New method for quantitative determination of serum proteins separated by paper electrophoresis

Mejbaum-Katzenellenbogen, W; Dobryszycka, W

doi:10.1016/0009-8981(59)90158-5

Cited by 189 publications

(53 citation statements)

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“…All protein preparations were checked for purity (Ͼ95%) by SDS-polyacrylamide gel electrophoresis (21), and for cyclic transhydrogenation activity (10,13,16). Protein concentrations were determined using the microtannin assay (22) (16,24).…”

Section: Methodsmentioning

confidence: 99%

Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

Venning

Peake²,

Quirk

et al. 2000

Journal of Biological Chemistry

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Transhydrogenase, which is found in the inner membranes of animal mitochondria and the cytoplasmic membranes of some bacteria, catalyzes the reaction shown below.A single proton is translocated across the membrane, from the p-aqueous phase (the "outside" of intact mitochondria and bacteria) to the n-aqueous phase (the "inside"), for each hydride equivalent transferred from NADH to NADP ϩ . Under most conditions, this is the in vivo direction; the reaction is driven by the proton electrochemical gradient (⌬p) resulting from the action of respiratory (or photosynthetic) electron transport. For recent reviews, see Refs. 1-3.The gross structure of transhydrogenases from different species is strikingly invariant. The enzyme has three components; dI and dIII, which bind NAD(H) and NADP(H), respectively, protrude from the membrane (on the cytoplasmic side in bacteria, and the matrix side in mitochondria), and dII spans the membrane. Crystal structures of the dIII components of human and bovine transhydrogenases were recently described (4 -6), and the solution structure of the Rhodospirillum rubrum equivalent was determined by NMR.1 The basic fold of dIII is similar to the classical, dinucleotide-binding domain of lactate dehydrogenase, but NADP ϩ is bound with an unusual, "reversed" orientation. The nicotinamide ring of the bound NADP ϩ is exposed on a ridge of dIII. A homology model of dI (8), based on the crystal structure of the sequence-related alanine dehydrogenase (9), suggests that the nicotinamide ring of NADH is located in a deep cleft. It was proposed that, in the complete transhydrogenase, the ridge of dIII inserts into the cleft of dI to bring the nicotinamide rings of the two nucleotides into apposition to effect direct hydride transfer (5, 6). The protruding helix-D/loop-D of dIII is thought to interact with the membrane-spanning, dII and, together with the adjacent, lidlike loop E, which passes over the bound nucleotide, might be responsible for the energy transmission. The proton translocation steps during turnover are probably coupled specifically to changes in the mode of NADP(H) binding (1,5,10).Kinetic studies of transhydrogenase have also developed, following discoveries that fragments of the protein retain their nucleotide binding and some catalytic capacity. Thus, recombinant dI and dIII proteins, which bind their cognate nucleotides, have now been isolated from a number of species (11-16); mixtures of these proteins, even from different species, catalyze transhydrogenation reactions, albeit with properties that are modified relative to those of the complete enzyme. Transient state experiments, in particular, have revealed useful information on the hydride transfer step (17)(18)(19). Without exception, experiments with dI⅐dIII complexes have been carried out with nucleotide analogues having altered absorbance spectra to facilitate measurement of the rate of reaction. In this report we describe transient state experiments with the physiological nucleotides. A procedure is described, in which singl...

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Section: Methodsmentioning

confidence: 99%

Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

Venning

Peake²,

Quirk

et al. 2000

Journal of Biological Chemistry

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“…One exception was that, in domain III preparation, during the cell-sonication step, the medium was supplemented with 50 µM NADP ϩ , and thereafter all buffers used in the purification were supplemented with 2 µM NADP ϩ . The purity of the two proteins was always confirmed by SDS/PAGE [13], and the protein concentrations were determined using the microtannin assay [14]. The concentrations are expressed with reference to protein monomers.…”

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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“…Protein (0.6 ml) was precipitated by addition of 0.3 ml 14% perchloric acid during continuous stirring, and kept for 10 min on ice. The solution was adjusted to pH 7.0 with 0.45 ml 1 M KOH, 1 M KHCO,, kept on ice for 15 niin and stored at -20°C for at least 1 h. The top clear layer (0.8 ml) was then centrifuged to remove any remaining precipitate, and used for estimation of nucleotide by fluorescence spectroscopy at excitation and emission wave- Protein concentration was routinely determined by the microtannin assay with bovine serum albumin as standard [29]. In comparisons, the bicinchoninic acid assay 1301 gave concentrations of domain I11 protein 20% lower than the microtannin assay.…”

Section: Methodsmentioning

confidence: 99%

Properties of the Purified, Recombinant, NADP(H)‐Binding Domain III of the Proton‐Translocating Nicotinamide Nucleotide Transhydrogenase from Rhodospirillum Rubrum

Diggle

Bizouarn

Cotton

et al. 1996

European Journal of Biochemistry

169

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Transhydrogenase comprises three domains. Domains I and I11 are peripheral to the membrane and possess the NAD(H)-and NADP(H)-binding sites, respectively, and domain 11 spans the membrane. Domain TIT of transhydrogenase from Rhodospirillum rubrum was expressed at high levels in Escherichia coli, and purified. The purified protein was associated with substoichiometric quantities of tightly bound NADP' and NADPH. Fluorescence spectra of the domain 111 protein revealed emissions due to Tyr residues. Energy transfer was detected between Tyr residue(s) and the bound NADPH, indicating that the amino acid residue(s) and the nucleotide are spatially close. The rate constants for NADP' release and NADPH release from domain Ill were 0.03 SKI and S.6X10-4s-1, respectively. In the absence of domain I1 a mixture of the recombinant domain 111 protein, plus the previously described recombinant domain 1 protein, catalysed reduction of acetylpyridine-adenine dinucleotide (AcPdAD') by NADPH (reverse transhydrogenation) at a rate that was limited by the release of NADP' from domain 111. Similarly, the mixture catalysed reduction of thio-NADP' by NADH (forward transhydrogenation) at a rate limited by release of thio-NADPH from domain 111. The mixture also catalysed very rapid reduction of AcPdAD' by NADH, probably by way of a cyclic reaction mediated by the tightly bound NADP(H). Measurement of the rates of the transhydrogenation reactions during titrations of domain I with domain 111 and vice versa indicated (a) that during reduction of AcPdAD' by NADPH, a single domain I protein can visit and transfer H ~ equivalents to about 60 domain 111 proteins during the time taken for a single domain I11 to release its NADP ' , whereas (b) the cyclic reaction is rapid on the timescale of formation and breakdown of the domain I . 111 complex. The rate of the hydride transfer reaction was similar in thc domain I I I11 complex to that in the complete membrane-bound transhydrogenase, but the rates of forward and reverse transhydrogenation were much slower in the I . 111 complex due to the greatly decreased rates of release of NADP' and NADPH. It is concluded that, in the complete enzyme, conforniational changes in the membrane-spanning domain 11, which result from proton translocation, lead to changes in the binding affinity of domain 111 for NADP' and for NADPH.

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New method for quantitative determination of serum proteins separated by paper electrophoresis

Cited by 189 publications

References 1 publication

Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

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

Properties of the Purified, Recombinant, NADP(H)‐Binding Domain III of the Proton‐Translocating Nicotinamide Nucleotide Transhydrogenase from Rhodospirillum Rubrum

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