Fingerprint patterns from laser-induced azido photochemistry of spin-labeled photoaffinity ATP analogs in matrix-assisted laser desorption/ionization mass spectrometry.
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The photochemical reaction of azide derivatives induced by ultraviolet (UV) laser in matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is reported. A novel synthesized class of azide aromatic derivatives, spin-labeled photoaffinity non-nucleoside adenosine triphosphate (ATP) analogs which are useful probes in study of muscle contraction mechanism, is used in this investigation. In the negative ion MALDI spectra of these ATP analogs, "fingerprint" peaks corresponding to [M - 10 - 1]-, [M - 12 - 1]-, [M - 16 - 1]-, [M - 26 - 1]-, [M - 28 - 1]-, [M - 41 - 1]-, and [M - 42 - 1]- were observed with relative intensities depending on the MALDI matrix. Only the [M - 16 - 1]- is present in the similar mass spectra of the analog in which the azido group is replaced by a hydrogen. A model is suggested for the photochemical reactions of azide derivatives under UV laser irradiation. The photoreaction fingerprint information is diagnostically useful in characterization of azido compounds, especially for spin-labeled photoaffinity non-nucleoside ATP analogs. (+info)
Mapping of ATP binding regions in poly(A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases.
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We have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross-linking of recombinant bovine and yeast poly(A) polymerases to 8-azido-ATP. Mn2+ strongly stimulated this reaction due to a 50-fold lower Ki for 8-azido-ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross-linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8-azido-ATP cross-linking were mapped in different poly(A) polymerases by CNBr-cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross-link in Schizosaccharomyces pombe poly(A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167-177). Database searches with sequences surrounding the cross-link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide-binding fold among these families of enzymes. Mutations in the region of the "helical turn motif" (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide-binding helix of bovine poly(A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases. (+info)
Binding of transcription termination protein nun to nascent RNA and template DNA.
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The amino-terminal arginine-rich motif of coliphage HK022 Nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with RNA polymerase (RNAP) and blocks transcription elongation. RNA binding is inhibited by zinc (Zn2+) and stimulated by Escherichia coli NusA. To study these interactions, the Nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker. The carboxyl terminus contacted NusA and made Zn2+-dependent intramolecular contacts. When Nun was added to a paused transcription elongation complex, it cross-linked to the DNA template. Nun may arrest transcription by anchoring RNAP to DNA. (+info)
ATP binding properties of the nucleotide-binding folds of SUR1.
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Pancreatic beta cell ATP-sensitive potassium (K(ATP)) channels regulate glucose-induced insulin secretion. The activity of the K(ATP) channel, composed of SUR1 and Kir6.2 subunits, is regulated by intracellular ATP and ADP, but the molecular mechanism is not clear. To distinguish the ATP binding properties of the two nucleotide-binding folds (NBFs) of SUR1, we prepared antibodies against NBF1 and NBF2, and the tryptic fragment of SUR1 was immunoprecipitated after photoaffinity labeling with 8-azido-[(32)P]ATP. The 35-kDa fragment was strongly labeled with 5 microM 8-azido-[(32)P]ATP even in the absence of Mg(2+) and was immunoprecipitated with the antibody against NBF1. The 65-kDa fragment labeled with 100 microM 8-azido-[alpha-(32)P]ATP in the presence of Mg(2+) was immunoprecipitated with anti-NBF2 and anti-C terminus antibodies. These results indicate that NBF1 of SUR1 binds 8-azido-ATP strongly in a magnesium-independent manner and that NBF2 binds 8-azido-ATP weakly in a magnesium-dependent manner. Furthermore, the 65-kDa tryptic fragment was not photoaffinity-labeled with 8-azido-[gamma-(32)P]ATP at 37 degrees C, whereas the 35-kDa tryptic fragment was, suggesting that NBF2 of SUR1 may have ATPase activity and that NBF1 has none or little. (+info)
Glucose modulates vitamin C transport in adult human small intestinal brush border membrane vesicles.
