)Sporadic hemiplegic migraine type 2 (SHM2) and familial hemiplegic migraine type 2 (FHM2) are rare forms of hemiplegic migraine caused by mutations in the Na(+),K(+)-ATPase alpha2 gene. Today, more than 70 different mutations have been linked to SHM2/FHM2, randomly dispersed over the gene. For many of these mutations, functional studies have not been performed. Here, we report the functional characterization of nine SHM2/FHM2 linked mutants that were produced in Spodoptera frugiperda (Sf)9 insect cells. We determined ouabain binding characteristics, apparent Na(+) and K(+) affinities, and maximum ATPase activity. Whereas membranes containing T345A, R834Q or R879W possessed ATPase activity significantly higher than control membranes, P796S, M829R, R834X, del 935-940 ins Ile, R937P and D999H membranes showed significant loss of ATPase activity compared to wild type enzyme. Further analysis revealed that T345A and R879W showed no changes for any of the parameters tested, whereas mutant R834Q possessed significantly decreased Na(+) and increased K(+) apparent affinities as well as decreased ATPase activity and ouabain binding. We hypothesize that the majority of the mutations studied here influence interdomain interactions by affecting formation of hydrogen bond networks or interference with the C-terminal ion pathway necessary for catalytic activity of Na(+),K(+)-ATPase, resulting in decreased functionality of astrocytes at the synaptic cleft expressing these mutants

Extracellular acidification induces ROS- and mPTP-mediated death in HEK293 cells

Contains fulltext : 184340.pdf (publisher's version ) (Open Access)The extracellular pH (pHe) is a key determinant of the cellular (micro)environment and needs to be maintained within strict boundaries to allow normal cell function. Here we used HEK293 cells to study the effects of pHe acidification (24h), induced by mitochondrial inhibitors (rotenone, antimycin A) and/or extracellular HCl addition. Lowering pHe from 7.2 to 5.8 reduced cell viability by 70% and was paralleled by a decrease in cytosolic pH (pHc), hyperpolarization of the mitochondrial membrane potential (Deltapsi), increased levels of hydroethidine-oxidizing ROS and stimulation of protein carbonylation. Co-treatment with the antioxidant alpha-tocopherol, the mitochondrial permeability transition pore (mPTP) desensitizer cyclosporin A and Necrostatin-1, a combined inhibitor of Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Indoleamine 2,3-dioxygenase (IDO), prevented acidification-induced cell death. In contrast, the caspase inhibitor zVAD.fmk and the ferroptosis inhibitor Ferrostatin-1 were ineffective. We conclude that extracellular acidification induces necroptotic cell death in HEK293 cells and that the latter involves intracellular acidification, mitochondrial functional impairment, increased ROS levels, mPTP opening and protein carbonylation. These findings suggest that acidosis of the extracellular environment (as observed in mitochondrial disorders, ischemia, acute inflammation and cancer) can induce cell death via a ROS- and mPTP opening-mediated pathogenic mechanism

The E1/E2-preference of gastric H,K-ATPase mutants.

Item does not contain fulltextGastric H,K-ATPase has, in the absence of ATP and added ions, a preference for the E(2) conformation. Mutations in the cation-binding pocket often result in a preference for the E(1)-conformation. This can be paralleled by the occurrence of K(+)-independent ATPase activity. These two phenomena could be separated by combined mutagenesis of several residues in and around the cation-binding pocket. Models of the three-dimensional structure of H,K-ATPase visualize the relationship between the E(1)/E(2) preference and the structure

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme.

Contains fulltext : 51463.pdf (publisher's version ) (Closed access)The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the beta(1) subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH(4)(+) as activating cation. The enzyme was also stimulated by Na(+), but in contrast to the rat enzyme, hardly by K(+). SCH 28080 inhibited the NH(4)(+)-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K(+)], and [Na(+)] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E(1)P to E(2)P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H(+)/Na(+) and/or H(+)/NH(4)(+) transport but not in Na(+)/K(+) transport

The beta-subunits of Na+,K+-ATPase and gastric H+,K+-ATPase have a high preference for their own alpha-subunit and affect the K+ affinity of these enzymes

Contains fulltext : 216410.pdf (Publisher’s version ) (Open Access