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M Iwatsubo

Publications and source records attributed to M Iwatsubo.

At least 19 recordsLinked to original sources
PubMed1980

Flavocytochrome b2 (Baker's yeast). Deuterium isotope effect studied by rapid-kinetic methods as a probe for the mechanism of electron transfer.

The use of DL-[2-2H]lactate in steady-state measurements of ferricyanide reduction by flavocytochrome b2 at 30 degrees C has previously yielded an isotope effect of 5 [F. Lederer (1974) Eur. J. Biochem. 46, 393--399]. We report here studies carried out at 5 degrees C with L-[2-2H]lactate, where flavin and heme reduction were observed in the stopped-flow apparatus, in the absence of acceptor. The generally biphasic reduction curves were analysed according to a new mathematical treatment which allowed us to derive microscopic constants from initial reduction rates. It has thus been possible to determine an isotope effect of 8 on flavin reduction, 6 on heme reduction, compared to 4 in the steady state. Consequently, two slightly rate-limiting steps occur after the first one where the alpha-hydrogen is abstracted. It has also been possible to calculate the substrate association and dissociation rate constants for intact enzyme. The studies were carried out in parallel on intact and cleaved cytochrome b2. The results suggest that proteolysis affects essentially the steps involved in flavin reduction, and not intramolecular electron transfer steps. Moreover, the experimental data obtained at low rates of electron entry have led us to reexamine a previously proposed scheme for electron transfer [Capeillère-Blandin, Bray, Iwatsubo and Labeyrie (1975) Eur. J. Biochem. 54, 549--566]. An alternative model based on computer-simulation studies will be presented in a paper in this journal.

Deuterium↗
PubMed1977

Pulse fluorimetry study of energy transfers between tryptophan residues and NADPH in beef liver glutamate dehydrogenase complexes.

A method is proposed to determine the rates of singlet energy transfers in an array of chromophores containing a finite number of donors and fluorescent acceptors. This method is based on measurements of transfer efficiency coupled with pulse fluorimetry. Three classes of donors can be distinguished which differ in their energy transfer rate. The rates of the first, the second and the third class are respectively greater than, of the order of, and smaller than the emission rate. The method is applied to the study of the energy transfers from tryptophan residues to NADPH, in ternary and quaternary glutamate dehydrogenase complexes. Practically, all these tryptophan residues belong to the first class. They can be divided into two subclasses having different transfer rate values. The distance between these residues and the NADPH site are of the order of 2.5 nm. In addition, the ligand binding induces a protein conformation change, leading to a fluorescence quenching of the tryptophanyl emission.

Chemical Phenomena↗
PubMed1977

[Study by a rapid mixing method of the interaction between a fluorescent cholinergic agonist and membrane fragments rich in cholinergic receptors from Torpedo marmorata].

The analysis by stopped-flow of the interaction of fluorescent agonist (C5DACho1) with the acetylcholine receptor in its membrane-bound form reveals several kinetic steps: a fast one, in the millisecond range, associated with the binding of C5DACho1 to a high affinity state and a "medium" and "slow" one, the last one representing possibly an isomerisation of the receptor molecule towards the high affinity state.

Acetylcholine↗
PubMed1976

[Rapid changes in the intensity of fluorescence observed in the presence of cholinergic agonists with Torpedo marmorata membrane fragments rich in cholinergic receptors and labelled with quinacrine].

Stopped-flow studies of receptor-rich membrane fragments from Torpedo marmorata labelled by quinacrine lead to the resolution (in the time scale of the millisecond to the second) of a fast increase of fluorescence intensity associated with the binding of typical agonists. The signal is reversible, does not take place with antagonists, has a Q10 of 1.4 and its amplitude decreases after pretreatment of the membrane fragment by dithiothreitol or ceruleotoxin.

Acetylcholine↗
PubMed1976

Pulse fluorimetry study of beef liver glutamate dehydrogenase reduced nicotinamide adenine dinucleotide phosphate complexes.

Single photon counting pulse fluorimetry has been used in order to study the two ternary complexes GDH-GTP-NADPH and GDH-L-glutamate-NADPH and the quaternary complex GDH-GTP-L-glutamate-NADPH. The fluorescence decay of the enzyme-bound NADPH is not monoexponential in any of these complexes. Moreover, it does not seem to be dependent on the coenzyme concentration. The experimental curves can be satisfactorily fitted with the sum of two exponentials, the relative amplitudes of which significantly depend on the complex studied. Thus, for dihydronicotinamide two possible environments might exist in the enzyme active sites. It is also shown that the fluorescence decay times of the enzyme are shortened by the bound NADPH.

Animals↗
PubMed1976

Mode of interaction between beta-lactam antibiotics and the exocellular DD-carboxypeptidase--transpeptidase from Streptomyces R39.

The exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R39 is inhibited by beta-lactam antibiotics according to the same general scheme of reaction as the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61. However, the values for the kinetic constants involved in the reaction are very different for the two enzymes and provide an explanation for the observation that the R39 enzyme is more sensitive to beta-lactam antibiotics than the R61 enzyme. Further, particular beta-lactams influence the kinetic constants to different extents depending on the source of the enzyme, so that a physical basis for the spectrum of antibiotic activity against particular enzyme systems is provided.