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The uptake of L-ascorbate (vitamin C) and its oxidized form, dehydro-L-ascorbic acid (DHAA), was evaluated in brush border membrane vesicles isolated from adult human duodenum, jejunum and ileum. Ascorbate was taken up along the entire length of the small intestine with a threefold higher initial uptake rate in distal than proximal segments. Ascorbate uptake was Na(+)-dependent, potential-sensitive and saturable (K(m), 200 micromol/L), whereas DHAA transport involved facilitated diffusion (K(m), 800 micromol/L). Pharmacologic experiments were conducted to characterize further these transport mechanisms. DHAA uptake was not mediated by the fructose carrier GLUT5, the uridine transporter or the 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive anion exchanger of the apical membrane. DIDS and sulfinpyrazone, an inhibitor of the urate/lactate exchanger, both significantly reduced the initial rate of ascorbate uptake. Acidic pH inhibited ascorbate uptake, and this effect was not due to a transmembrane proton gradient. Increasing concentrations of glucose in the transport media also significantly inhibited ascorbate uptake, but no effect of glucose was seen when glucose internalization was blocked by phlorizin. Preloading the vesicles with glucose inhibited ascorbate uptake similarly, indicating that glucose interferes with the ascorbate transporter from the internal side of the membrane. The results of this study suggest that DHAA crosses the apical membrane by facilitated diffusion, whereas ascorbate transport is a Na(+)-dependent, electrogenic process modulated by glucose. (+info)
Covalent modification of the catalytic sites of the H(+)-ATPase from chloroplasts, CF(0)F(1), with 2-azido-[alpha-(32)P]ADP: modification of the catalytic site 2 (loose) and the catalytic site 3 (open) impairs multi-site, but not uni-site catalysis of both ATP synthesis and ATP hydrolysis.
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The H(+)-ATPase from chloroplasts, CF(0)F(1), was isolated and purified. The enzyme contained one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-(32)P]AD(T)P leads to a tight binding of the azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and after UV-irradiation, the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-(32)P]ADP, the covalently bound label was found exclusively at beta-Tyr-362, i.e. binding occurs only to catalytic sites. Incubation conditions with 2-azido-[alpha-(32)P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, either to catalytic site 2 or to catalytic site 3. For measurements of the degree of inhibition by covalent modification, CF(0)F(1) was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential. The rate of ATP synthesis was 120 s(-1), and the rate of ATP hydrolysis was 20 s(-1), both measured under multi-site conditions. Covalent modification of either catalytic site 2 or catalytic site 3 inhibited both ATP synthesis and ATP hydrolysis, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that modification of one catalytic site, either site 2 or site 3, is sufficient to completely block multi-site ATP synthesis and ATP hydrolysis. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with covalently modified CF(0)F(1) and with non-modified CF(0)F(1). The result was that uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent modification of either catalytic site 2 or site 3. The results indicate cooperative interactions between catalytic nucleotide binding sites during multi-site catalysis, whereas neither uni-site ATP synthesis nor uni-site ATP hydrolysis require interaction with other sites. (+info)
Sensory rhodopsin II from the haloalkaliphilic natronobacterium pharaonis: light-activated proton transfer reactions.
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In the present work the light-activated proton transfer reactions of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) and those of the channel-mutants D75N-pSRII and F86D-pSRII are investigated using flash photolysis and black lipid membrane (BLM) techniques. Whereas the photocycle of the F86D-pSRII mutant is quite similar to that of the wild-type protein, the photocycle of D75N-pSRII consists of only two intermediates. The addition of external proton donors such as azide, or in the case of F86D-pSRII, imidazole, accelerates the reprotonation of the Schiff base, but not the turnover. The electrical measurements prove that pSRII and F86D-pSRII can function as outwardly directed proton pumps, whereas the mutation in the extracellular channel (D75N-pSRII) leads to an inwardly directed transient current. The almost negligible size of the photostationary current is explained by the long-lasting photocycle of about a second. Although the M decay, but not the photocycle turnover, of pSRII and F86D-pSRII is accelerated by the addition of azide, the photostationary current is considerably increased. It is discussed that in a two-photon process a late intermediate (N- and/or O-like species) is photoconverted back to the original resting state; thereby the long photocycle is cut short, giving rise to the large increase of the photostationary current. The results presented in this work indicate that the function to generate ion gradients across membranes is a general property of archaeal rhodopsins. (+info)
Lateral transport on cell membranes: mobility of concanavalin A receptors on myoblasts.
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We report measurements of the lateral mobility of fluorescent labeled concanavalin A receptor complexes on the plasma membrane of cultured myoblasts of rat. Transport rates were measured by observing the recovery of fluorescence in a small region of the cell surface initially photobleached irreversibly by an intense, focused laser light pulse. Under different conditions we measured effective diffusion coefficients of the receptor complexes in the range 8 x 10(-12) less than D less than 3 x 10(-11) cm2/sec which is two orders of magnitude lower than we found for a fluorescent lipid probe, D approximately (8 +/- 3) x 10(-9) cm2/sec. This large difference and the presence of apparently immobile concanavalin A receptors suggests that factors beyond the fluoidity of the phospholipid bilayer membrane matrix control the rate of lateral transport of the complexes. Effective mobilities of the complexes decrease with increases in the valence, dose, and occupation time of the lectin on the membrane. These properties imply an aggregation of the lectin-receptor complexes. Mobilities are not influenced by azide, colchicine or preincubation at low temperature. Cytochalasin B and low temperatures, during the time of measurement, decrease the lateral transport rate. (+info)