Acta physiologica Scandinavica. Supplementum最新文献

Prostaglandin D2 (PGD2) is the major cyclooxygenase metabolite of arachidonic acid released after stimulation of mast cells. Quantification of metabolites of PGD2 can be used as an objective indices of PGD2 production and hence mast cell activation in vivo. The aim of this thesis was to investigate the feasibility of measuring the primary urinary metabolite of PGD2, 9 alpha,11 beta-PGF2 with enzyme immunoassay (EIA). Measurements of 9 alpha,11 beta-PGF2 in urine made by EIA were compared with values obtained by negative ion chemical ionisation gas chromatography-mass spectrometry (NCI GC-MS), the gold standard method. Levels of 9 alpha,11 beta-PGF2, in urine samples measured by NCI GC-MS were consistently lower than those obtained by EIA. NCI GC-MS analysis revealed the presence of two additional dinor compounds, shorter metabolites of 9 alpha,11 beta-PGF2 in the urine. One of the compounds was identical to 9 alpha,11 beta-2,3-dinor-PGF2 which was generated by beta-oxidation of 9 alpha,11 beta-PGF2 and identified by electron impact (EI GC-MS). Thus, urinary 9 alpha,11 beta-PGF2 concentrations measured by EIA represent the sum of three PGD2 metabolites. For convenience sake, the metabolites are collectively referred to as 9 alpha,11 beta-PGF2 in the subsequent studies. A 3-fold increase in the urinary excretion of 9 alpha,11 beta-PGF2 was documented after allergen-induced bronchoconstriction in nine atopic asthmatics. This challenge was considered a positive control since it is unambiguous that mast cell activation occurs during the early phase of allergen-induced airway obstruction. Histamine-induced bronchoconstriction did not result in an increase in the levels of 9 alpha,11 beta-PGF2 demonstrating that PGD2 was not formed as a consequence of the bronchoconstriction per se. Moreover, bronchial challenge with lysine-aspirin in eight aspirin-intolerant asthmatics elicited bronchoconstriction and was accompanied by a significant increase in the urinary excretion of 9 alpha,11 beta-PGF2. Challenge with a higher dose of aspirin produced an even greater increase in 9 alpha,11 beta-PGF2 levels, indicating a dose-dependent release of PGD2 during aspirin-induced bronchoconstriction. The pattern of mediator release during the early (EAR) and late asthmatic response (LAR) to allergen was investigated by subjecting twelve mild atopic asthmatics to allergen challenge. Within one hour of the maximal bronchoconstrictor response, there was a significant increase in the urinary concentrations of the mast cell markers, 9 alpha,11 beta-PGF2 and N tau-methylhistamine, urinary metabolite of histamine, and the end product of the cysteinyl-leukotrienes, leukotriene (LT)E4. Levels of all three mediators were also significantly elevated above baseline during the LAR. Urinary levels of eosinophil protein X (EPX), a marker of eosinophil activation, remained unaltered during both the EAR and LAR. Preliminary evidence suggests a diurnal variation in the urinary excre

This review summarizes some of the structural information that has been obtained on the gastric H,K ATPase. Methods such as tryptic digestion, site specific labeling and in vitro translation combine to provide a ten membrane segment model with however reservations as to the full transmembrane nature of M5 or M6. Labeling this region with the thiophilic luminal face reagent omeprazole provided cogent evidence that cys 813 but not cys 822 was labeled. On the other hand, cysteine mutagenesis provided evidence that removal of cys 813 did not affect inhibition of Rb transport by omeprazole whereas removal of cys 822 although not affecting ATPase activity abolished omeprazole inhibition of transport. A model to reconcile these data is presented where M5 and M6 although intramembranal are not transmembrane hairpin structures. Analysis of the region of alpha beta interaction by tryptic digestion and WGA chromatography to define those fragments of alpha that remain beta associated shows that leu 853 to arg 922 in the TM7-loop are a major region of association with the beta subunit. Yeast two hybrid analysis, when combined with these data and those from a chimeric construct, indicates that the sequence Q 907 to R 922 is the important element of interaction in the alpha subunit and no other extracytoplasmic domain was found to interact. Two regions of the beta subunit interact with this region of the alpha subunit between Q64 and N130 as well as A156 and R188. Apparently the beta subunit is folded around a small region of the large extracytoplasmic loop between TM7 and TM8, closer to TM8.

