Test reactions for a stopped-flow apparatus

Tonomura, Ben’ichiro; Nakatani, Hiroshi; Ohnishi, Masatake; Yamaguchi-Ito, Junko; Kitano, Hiromi

doi:10.1016/0003-2697(78)90054-4

Cited by 276 publications

(187 citation statements)

References 12 publications

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“…Depending on urea concentration, the mixing dead time of this instrument was 0.8 -1.1 ms. The dead times were determined by a standard protocol for the reduction of 2,6-dichlorophenolindophenol by L-ascorbate (12). Each protein folding/unfolding reaction was measured at least eight times.…”

Section: Methodsmentioning

confidence: 99%

Kinetic Intermediate in the Folding of Human Prion Protein

Apetri

Surewicz

2002

Journal of Biological Chemistry

118

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Transmissible spongiform encephalopathies, or prion diseases, are a group of intriguing neurodegenerative disorders that include scrapie in sheep and goat, bovine spongiform encephalopathy in cattle, chronic waste disorder in deer and elk, and Creutzfeldt-Jacob disease in humans (1). These diseases are associated with conformational conversion of a normal prion protein, PrP C , 1 into a misfolded isoform, PrP Sc . According to the "protein-only" hypothesis (1), the transmission of the disease does not require nucleic acids, and the prion pathogen consists solely of PrP Sc . The latter conformer is believed to act as an infectious agent by catalyzing self-propagating conversion of endogenous PrP C into the pathogenic PrP Sc isoform. Although the ultimate proof for the protein-only hypothesis is still missing (2), the central role of prion protein in the pathogenesis of the disease is documented by a wealth of biochemical and genetic data (1).Cellular human prion protein, PrP C , is a glycoprotein that contains a disulfide bond, is N-glycosylated, and is attached to the plasma membrane by a glycosyl phosphatidylinositol anchor (1). NMR studies have shown that the recombinant prion protein in solution consists of a largely unordered N-terminal region and the folded C-terminal domain encompassing three ␣-helices and a short ␤-sheet (3-5). Recent crystallographic studies have captured the C-terminal part of PrP as a domainswapped dimer (6). This dimer, which is only marginally populated in solution and selectively crystallizes, is also ␣-helical, and its overall fold is similar to that of the monomer. The PrP C 3 PrP Sc conversion, which appears to occur without any covalent modifications, is believed to involve a major conformational change in the prion protein (1). In contrast to a largely ␣-helical PrP C , the pathogenic PrP Sc isoform is characterized by a high content of ␤-sheet structure (7, 8), partial resistance to proteolytic digestion, and a propensity to aggregate into insoluble amyloid-like fibrils and plaques (1). Despite intensive research, the molecular mechanism underlying the PrP C 3 PrP Sc conversion and prion propagation remains enigmatic. The key to understanding this mechanism is to elucidate the folding pathway(s) of the prion protein. Here we present evidence for a population of a kinetic intermediate during the folding of the human prion protein. This species may represent a crucial monomeric precursor on the pathway of prion protein conversion to the pathogenic PrP Sc isoform. EXPERIMENTAL PROCEDURESProtein Expression and Purification-The plasmid encoding huPrP-(90 -231) with an N-terminal linker containing a His 6 tail and a thrombin cleavage site was described previously (9, 10). The Y218W and F175W variants were constructed by site-directed mutagenesis using appropriate primers and the QuikChange kit (Stratagene). The proteins were expressed, cleaved with thrombin, and purified according to the previously described protocol (9).Equilibrium Unfolding in Urea-The equilibrium unfolding curves...

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Section: Methodsmentioning

confidence: 99%

Kinetic Intermediate in the Folding of Human Prion Protein

Apetri

Surewicz

2002

Journal of Biological Chemistry

118

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“…The instrument dead time, determined at 8°C and 24°C from the reaction of L-ascorbic acid with 2,6-dichlorophenolindophenol, as described [15], was 1.31 ms. The dependence of the pseudo first-order rate constant for reduction of the 2,6-dichlorophenolindophenol on the ascorbic acid concentration deviated from linearity when k app exceeded Ϸ700 s Ϫ1 (not shown), and this is considered to represent the performance limit of the instrument.…”

Section: Methodsmentioning

confidence: 99%

Stopped‐flow kinetics of hydride transfer between nucleotides by recombinant domains of proton‐translocating transhydrogenase

