Conclusions

Orr, Mary

doi:10.1093/acprof:oso/9780199258581.003.0009

Cited by 2 publications

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“…The chromatographic separation of the components of the glutamate synthase reaction was effected using a MonoQ anion-exchange column connected to the FPLC apparatus. The column was equilibrated in 10 mM triethanolamine/Cl, pH 7.7 (Orr and Blanchard, 1984). After injection of the sample, the column was rinsed with 4 ml 10 mM triethanolamine/Cl, pH 7.7.…”

Section: Determination Of the Stereospecificity Of Oxidation Of ( 4 Smentioning

confidence: 99%

The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense

Vanoni

Nuzzi

Rescigno

et al. 1991

European Journal of Biochemistry

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The reactions catalyzed by glutamate synthase from Azospirillum hrasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A . hrasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillurn is specific for the transfer of the 4 s hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent.The data obtained with glutamate synthase from A . hrasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillurn is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Glutamate synthase catalyzes the transfer of the amide group of L-glutamine to the C2 of 2-oxoglutarate, yielding two molecules of glutamate. The reducing equivalents are provided by reduced pyridine nucleotides in microbial glutamate synthases, or by reduced ferredoxin in photosynthetic microorganisms and in the chloroplast enzyme (Vanoni et al., 1991).Glutamate synthase is especially relevant in diazotrophs, since with glutamine synthetase, it forms the major route of ammonia assimilation at low intracellular levels of ammonia. The bacterial enzyme has been purified to homogeneity from a number of enteric bacteria, but only from two diazotrophs, namely Klehsiella aerogenes and Azospirillurn brasilense. The enzyme from A. brasilense shares several properties with that isolated from other bacterial sources (Ratti et al., 1985). It is an iron-sulfur flavoprotein, which contains 1 mol FAD, 1 mol FMN, about 8 atoms non-heme iron and about 8 atoms acidlabile sulfur/l95 kDa protomer. The protomer is formed by two dissimilar subunits of approximately 140 kDa and approximately 55 kDa for the c i and / I subunits, respectively.Bacterial glutamate synthases are specific for NADPH. Lglutamine, and 2-oxoglutarate. At alkaline pH, they can carry

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Section: Determination Of the Stereospecificity Of Oxidation Of ( 4 Smentioning

confidence: 99%

The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense

Vanoni

Nuzzi

Rescigno

et al. 1991

European Journal of Biochemistry

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“…A non-ex ponential relationship has been found for heat injury in the sclerotia of myxomycetes (97). Orr (42,159,160) has compared heat-death curves in a number of different animals.…”

Section: Effect Of High Temperaturesmentioning

confidence: 99%

“…As is known, the temperature coefficients of various physiological proc esses in a given organism may differ greatly in magnitude, and this point has been studied systematically in the frog (41,42). Moreover, the various phases of a physiological activity may have temperature coefficients of different values.…”

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Physiological Aspects of Heat and Cold

Belehradek¹

1957

Annu. Rev. Physiol.

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The author of this chapter has been invited to revise the literature on the general physiological aspects of the action of temperature for the 20 years which have elapsed since the publication of the first edition of his relevant book (1). Well over 3,000 papers in this field have been published since then, and only a small fraction can be mentioned here. These should be regarded as a representative sample illustrating the general trends prevailing during this period. The two most important are (a) a descriptive study of the temperature response, requirements, and tolerance of various physiological properties and functions, and (b) an attempt to obtain a closer insight into the intimate cellular chemomechanisms by analytic studies of the responses of living systems to temperature variations. THERMIC PROPERTIESThe effect of body size upon the time required to attain equilibrium with the external temperature has been illustrated by experiments on the fruit fly (Drosophila), where 30 secs. suffice to stabilize the wing rhythm at a new temperature (2), and on bacteria in which flash-heat exposures of 0.5 sec. duration were applied, when only 10-5 sec. were lost in heat conduc tion (3; cf. also 164). The combined effect of body size, coloration, radiation, and air current in medium-sized insects was studied, and the results show that the excess temperature varies with from 0.4 to 1.0 power of the body size, compared with 0.4 to 0.7 power in control experiments with spherical plasticine models (4). As regards poikilothermic animals in general, several good reviews are available (see S, 6, 7, 18).The specific heat of the sickle-shaped human erythrocyte was found to be normal, so the condition cannot therefore be attributed to abnormal crystallization of haemoglobin. On the other hand, the specific heat of normal red cells decreases somewhat on slight heating (8). RATE PROCESSESTemperature coefficients.-To describe the extent of the response to temperature variations of physiological rate processes, three kinds of temperature coefficient mainly are used, namely, Q10, J.I. (otherwise written as E, or A), and b [in the b, a-rule (cf. 1)]. Most authors apply these constants merely as a means of description without accepting the theoretical implica-1 The survey of the literature pertaining to this review was completed in May, 1956. 59 Annu. Rev. Physiol. 1957.19:59-82. Downloaded from www.annualreviews.org by Case Western Reserve University on 11/02/14. For personal use only. Quick links to online content Further ANNUAL REVIEWS B E LEHR A DEKtions attached to anyone of them ; for this purpose QID is still most widely used despite its recognized drawbacks, as it is the easiest to calculate. The somewhat more complicated b, a-rule, calculating the temperature from a specific zero point (a) upwards and using the log/log representation, has been recently applied by various authors to the reproduction rate of ciliates (9, 10); to the rate 0f respiration in bacteria (11, 12), sliced mammalian tissue (13), and insects (14); to the ...

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Conclusions

Cited by 2 publications

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The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense

The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense

Physiological Aspects of Heat and Cold

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