Carboxypeptidases↗
PubMed1975

Kinetics of interaction between the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61 and beta-lactam antibiotics. A choice of models.

The simplest model for the interaction between the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61 and beta-lactam antibiotics involves the three following steps: (a) the formation of a reversible equimolar enzyme - antibiotic complex; (b) the irreversible transformation of this complex into a modified enzyme - antibiotic complex; and (c) the breakdown of this latter complex and the concomitant release of a regenerated enzyme and a modified antibiotic molecule. The dissociation constant for step 1 and the rate constants for steps 2 and 3 were measured with various beta-lactam antibiotics. With antibiotic such as benzylpenicillin, which behaves as a good 'substrate', steps 1 and 2 occur at enzymic velocities, whereas step 3 occurs at a very low velocity and hence is responsible for the low efficiency of the overall process.

Acyltransferases↗
PubMed1975

Binding studies of NADPH to NADP-specific L-glutamate dehydrogenase from Saccharomyces cerevisiae.

Optical characteristics of enzyme-reduced coenzyme complexes of yeast NADP-specific glutamate dehydrogenase have been investigated in the presence and absence of product (L-glutamate) and in the presence or absence of phosphate. The phosphate effect, pointed out in a previous work, is found again: inorganic phosphate (Pi) destabilizes the binary complex (E - NADPH), the dissociation constant of which is equal to 14 muM, a value much higher than that determined in Tris-HCl buffer: Kd = 0.9 muM. Concerning the role of phosphate some assumptions are drawn up with respect to a similar behaviour of Pi toward yeast glutamate dehydrogenase and ADP toward the beef liver enzyme. In the same way, L-glutamate induces a stabilization of the binary complex; this latter effect is unchanged in the presence of phosphate, yet it is less marked than in the case of beef liver glutamate dehydrogenase. Protein fluorescence, nucleotide fluorescence and circular dichroism measurements allowed the determination of three identical and independent NADPH binding sites per hexameric active unit. In analogy with beef liver enzyme, it seems that yeast glutamate dehydrogenase is a good model to study anticooperativity in ligand binding.

Binding Sites↗
PubMed1975

A deactivating conformational change induced by reduced nicotinamide-adenine dinucleotide phosphate in a Neurospora glutamate dehydrogenase.

Stopped-flow fluorescence techniques have been used to observe the formation of the binary comples of E-NADPH. At pH 7.5 there is a protein conformational change after the formation of the binary complex. This conformational change can be detected by a decrease in the fluorescence intensity of the complex at 350 nm and by an increase in its fluorescence intensity at 450 nm.

Fluorescence↗
PubMed1975

Flavocytochrome b2: kinetic studies by absorbance and electron-paramagnetic-resonance spectroscopy of electron distribution among prosthetic groups.

The reduction by L-lactate of the prosthetic groups of flavocytochrome b2 (L-lactate cytochrome c oxidoreductase from aerobic yeast, a tetrameric molecule containing one haem and one flavin mononucleotide per protomer) was reinvestigated. It was confirmed that the enzyme ultimately takes up 3 electrons per protomer from this 2-electron donor. Stopped-flow absorbance data at an haem isosbestic point to follow the oxidized flavin and in a haem band indicate that, under the conditions used, haem and flavin reduction time courses are indistinguishable, both being biphasic (phases I and II). Comparison with electron paramagnetic resonance data (Fe3+ haem and flavosemiquinone signals) led to a complete description at 24 degrees C of the time courses of the various reduction states of the prosthetic groups. It has been previously demonstrated (Morton and Sturtevant, 1964) that, after the formation of the enzyme-substrate complex, the electron transfer to the enzyme takes place as the first and rate-limiting step of the turnover. In the present study, an initial burst of fully reduced flavin, of small amplitude, is detected at the very beginning of phase I (before 6 ms). The redox forms which accumulate thereafter till the end of phase I (30-35 ms) are the reduced haem (up to 80%), the flavin semiquinone (up to 50%) and the fully reduced flavin (from 25% up to 35%); the total of electrons distributed at the end of phase I is about 2 per protomer meaning that, in this phase, each enzyme site acts as a 2-electron and not a 3-electron acceptor. A 2-electron flow as the limiting step during phase I with the rate constant kI accounts for the steady-state electron flow during catalysis. Phase I is followed by the much slower phase II which corresponds to the entry of the third electron and cannot be involved in the turnover. The interpretation of the results are given as a scheme, with the proper rate constants, allowing a satisfactory fitting of experimental data by simulation. Among the elementary steps required are a rapid distribution of one electron from reduced flavin to the haem, a rapid interprotomers dismutation between couples of flavin semiquinone regenerating two oxidized flavin per tetramer. The very low reactivity of the latter for the entry of the third electron per protomer is tentatively explained by the occurrence of a slow additional step limiting the final reduction reaction. It was observed that, over phase I and the beginning of phase II, from 15 to 200 ms, all the redox species remain apparently under equilibrium conditions. Parallel studies (titrations of flavocytochrome b2 by L-lactate) showed that the set of equilibrium parameters relative to haem and flavin species is significantly different in the "final" equilibrium (after 30 s) from that in the time interval 15-200 ms. Such an anomaly suggests a conformation change takes place very slowly in the molecule after the acceptance of the first two electrons per protomer.

Cytochromes↗