The oligomeric state of the proteolipid subunit of V-ATPase from Saccharomyces cerevisiae was studied using hemagglutinine (HA) epitope-tag. Like with several other highly hydrophobic proteins, the proteolipid tends to aggregate in the presence of sodium dodecyl sulfate (SDS). We observed that the oligomeric state of the proteolipid predetermined its tendency for aggregation. Recently we discovered a novel V-ATPase subunit, denoted as M16 for the mammalian enzyme and Vma10p for the yeast enzyme, that is homologous to the b subunit of the membrane sector of F-ATPases. It is assumed that the structure of Vma10p resembles that of subunit b which is basically two anti parallel helices. We mutated the VMA10 gene to change charges on the protein in helices and to introduce helix braking instead of helix forming amino acids. The functionality of the mutated VMA10 was analyzed by growing the transformed yeast cells on a YPD medium buffered at pH 7.5. Two inactive site-directed mutants we used for obtaining second-site suppressors. Mutagenesis with EMS was utilized to get an equal chance of obtaining intra and extragene second-site suppressors. To our surprise the number of colonies that grew at pH 7.5 was too large to account for mutations in V-ATPase subunits. Apparently, mutations that are situated in genes that do not encode V-ATPase subunits could reverse the phenotype of V-ATPase null mutations resulting in growth at pH 7.5. The large number of colonies that grew at pH 7.5 after EMS treatment suggest a big complex with multiple subunits as a target for mutagenesis. The observed phenomenon is very intriguing. If the responsible protein complex is identified, it may shed light on an important and novel cell biology subject.

The complex functions of the Na,K-ATPase can be described by reaction cycles based on the generally accepted "Post-Albers cycle". By appropriate experimental conditions various, partly overlapping partial reactions may be isolated which allow the investigation of specific reaction steps and their succession. From kinetic analysis rate constants and dielectric properties may be determined which characterize the function of the ion pump and allow the formulation of constraints with respect to structure-function relations. This is exemplified by two partial reactions which comprise (1) the ATP-driven Na+ transport, and (2) binding of Na+ ions to the cytoplasm sites. Equilibrium Na+ titration experiments were performed using the fluorescent dyes RH421 and FITC. Fluorescence changes upon addition of Na+ in the presence of various Mg2+ concentrations were similar and the half-saturation concentrations determined were almost identical. As RH421 responds to binding of Na+ to the neutral site whereas FITC monitors conformational changes, this result implies that electrogenic biding of the third Na+ is a trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied with a fast occlusion of the Na+ ions and followed by the conformational transition E1/E2 of the protein. Current transients produced by the Na,K-ATPase could be induced by ATP-concentration jumps using DMB-caged ATP. The dependence of the maximum of the current transients on concentration of ADP was reproduced by mathematical simulations. They fit the data well on the assumption that the rate-limiting reaction step of the Na(+)-translocation partial reaction is the conformational transition E1/E2.

Cotransporters are a major class of membrane transport proteins that are responsible for the accumulation of nutrients, neurotransmitters, osmolytes and ions in cells from bacteria to man. The energy for solute accumulation comes from the proton and/or sodium electrochemical gradients that exist across cell membranes. A major problem in biology is how transport is coupled to these electrochemical potential gradients. The primary example of this class of membrane proteins is the intestinal brush border Na+/glucose cotransporter (SGLT1), first described by Bob Crane in 1960. Over 35 members of the SGLT1 gene family have been identified in animal cells, yeast and bacteria, and all share a common core structure of 13 transmembrane (TM) helices. Electrophysiological techniques have been used to examine the function of several family members, chimeras and mutants expressed in heterologous systems such as Xenopus laevis oocytes. These have revealed that cotransporters are multi-functional proteins: they are responsible for 1). uncoupled passive Na+ transport (Na+ uniport); 2). down-hill water transport in the absence of substrate; 3). Na+/substrate cotransport; and 4). Na+/substrate/water cotransport. The sugar binding and translocation pathway is formed by 4 TM helices near the C-terminal of the protein, helices 10-13. We propose that the N-terminal domains of SGLT1 are responsible for Na+ binding and/or translocation, and that Na+/glucose cotransport results from interactions between the N- and C-terminal domains of the protein.