Cross-linking of two beta subunits in the closed conformation in F1-ATPase. (1/2265)

In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP.  (+info)

Activation of Go-proteins by membrane depolarization traced by in situ photoaffinity labeling of galphao-proteins with [alpha32P]GTP-azidoanilide. (2/2265)

Evidence for depolarization-induced activation of G-proteins in membranes of rat brain synaptoneurosomes has been previously reported (Cohen-Armon, M., and Sokolovsky, M. (1991) J. Biol. Chem. 266, 2595-2605; Cohen-Armon, M., and Sokolovsky, M. (1993) J. Biol. Chem. 268, 9824-9838). In the present work we identify the activated G-proteins as Go-proteins by tracing their depolarization-induced in situ photoaffinity labeling with [alpha32P]GTP-azidoanilide (GTPAA). Labeled GTPAA was introduced into transiently permeabilized rat brain-stem synaptoneurosomes. The resealed synaptoneurosomes, while being UV-irradiated, were depolarized. Relative to synaptoneurosomes at resting potential, the covalent binding of [alpha32P]GTPAA to Galphao1- and Galphao3-proteins, but not to Galphao2- isoforms, was enhanced by 5- to 7-fold in depolarized synaptoneurosomes, thereby implying an accelerated exchange of GDP for [alpha32P]GTPAA. Their depolarization-induced photoaffinity labeling was independent of stimulation of Go-protein-coupled receptors and could be reversed by membrane repolarization, thus excluding induction by transmitters release. It was, however, dependent on depolarization-induced activation of the voltage-gated sodium channels (VGSC), regardless of Na+ current. The alpha subunit of VGSC was cross-linked and co-immunoprecipitated with Galphao-proteins in depolarized brain-stem and cortical synaptoneurosomes. VGSC alpha subunit most efficiently cross-linked with guanosine 5'-O-2-thiodiphosphate-bound rather than to guanosine 5'-O-(3-thiotriphosphate)-bound Galphao-proteins in isolated synaptoneurosomal membranes. These findings support a possible involvement of VGSC in depolarization-induced activation of Go-proteins.  (+info)

Identification of a GABAB receptor subunit, gb2, required for functional GABAB receptor activity. (3/2265)

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant gamma-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situ hybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+ channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.  (+info)

Resolution of the paradox of red cell shape changes in low and high pH. (4/2265)

The molecular basis of cell shape regulation in acidic pH was investigated in human erythrocytes. Intact erythrocytes maintain normal shape in the cell pH range 6.3-7.9, but invaginate at lower pH values. However, consistent with predicted pH-dependent changes in the erythrocyte membrane skeleton, isolated erythrocyte membranes evaginate in acidic pH. Moreover, intact cells evaginate at pH greater than 7.9, but isolated membranes invaginate in this condition. Labeling with the hydrophobic, photoactivatable probe 5-[125I]iodonaphthyl-1-azide demonstrated pH-dependent hydrophobic insertion of an amphitropic protein into membranes of intact cells but not into isolated membranes. Based on molecular weight and on reconstitution experiments using stripped inside-out vesicles, the most likely candidate for the variably labeled protein is glyceraldehyde-3-phosphate dehydrogenase. Resealing of isolated membranes reconstituted both the shape changes and the hydrophobic labeling profile seen in intact cells. This observation appears to resolve the paradox of the contradictory pH dependence of shape changes of intact cells and isolated membranes. In intact erythrocytes, the demonstrated protein-membrane interaction would oppose pH-dependent shape effects of the spectrin membrane skeleton, stabilizing cell shape in moderately abnormal pH. Stabilization of erythrocyte shape in moderately acidic pH may prevent inappropriate red cell destruction in the spleen.  (+info)

Analysis of the membrane-interacting domains of myelin basic protein by hydrophobic photolabeling. (5/2265)

Myelin basic protein is a water soluble membrane protein which interacts with acidic lipids through some type of hydrophobic interaction in addition to electrostatic interactions. Here we show that it can be labeled from within the lipid bilayer when bound to acidic lipids with the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID) and by two lipid photolabels. The latter included one with the reactive group near the apolar/polar interface and one with the reactive group linked to an acyl chain to position it deeper in the bilayer. The regions of the protein which interact hydrophobically with lipid to the greatest extent were determined by cleaving the TID-labeled myelin basic protein (MBP) with cathepsin D into peptides 1-43, 44-89, and 90-170. All three peptides from lipid-bound protein were labeled much more than peptides from the protein labeled in solution. However, the peptide labeling pattern was similar for both environments. The two peptides in the N-terminal half were labeled similarly and about twice as much as the C-terminal peptide indicating that the N-terminal half interacts hydrophobically with lipid more than the C-terminal half. MBP can be modified post-translationally in vivo, including by deamidation, which may alter its interactions with lipid. However, deamidation had no effect on the TID labeling of MBP or on the labeling pattern of the cathepsin D peptides. The site of deamidation has been reported to be in the C-terminal half, and its lack of effect on hydrophobic interactions of MBP with lipid are consistent with the conclusion that the N-terminal half interacts hydrophobically more than the C-terminal half. Since other studies of the interaction of isolated N-terminal and C-terminal peptides with lipid also indicate that the N-terminal half interacts hydrophobically with lipid more than the C-terminal half, these results from photolabeling of the intact protein suggest that the N-terminal half of the intact protein interacts with lipid in a similar way as the isolated peptide. The similar behavior of the intact protein to that of its isolated peptides suggests that when the purified protein binds to acidic lipids, it is in a conformation which allows both halves of the protein to interact independently with the lipid bilayer. That is, it does not form a hydrophobic domain made up from different parts of the protein.  (+info)

