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E C Slater

Publications and source records attributed to E C Slater.

At least 37 records · Page 2Linked to original sources
PubMed1981

A new species of bound ubisemiquinone anion in QH2: cytochrome c oxidoreductase.

Using a combination of EPR and low temperature diffuse reflectance spectroscopy, a new species of semiquinone anion has been detected in QH2:cytochrome c oxidoreductase in submitochondrial particles under conditions of oxidant-induced extra reduction of cytochrome b. In contrast to the previously detected semiquinone anion, this new species is insensitive to antimycin but sensitive to treatment with 2,3-dimercaptopropanol and O2. The two species can easily be distinguished on the basis of their respective EPR properties since they differ in g-value, line width, and microwave power saturation behavior. It is concluded that the two species of semiquinone anion are bound to different domains on QH2:cytochrome c oxidoreductase. The existence of two different semiquinone anions in the enzyme strongly supports a mechanism of electron flow as proposed in the Q-cycle.

Animals↗
PubMed1981

Kinetics of cytochrome b reduction in submitochondrial particles.

(1) In agreement with Eisenbach and Gutman (Eisenbach, M. and Gutman, M. (1975) Eur. J. Biochem. 52, 107--116) the reduction of cytochrome b in beef-heart submitochondrial particles by succinate in the presence of antimycin was found to be biphasic, the relative amounts of fast and slow phases being dependent on the redox state of a compound located on the oxygen side of the antimycin block. (2) HQNO is a concentration sufficiently large to saturate the specific antimycin- and HQNO-binding sites can substitute for antimycin in these experiments. (3) The rate of the slow phase of the reduction of cytochrome b is decreased under anaerobic conditions and after pretreatment with 2,3-dimercaptopropanol (BAL). (4) In the presence of antimycin and cyanide, cytochrome b-562 is, to some extent, preferentially reduced in the rapid phase and b-566 in the slow phase. (5) The previously proposed regulatory effects of redox-sensitive components X and Y on the redox level and reduction kinetics, respectively, of cytochrome b are ascribed to the role of the Fe-S protein, when it is oxidized, in producing the reductant of cytochrome b by oxidation of QH2, and by the fact that when QH2 is bound to it, the reduced Fe-S protein cannot be oxidized by its natural oxidant, cytochrome c.

Animals↗
PubMed1981

Purification and some properties of the soluble hydrogenase from Chromatium vinosum.

A routine procedure for the growth and harvesting of large (600 1) batches of Chromatium vinosum and the isolation of hydrogenase (hydrogen: (acceptor) oxidoreductase, EC 1.12.-.-) are described. The enzyme is pure according to polyacrylamide gel electrophoresis, has a molecular weight of 61,000-63,000 and consists of a single polypeptide chain. The enzyme is stable in air but not active. Activity is obtained only after complete removal of oxygen. EPR spectroscopy at 9 GHz shows a signal indicative for a [4Fe-4S]3+(3+,2+) cluster and in addition a rather complex signal of unknown origin. This additional signal completely disappears upon removal of oxygen, by incubation with 2-mercaptoethanol or by reduction with ferrocytochrome c. No EPR signals are detected in the enzyme reduced with H2 or dithionite. The intensity of the EPR signal of the [4Fe-4S] cluster corresponds to one-quarter of the enzyme concentration, both in the untreated as well as in the He- or N2-activated enzyme. If the enzyme is activated under He and then brought in contact with air the signal increases 4-fold and represents about one free spin/enzyme molecule. When measured at 35 GHz the line shape and peak positions of the additional signal change, indicating that the signal is not originating from a simple S = 1/2 system. None of the inhibitors of the hydrogenase activity has any effect on the shape or intensity o the EPR signal fo the Fe-S cluster, 2H2O also has no effect. All EPR signals disappear after reduction with NADH or ascorbate in the presence of phenazine methosulphate. It is suggested that the Fe-S cluster is not the primary site of interaction of H2 with the enzyme.

Chromatium↗
PubMed1980

Identification of the BAL-labile factor.

