Apo-azurin folds via an intermediate that resembles the molten-globule

The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates. Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A). The guanidinium-induced unfolding free energies (DeltaG(U-N)(H2O)) of the C112S and C112A mutants were measured to 36.8 +/- 1 kJ mole(-1) and 26.1 +/- 1 kJ mole(-1), respectively, and the m-value of the transition to 23.5 +/- 0.7 kJ mole(-1) M(-1). The difference in folding free energy (DeltaDeltaG(U-N)(H2O)) is largely attributed to the intramolecular hydrogen bonding properties of the serine Ogamma in the C112S mutant, which is lacking in the C112A structure. Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed Delta DeltaG(U-N)(H2O). This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state. In both mutants we also noted a burst-phase intermediate. Because this intermediate was capable of binding 1-anilinonaphtalene-8-sulfonate (ANS), as were an acid-induced species at pH 2.6, we ascribe it molten globule-like status. However, despite the presence of an intermediate, the folding of apo-azurin C112S is well approximated by a two-state kinetic mechanism.     

Reactivation of formyl peptide receptors triggers the neutrophil NADPH-oxidase but not a transient rise in intracellular calcium

In neutrophils, coupling of chemoattractants to their cell surface receptor at low temperature (相似文献   

Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme

Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called "occluded" intermediary state of the reaction cycle of the intact enzyme. Despite a K(d) in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP(+) is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2'-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD(+) by NADH, indicating that 3-acetylpyridine-NAD(+) and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2'-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.     

Irreversible binding and activity control of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii at an anionic lipid bilayer surface

1,2-Diacylglycerol 3-glucosyltransferase is associated with the membrane surface catalyzing the synthesis of the major nonbilayer-prone lipid alpha-monoglucosyl diacylglycerol (MGlcDAG) from 1,2-DAG in the cell wall-less Acholeplasma laidlawii. Phosphatidylglycerol (PG), but not neutral or zwitterionic lipids, seems to be essential for an active conformation and function of the enzyme. Surface plasmon resonance analysis was employed to study association of the enzyme with lipid bilayers. Binding kinetics could be well fitted only to a two-state model, implying also a (second) conformational step. The enzyme bound less efficiently to liposomes containing only zwitterionic lipids, whereas increasing molar fractions of the anionic PG or cardiolipin (CL) strongly promoted binding by improved association (k(a1)), and especially a decreased rate of return (k(d2)) from the second state. This yielded a very low overall dissociation constant (K(D)), corresponding to an essentially irreversible membrane association. Both liposome binding and consecutive activity of the enzyme correlated with the PG concentration. The importance of the electrostatic interactions with anionic lipids was shown by quenching of both binding and activity with increasing NaCl concentrations, and corroborated in vivo for an active enzyme-green fluorescent protein hybrid in Escherichia coli. Nonbilayer-prone lipids substantially enhanced enzyme-liposome binding by promoting a changed conformation (decreasing k(d2)), similar to the anionic lipids, indicating the importance of hydrophobic interactions and a curvature packing stress. For CL and the nonbilayer lipids, effects on enzyme binding and consecutive activity were not correlated, suggesting a separate lipid control of activity. Similar features were recorded with polylysine (cationic) and polyglutamate (anionic) peptides present, but here probably dependent on the selective charge interactions with the enzyme N- and C-domains, respectively. A lipid-dependent conformational change and PG association of the enzyme were verified by circular dichroism, intrinsic tryptophan, and pyrene-probe fluorescence analyses, respectively. It is concluded that an electrostatic association of the enzyme with the membrane surface is accompanied by hydrophobic interactions and a conformational change. However, specific lipids, the curvature packing stress, and proteins or small molecules bound to the enzyme can modulate the activity of the bound A. laidlawii MGlcDAG synthase.     

Roles of individual amino acids in helix 14 of the membrane domain of proton-translocating transhydrogenase from Escherichia coli as deduced from cysteine mutagenesis

Proton-translocating nicotinamide nucleotide transhydrogenase is a membrane-bound protein composed of three domains: the hydrophilic NAD(H)-binding domain, the hydrophilic NADP(H)-binding domain, and the hydrophobic membrane domain. The latter harbors the proton channel. In Escherichia coli transhydrogenase, the membrane domain is composed of 13 transmembrane alpha helices, of which especially helices 13 and 14 contain conserved residues. To characterize the roles of the individual residues betaLeu240 to betaSer260 in helix 14, these were mutated as single mutants to cysteines in the cysteine-free background, and in the case of betaGly245, betaGly249, and betaGly252, also to leucines. In addition to the residues forming the helix, residues betaAsn238 and betaAsp239 were also mutated. Except for betaI242C, all mutants were normally expressed, purified, and characterized with respect to, e.g., catalytic activities and proton pumping. The results show that mutation of the conserved glycines betaGly245, betaGly249, and betaGly252, located on the same face of the helix, led to a general inhibition of all activities, especially in the case of betaGly252, suggesting a role of these glycines in helix-helix interactions. In contrast, mutation of the conserved serines betaSer250, betaSer251, and betaSer256 led to enhanced activities of all reactions, including the cyclic reaction which was mediated by bound NADP(H). Mutation of the remaining residues resulted in intermediate inhibitory effects. The results strongly support an important regulatory role of the membrane domain on the NADP(H)-binding site.     