Selective inhibition of MDR1 P-glycoprotein-mediated transport by the acridone carboxamide derivative GG918. (6/2265)

The acridone carboxamide derivative GG918 (N-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-pheny l}-9,10dihydro-5-methoxy-9-oxo-4-acridine carboxamide) is a potent inhibitor of MDR1 P-glycoprotein-mediated multidrug resistance. Direct measurements of ATP-dependent MDR1 P-glycoprotein-mediated transport in plasma membrane vesicles from human and rat hepatocyte canalicular membranes indicated 50% inhibition at GG918 concentrations between 8 nM and 80 nM using N-pentyl-[3H]quinidinium, ['4C]doxorubicin and [3H]daunorubicin as substrates. The inhibition constant K for GG918 was 35 nM in rat hepatocyte canalicular membrane vesicles with [3H]daunorubicin as the substrate. Photoaffinity labelling of canalicular and recombinant rat Mdr1b P-glycoprotein by [3H]azidopine was suppressed by 10 muM and 40 muM GG918. The high selectivity of GG918-induced inhibition was demonstrated in canalicular membrane vesicles and by analysis of the hepatobiliary elimination in rats using [3H]daunorubicin, [3H]taurocholate and [3H]cysteinyl leukotrienes as substrates for three distinct ATP-dependent export pumps. Almost complete inhibition of [3H]daunorubicin transport was observed at GG918 concentrations that did not affect the other hepatocyte canalicular export pumps. The high potency and selectivity of GG918 for the inhibition of human MDR1 and rat Mdr1b P-glycoprotein may serve to interfere with this type of multidrug resistance and provides a tool for studies on the function of these ATP-dependent transport proteins.  (+info)

Influence of bioenergetic stress on heat shock protein gene expression in nucleated red blood cells of fish. (7/2265)

The physiological and biochemical signals that induce stress protein (HSP) synthesis remain conjectural. In this study, we used the nucleated red blood cells from rainbow trout, Oncorhynchus mykiss, to address the interaction between energy status and HSP gene expression. Heat shock (25 degrees C) did not significantly affect ATP levels but resulted in an increase in HSP70 mRNA. Hypoxia alone did not induce HSP transcription in these cells despite a significant depression in ATP. Inhibition of oxidative phosphorylation with azide, in the absence of thermal stress, decreased ATP by 56% and increased lactate production by 62% but did not induce HSP gene transcription. Inhibition of oxidative phosphorylation and glycolysis with azide and iodoacetic acid respectively, decreased ATP by 79% and prevented lactate production, but did not induce either HSP70 or HSP30 gene transcription in these cells. This study demonstrates that a reduction in the cellular energy status will not induce stress protein gene transcription in rainbow trout red blood cells and may, in fact, limit induction during extreme metabolic inhibition.  (+info)

Influence of different types of effectors on the kinetic parameters of suicide inactivation of catalase by hydrogen peroxide. (8/2265)

The effects of cyanide and azide ions (class A), sodium-n-dodecyl sulphate (SDS) and 2-mercaptoethanol (class B), 3-aminotriazole (class C) and NADPH (class D) on the initial activity (ai), inactivation rate constant (ki) and the partition ratio (r) of bovine liver catalase reaction with its suicide substrate, hydrogen peroxide, were studied in 50 mM sodium phosphate buffer, pH 7.0 at 27 degrees C. The above kinetic parameters were determined by processing the progress curve data. In class A, which contains fast and reversible inhibitors of catalase, a proportional decrease in ai and ki was observed by inhibitors, so that the r remained constant. In class B, which contains slow and irreversible inactivators, a decrease in ai and constancy of ki and r were observed when catalase was incubated in the presence of such inactivators for a determined time. In class C, containing effector which can combine with intermediate compound I, ai was relatively unchanged but an increase in ki and a decrease in r were observed. In class D, containing effector which reduces compound I to ferricatalase, ai was not affected significantly but some decrease in ki was detected which was linked with an increase in r. These results demonstrate that different classes of effectors affect the determined kinetic parameters of catalase in various ways. Thus, determination of such parameters by simple kinetic experiments can be carried out for classification of the agents which have an effect on the kinetics of catalase.  (+info)