One of us has previously reported that treatment of the Keilin and Hartree heart-muscle preparation with 2,3-dimercaptopropanol (BAL), in the presence of air, leads to the complete inactivation of the succinate oxidase system with little if any effect on the activities of succinate dehydrogenase (until more than half the BAL was oxidized) or cytochrome c oxidase. The inactivation of the complete succinate oxidase system requires the oxidation of BAL by air in the presence of the enzyme. It is not caused by H2O2 or BAL disulphides produced during the oxidation of BAL. Spectroscopic studies identified the block as lying between cytochromes b and c. It was suggested that a BAL-labile factor is present which transfers electrons from cytochrome b to cytochrome c and which is destroyed by coupled oxidation with BAL. The factor is also required for NADH oxidation. Subsequent work showed it is not identical with cytochrome c1 (ref. 4), myoglobin present in the preparation or the antimycin-binding site. We report here that this factor is identical to the iron-sulphur protein in the central portion of the respiratory chain first identified by Rieske.

Cell-Free System↗
PubMed1980

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP.

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F1) as the triphosphate, either in the absence or presence of Mg2+, label covalently bound is not hydrolysed. 2. In the presence of Mg2+ the nitreno-ATP is bound to both the alpha and beta subunits, mainly (63%) to the alpha subunits. 3. After successive photolabelling of F1 with 8-azido-ATP (no Mg2+) and 8-azido-ADP (with Mg2+) 4 mol label is bound to F1, 2 mol to the alpha and 2 mol to the beta subunits. 4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F1 previously labelled with 8-nitreno-ADP. It is concluded that binding to the alpha-subunits hinders binding to the beta subunits. 5. F1 that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F1. 6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F1.

Adenine Nucleotides↗
PubMed1980

Subunit interactions in glyceraldehyde-3-phosphate dehydrogenases. Their involvement in nucleotide binding and cooperativity.

1. The hybridization of rabbit muscle and yeast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) was used to study the involvement of subunit interactions in NAD+ and NADH binding by these enzymes. 2. In the presence of 1 mM NAD+ or NADH no hybrid formation was observed with our preparations of the two enzymes. 3. The inhibition by NADH of the hybrid formation is shown to be a consequence of an unfavourable equilibrium of the hybridization process in the presence of NADH. 4. The inhibition by NAD+ of the hybrid formation is shown to be a consequence of both a shift in the equilibrium, as in the case of NADH, and a decrease in the rate of the dissociation of the enzymes. 5. The dimer of the yeast enzyme binds NAD+ or NADH with equal affinity irrespective of whether it is combined with another yeast dimer in the yeast tetramer or with a rabbit muscle dimer in the hybrid. 6. The binding of NAD+ and NADH to the dimer of the rabbit muscle enzyme is stronger in the rabbit muscle tetramer than in the hybrid; this explains the shift in the equilibrium of the hybridization process caused by these nucleotides. 7. Alkylation of the rabbit muscle enzyme with iodoacetate does not influence the hydridization process in the absence of nucleotides. 8. After alkylation of the rabbit muscle enzyme NADH cannot cause a large shift in the equilibrium of the hybridization process. 9. In accordance with this it was found that the binding of NADH (and NAD+) to the rabbit muscle enzyme is weakened by alkylation, whereas the binding of NADH to the alkylated rabbit muscle subunits is not affected strongly by the hydridization. 10. An attempt is made to combine the effects of nucleotides on the hybridization properties of the yeast enzyme and the alkylated or unalkylated rabbit muscle enzymes with the binding properties of all tetrameric species involved in the hybridization processes in a thermodynamic description of nucleotide binding and subunit interactions.

Animals↗
PubMed1980

Binding of ADP to beef-heart mitochondrial ATPase (F1).

1. ADP binding to beef-heart mitochondrial ATPase (F1), in the absence of Mg2+, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure. 2. Since during the binding experiments the 'tightly' bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters. 3. The binding data show that only one tight binding site (Kd about 0.5 microM) for ADP is present. 4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter is not less than about 50 microM.

Adenosine Diphosphate↗
PubMed1980

The pre-steady state and steady-state kinetics of the ATPase activity of mitochondrial F1.