Venning¹,

Bizouarn²,

Cotton³

et al. 1998

European Journal of Biochemistry

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Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD ϩ /NADH, and domain III for NADP ϩ /NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped-flow spectrophotometer, there was a biphasic burst of hydride transfer from NADPH to the NAD ϩ analogue, acetylpyridine adenine dinucleotide (AcPdAD ϩ ). The burst, corresponding to a single turnover of domain III, precedes the onset of steady state, which is limited by very slow release of product NADP ϩ (kϷ0.03 s Ϫ1 ). Phase A of the burst (kϷ600 s Ϫ1 ) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (kϷ10Ϫ50 s Ϫ1 ), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I:III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I:III complex, are directly coupled to proton binding or Keywords : transhydrogenase; stopped flow; proton translocation; recombinant protein; membrane protein.Transhydrogenase couples the transfer of reducing equivalents (hydride ion equivalents) between NAD(H) and NADP(H) to the translocation of protons across a membrane (for reviews,(1) The enzyme is found in animal mitochondria and in bacteria. Probably, under most physiological conditions, the reaction is driven to the right, towards NADPH formation, by the proton motive force generated by the respiratory (or photosynthetic) electron-transport chain.Transhydrogenase has three domains. Domains I and III protrude from the membrane and possess the nucleotide-binding sites ; domain I for NAD ϩ and NADH, and domain III for NADP ϩ and NADPH [1Ϫ3]. Domain II spans the membrane and might comprise 10Ϫ12 transmembrane helices [4].Recombinant forms of domain I from Rhodospirillum rubrum [5,6] and Escherichia coli [7,8] and purified. The isolated domains bind their respective nucleotides with high specificity and affinity [5Ϫ10, 11]. A mixture of the two domains from the R. rubrum protein catalyses transhydrogenation, even in the absence of the membrane-spanning domain II [6,9], but the rates of the 'forward' and 'reverse' transhydrogenation reactions catalysed by the mixture are very slow Ϫ they are profoundly limited by release of product NADPH (k off Ϸ 5ϫ10 Ϫ4 s Ϫ1 ) and NADP ϩ (k off Ϸ 0.03 s Ϫ1 ), respectively, from domain III [9]. Evidently, in the complete enzyme, release of NADP(H) is accelerated by domain II. The mixture of domains I and III...

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“…All quoted concentrations are final concentrations after mixing the two reagents. The mixing dead time was determined to be 6.8 Ϯ 0.5 ms using the dichloroindophenol and ascorbic acid test reaction (31). Both excitation and emission wavelengths were set at 680 nm, and the time-dependent change in scattering light at a 90°angle, perpendicular to the beam, was recorded (32).…”

Section: Preparation Of Holo and Apo Pea Seed Ferritin And The Ep-mentioning

confidence: 99%

Protein Association and Dissociation Regulated by Ferric Ion

et al. 2009

Journal of Biological Chemistry

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Iron stored in phytoferritin plays an important role in the germination and early growth of seedlings. The protein is located in the amyloplast where it stores large amounts of iron as a hydrated ferric oxide mineral core within its shell-like structure. The present work was undertaken to study alternate mechanisms of core formation in pea seed ferritin (PSF). The data reveal a new mechanism for mineral core formation in PSF involving the binding and oxidation of iron at the extension peptide (EP) located on the outer surface of the protein shell. This binding induces aggregation of the protein into large assemblies of ϳ400 monomers. The bound iron is gradually translocated to the mineral core during which time the protein dissociates back into its monomeric state. Either the oxidative addition of Fe 2؉ to the apoprotein to form Fe 3؉ or the direct addition of Fe 3؉ to apoPSF causes protein aggregation once the binding capacity of the 24 ferroxidase centers (48 Fe 3؉ /shell) is exceeded. When the EP is enzymatically deleted from PSF, aggregation is not observed, and the rate of iron oxidation is significantly reduced, demonstrating that the EP is a critical structural component for iron binding, oxidation, and protein aggregation. These data point to a functional role for the extension peptide as an iron binding and ferroxidase center that contributes to mineralization of the iron core. As the iron core grows larger, the new pathway becomes less important, and Fe 2؉ oxidation and deposition occurs directly on the surface of the iron core.The chemistry of iron and oxygen in a number of non-heme di-iron proteins has been a subject of intense interest because of their varied roles in oxygen activation and catalysis, substrate hydrolysis, oxygen transport, redox reactions, H 2 O 2 , and iron detoxification and iron storage. Di-iron centers have similar structural motifs consisting of a combination of carboxylate and histidine ligands that either bind or bridge the two metal ions of the di-nuclear active site; di-iron proteins containing these centers include methane monooxygenase, ribonucleotide reductase, rubrerythrin, stearoyl desaturase, purple acid phosphatase, hemerythrin, the Dps proteins, and ferritins (1-6). Despite their similar di-nuclear centers, each of these proteins fulfills a distinct biological role that seems to be mediated by the nature of the first and second coordination sphere of the di-iron center.Ferritins are a class of intracellular iron storage and detoxification proteins that facilitate the oxidation of iron by molecular oxygen or hydrogen peroxide to form a hydrous ferric oxide mineral core within their interiors (1-3). Rapid Fe 2ϩ oxidation occurs at the di-iron ferroxidase center located on the H-subunit of the mammalian protein. Unlike the H-chain, the more acidic L-subunit lacks the ferroxidase center but contains a putative nucleation site responsible for slower iron oxidation and mineralization (7). The shapes of both the H-and L-subunit are nearly cylindrical and composed of a four...

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Test reactions for a stopped-flow apparatus

Cited by 276 publications

References 12 publications

Kinetic Intermediate in the Folding of Human Prion Protein

Kinetic Intermediate in the Folding of Human Prion Protein

Stopped‐flow kinetics of hydride transfer between nucleotides by recombinant domains of proton‐translocating transhydrogenase

Protein Association and Dissociation Regulated by Ferric Ion

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