Separation of native and latent forms of human antithrombin by hydrophobic interaction high-performance liquid chromatography

Hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) was utilized for the separation of native human antithrombin (AT) and a partially denaturated form of AT, known as the latent form (L-AT). The AT used in this study is commercially available (Atenativ, Pharmacia & Upjohn, Sweden) and contains albumin as the main stabilizer. The AT was reconstituted and heat treated in order to generate L-AT. This latent form of AT has been shown to exhibit a strong antiangiogenic activity and also to suppress tumor growth. The HPLC system included a TSK Phenyl 5PW column and a segmented gradient, 4.5-0 mol/L sodium chloride. Antithrombin was eluted at about 13 min, and L-AT, at 30 min, corresponding to about 4.2 and 1.6 mol/L sodium chloride, respectively. A reference sample gave 42% L-AT when analyzed by the HIC method and 41% L-AT when analyzed by the heparin affinity chromatography method. The resolution between AT and L-AT was higher with the HIC method than with the heparin affinity method. Incubation of Atenativ at 45 degrees C for 15 h gave about 18% L-AT and was shown by native polyacrylamide gel electrophoresis to contain only monomeric AT. A good resolution between AT and L-AT, but not between albumin and L-AT, was also achieved by a linear gradient of 2-0 mol/L ammonium sulfate, in 25 mmol/L Tris/HCl, pH 8.0.     

Dose-response relationship for parathyroid adenoma after exposure to ionizing radiation in infancy

Several authors have suggested that there is an excess risk of hyperparathyroidism, adenomas or hyperplasia after exposure to ionizing radiation. There is still, however, some uncertainty about this association, because these diseases are often asymptomatic and escape clinical detection if not specially searched for. This study is based on a pooled Swedish cohort of 27,925 persons with skin hemangiomas. The majority received radiation treatment in infancy between 1920 and 1965 in Stockholm and Gothenburg. The mean age at treatment was 6 months and the median thyroid dose was 0.20 Gy (range 0-28.5 Gy). Record linkage with the Swedish Cancer Register for the period 1958-1997 gave 43 cases of parathyroid adenoma in the cohort. Analyses of excess relative risk (ERR) models were performed using Poisson regression methods. Clinical records were scrutinized to determine if the childhood radiation exposure was known (biased cases) at the time of diagnosis. Seven of the cases of parathyroid adenoma were classified as biased cases. The standardized incidence ratio (SIR) was 2.10 (95% confidence interval 1.52-2.82) when all cases were included and 1.76 (95% CI 1.23-2.43) with the biased cases excluded. A linear dose-response model with stratification for sex fitted the data best. The ERR per gray was 3.84 (95% CI 1.56-8.99) with all cases and 1.56 (95% CI 0.36-4.45) with the biased cases excluded. There was a significant difference in the ERR per gray between the two subcohorts, probably because of different diagnostic activity in the regions. Our findings confirm that there is a dose-response relationship for radiation-induced parathyroid adenomas.     

Activation of the neutrophil nicotinamide adenine dinucleotide phosphate oxidase by galectin-1

Galectins are a group of lactose-binding proteins widely distributed in nature. Twelve mammalian galectins have so far been identified, but their functions are to a large extent unknown. In this work we study galectin-1 in its interaction with human neutrophils, with regard to both cell surface binding and activation of the superoxide-producing NADPH-oxidase. We show that galectin-1 is able to activate the neutrophil NADPH-oxidase, provided that the cells have been primed by extravasation from the blood into the tissue, an activation pattern that is similar to that of galectin-3. Using in vitro priming protocols, the galectin-1 responsiveness was found to correlate to granule mobilization and galectin-1 binding to the cells, suggesting the presence of granule-localized receptors that are up-regulated to the cell surface upon priming. By galectin-1 overlay of fractionated neutrophils we identified potential galectin-1 receptor candidates localized in the membranes of the secretory vesicle and gelatinase granules. The binding of galectin-1 and galectin-3 to neutrophil proteins was compared, as were the dose dependencies for activation by the two lectins. The results suggest that, although similarities are found between the two galectins, they appear to activate the NADPH-oxidase using different receptors. In conclusion, galectin-1 appears to have proinflammatory functions, mediated through activation of the neutrophil respiratory burst.     

Overlapping and specific patterns of GDNF,c-ret and GFR alpha mRNA expression in the developing chicken retina

GDNF and the GDNF receptors, c-Ret, GFR alpha 1 and 2 mRNA is expressed in the developing chicken retina. GDNF labelling was mainly found in embryonic day 4-5 retina but weak labelling could also be found over scattered retinal cells at later stages. c-ret labelling was found over ganglion cells, amacrine and horizontal cells; the preferred GDNF receptor (GFR alpha 1) over amacrine and horizontal cells; and the less preferred GDNF receptor (GFR alpha 2) over ganglion cells, amacrine cells and photoreceptors.     

Enantiomeric separations of amino alcohols by packed-column SFC on Hypercarb with L-(+)-tartaric acid as chiral selector

The use of L-(+)-tartaric acid as a chiral mobile phase additive (CMPA) has been investigated in a packed-column SFC system. The CMPA, carbon dioxide, and methanol, containing a high concentration of aliphatic amine additive, were used as the mobile phase and Hypercarb as support [Gyllenhaal O., Karlsson A., SFC of metoprolol and other amino alcohols on Hypercarb (in preparation)]. Good enantioselectivities were obtained for tertiary amine homologues of 2-amino alcohols, used as beta-adrenoreceptor-blocking drugs. Moderate selectivities were observed for aromatic compounds having a second substituent in the ortho-position. The overall retention was influenced by the aromaticity of the analytes as well as the presence of free electron pairs in the molecule. Increased concentrations of CMPA gave higher retention and also increased the enantioselectivity. The practical utility of this present enantioselective system was demonstrated on one batch of (S)-metoprolol that was N-methylated with methyl iodide. The enantiomeric separation was accomplished within 10 min.