1. The lag time before maximum velocity of ATP hydrolysis is reached upon mixing ATP with F1 is much greater than can be explained by a simple Michaelis-Menten mechanism, and must be due to an activation reaction. The lag time is dependent on the concentration of MgATP (half-maximal at 30 microM) and is equal to 30 ms at infinite MgATP concentration. The initial rate of hydrolysis by nucleotide-depleted F1 is much greater than with normal F1. It is tentatively suggested that the activation reaction with normal preparations is due to replacement of firmly bound ADP by MaATP. 2. After the initial time lag, the reaction follows very closely first-order kinetics provided that the concentration of MgATP is much less than the Km and the reaction is completed within 2 s. This is not expected if the dissociation constant of the enzyme-MgADP complex, an intermediate in the enzymic reaction, is much lower than the Km as has been reported in the literature. The value of V/Km, calculated from the exponential decay, is very close to that calculated from independent measurements of V and Km. 3. The low values for Ki(ADP) reported in the literature were found to be due to a slow (in the order of seconds) formation of an inhibited MgADP-enzyme complex. Dissipation of this inhibited complex by ATP requires seconds. The dissociation constant of the MgADP-enzyme complex that is an intermediate in the enzyme reaction was found to be 150 microM. 4. ADP but not ATP becomes firmly bound to nucleotide-depleted F1 in the absence of Mg2+.

Adenosine Diphosphate↗
PubMed1979

Subunit interactions in rabbit-muscle glyceraldehyde-phosphate dehydrogenase, as measured by NAD+ and NADH binding.

1. The binding parameters for NADH and NAD+ to rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been measured by quenching of the flourescence of the protein and the NADH. 2. The fact that the degree of protein fluorescence quenching by bound NAD+ or NADH, excited at 285 nm and measured at 340 nm ('blue' tryptophans), is not linearly related to the saturation functions of these nucleotides, leads to a slight overestimation of the interaction energy and an underestimation of the concentration of sites, if linearity is assumed. 3. This is also the case for NADH, but not for NAD+, when the protein fluorescence is excited at 305 nm and measured at 390 nm ('red' tryptophans). 4. The binding of NAD+ can be described by a model in which the binding of NAD+, via negative interactions within the dimer, induces weaker binding sites, with the result that the microscopic dissociation constant is 0.08 microM at low saturation and 0.18 microM for the holoenzyme. 5. The binding of NADH can be described on the basis of the same model, the dissociation constant at low saturation being 0.5 microM and of the holoenzyme 1.0 microM. 6. The fluorescence of bound NADH is not sensitive to the conformational changes that cause the decrease in affinity of bound NAD+ or NADH. 7. The binding of NAD+ to the 3-phosphoglyceroyl enzyme can be described by a dissociation constant that is at least two orders of magnitude greater than the dissociation constants of the unacylated enzyme. The affinity of NAD+ to this form of the enzyme is in agreement with the Ki calculated from product inhibition by NAD+ of the reductive dephosphorylation of 1,3-diphosphoglycerate.

Acylation↗
PubMed1979

Ferrocyanide as electron donor to cytochrome aa3. Cytochrome c requirement for oxygen uptake.

1. In the absence of cytochrome c, ferrocyanide or ferrous sulphate reduces cytochrome c oxidase (EC 1.9.3.1), but no continuous oxygen uptake ensues, as it does with N,N,N',N'-tetramethyl-p-phenylenediamine or reduced phenazine methosulphate as reductants, unless a substoichiometric amount of cytochrome c or an excess of clupein is present. Cytochrome c cannot be replaced by porphyrin cytochrome c. 2. Cytochrome c, porphyrin cytochrome c and clupein all stimulate the reduction of cytochrome aa3 by ferrocyanide. 3. A model is proposed to explain these findings in which a high-affinity site for cytochrome c on the oxidase regulates the access of hydrophilic electron donors to a low-affinity site, and reduction via the high-affinity site is required for continuous oxygen uptake. 4. Furthermore, it is shown that upon reaction of oxidase with ferrocyanide, cyano-oxidase is formed.

Cyanides↗
PubMed1978

Preparation and properties of rabbit-muscle glyceraldehyde-phosphate dehydrogenase with equal binding parameters for the third and fourth NAD+ molecules.

1. A method of preparing rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) is described which yields a preparation differing in important respects from those previously described and resembling the enzyme isolated from sturgeon muscle. 2. Direct binding measurements at 25 degrees C by equilibrium gel filtration fit dissociation constants for the first two molecules that are too low to be measured by this technique and 0.9 micrometer for the third and fourth molecules. The dissociation constant of the fourth molecule is much lower than that previously reported for the rabbit-muscle enzyme. 3. In contrast to previous results with the rabbit-muscle enzyme, the increase in absorbance at 360 nm between three and four molecules of NAD+ bound to the enzyme was, within experimental error, the same as that with each of the first three molecules. 4. Data on the quenching of the protein fluorescence by NAD+ at 15 degrees C at different enzyme concentrations closely fit dissociation constants of 0.028 micrometer for the first two molecules and 0.27 micrometer for the third and fourth molecules.

Animals↗
PubMed1978

Chaotropic resolution of high molecular weight (type I) NADH dehydrogenase, and reassociation of flavin-rich (type II) and flavin-poor subunits.

1. Type-I NADH dehydrogenase (Complex I) was solubilized and dissociated into subunits by NaClO4. NADH slows the dissociation. On subsequent stepwise addition of (NH4)2SO4 the dissociation is partly reversed, as is to be expected from the opposing effects of ClO-4 and SO-24, which are on the salting-in and salting-out sides, respectively, of the lyotropic series. 2. In consequence, the aggregates of subunits that are separated by (NH4)2-SO4 fractionation consist of randomly associated subunits as well as fragments of Type I enzyme. The fraction precipitating at 27% satd. (NH4)2SO4 is flavin-poor, that remaining soluble at 55% satd. (NH4)2SO4 flavin-rich and those separating between 27 and 55% satd. (NH4)2SO4 intermediate in composition. 3. The fraction remaining soluble at 55% satd. (NH4)2SO4 contains the purified low-molecular-weight iron-sulphur flavoprotein (Type-II dehydrogenase). It is a dimer consisting of one molecule of FMN, one 28-kilodalton and one 56-kilodalton subunit per protomer. Work of others indicates that it contains 4 Fe and 4 acid-labile S atoms per molecule of FMN. Sometimes the fraction remaining soluble at 55% satd. (NH4)2SO4 contained an additional small subunit (12 kilodaltons) and four additional Fe and acid labile S atoms per protomer. The sedimentation coefficients (s020,w) of the two preparations were 5.3 and 6.6 S, respectively, with calculated frictional ratios of 1.5 and 1.24, respectively. 4. The intermediate fractions are mixtures of the various subunits present in Complex I. Specifically a fraction separating at 55% satd. (NH4)2SO4 was found to be a mixture of two fragments, the pure iron-sulphur flavoprotein and a 26-S fragment that contained per protomer four subunits of 12 kilodaltons, one each of 28, 32, 56 and 77 kilodaltons, one molecule of FMN and 20 Fe and acid-labile S atoms. It was probably tetrameric or even larger. 5. The oxidoreductase activity of the intermediate fractions is dependent on the protein concentration, the activity with ferricyanide increasing and that with ferricytochrome c decreasing with increasing protein concentration. This is interpreted as an increased association of subunits present in the intermediate fractions. Similar results are obtained when flavin-rich and flavin-poor fractions are mixed. The association is cooperative. NADH favours the association of the subunits. 6. Association of the subunits is accompanied by a 10-fold increase in k2 (rate constant for intramolecular electron flow), a 10-fold decrease of the accessibility of ferricyanide to the reduced enzyme and a 10(4)-fold decrease of the accessibility of ferricytochrome c. The Ks (NADH) is also decreased. Although the changes are in the direction to be expected from a conversion of Type II enzyme to Type I, the value of k2 is still much less than in the latter enzyme.

Flavin Mononucleotide↗