Quaternary Structure and Enzymic Properties of Beef Liver Glutamate Dehydrogenase

Krause, Jobst; Markau, K.; Minssen, M.; Sund, Horst

doi:10.1007/978-3-642-49974-6_25

Cited by 14 publications

(5 citation statements)

References 33 publications

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“…The importance of hydrophobic interactions not only for the reversible association but also for the stabilisation of the quaternary structure within the unimer also explains the strong dissociating effect of even small concentrations of sodium dodecylsulfate [7]. The associating effect of benzene and its derivatives [21] under these aspects can be explained by the following hypothesis.…”

Section: Discussionmentioning

confidence: 99%

“…The association is unlimited and the equilibrium constants of subsequent steps of association are all equal (open equilibrium). The deviation of the apparent weight-average molecular weight from the true molecular weight can be calculated by the introduction of a second virial coefficient for which a value of 8 x l o p 9 nmol x 1 x g-2 independent of the association state was found [7,8]. The associated particles can be described as long rods with a diameter of 8.4 nm, the length of the oligomer being 12.6 nm Among the proteins which are composed of more than one polypeptide chain, there are only a few which exhibit an association-dissociation equilibrium.…”

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Studies of Glutamate Dehydrogenase

Gauper

Markau

Sund

1974

European Journal of Biochemistry

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The association-dissociation equilibrium of beef-liver glutamate dehydrogenase was investigated in phosphate buffer of different ionic strengths and in the presence of the organic solvents ethylene glycol and dioxane. For some of the solvents the temperature dependence of the equilibrium constant was analyzed. The dependence of the apparent weight-average molecular weight on enzyme concentration shows always the same general shape as that found earlier in 0.067 M phosphate buffer at pH 7.6. Each curve can be fitted assuming an open association-dissociation equilibrium with one single equilibrium constant for all steps and with one single value for the second virial coefficient. Within the limit of the experimental error, the second virial coefficient is found to be the same for all systems (8 -9 nmol x 1 x gp2), whereas the equilibrium constants vary by orders of magnitude.At low ionic strength a very small temperature dependence was found with a reduced association at higher temperatures. At high ionic strength a temperature maximum of the equilibrium constant is observed. Preferential phosphate binding is indicated by the high values of the refractive index increment.The organic solvents investigated here (ethylene glycol, dioxane) cause a dissociation into the unimers (oligomers). The equilibrium constant decreases from 9 x lo5 M-' i n aqueous phosphate buffer to 5.2 x lo3 M-' i n 6 M ethylene glycol and to 2.4 x lo5 M-l in 2 M ethylene glycol. The effects obtained with 5 % dioxane (0.57 M) are similar to that with 2 M ethylene glycol. In both cases a sharp decrease of the association with increasing temperature is observed.The reaction enthalpy and the reaction entropy of the association reaction decrease with decreasing temperature in all solvents, the enthalpy being positive in the absence and negative in the presence of organic solvents. The free enthalpy of reaction, however, increases with increasing temperature in buffers of high and low ionic strengths but decreases in the presence of organic solvents. The association reaction at high ionic strength appears mainly to be entropydriven whereas in the presence of the organic solvents the reaction seems to be energy-driven at all temperatures, It is suggested that the organic solvents interfere with the hydrophobic interactions between the unimers. The positive reaction enthalpy in the presence of high salt concentrations indicates that the attractive electrostatic forces are largely shielded, whereas shielding is imperfect for the repulsive forces which even seem to be enhanced. This could be due to a preferential binding of anions which would tend to increase the negative net charge and could explain the difference observed between the effects of phosphate and chloride solutions of identical ionic strength. ~-This work has been described in a preliminary report [1,2]. The previous paper of this series has appeared in this Enzyme. Glutamate dehydrogenase or L-glutamate : NAD(P) oxidoreductase (deaminating) (EC 1.4.1.3).journal [3].Eur. J. Biochem. 49 (19...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Studies of Glutamate Dehydrogenase

Gauper

Markau

Sund

1974

European Journal of Biochemistry

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“…ADP is known to activate the glutamate dehydrogenase (GluDH EC 1.4.1.3) reaction above pH 7 [2,3] whereas below this pH ADP is inhibitory. Therefore, it has been postulated that at low pH values, ADP instead of binding to an activator site binds to the inhibitor site which at high pH values is used by GTP [4] Since NAD' shows a strong self-activating effect [5], the activation by ADP is dependent on NAD' concentration.…”

Section: Introductionmentioning

confidence: 99%

Kinetic studies on the mechanism of the action of ADP on the glutamate dehydrogenase reaction

1972

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“…The RF values are the means of four runs using bromophenol blue as reference (marked before staining with a copper wire). The following marker proteins were used: serum albumin ( 0 , molecular weight 69000 [23]), glutamic acid dehydrogenase (A, molecular weight 55000 [24]), transferrin (0, molecular weight 78 000 [25]) and catalase (A, molecular weight 61000 [26]). The molecular weights of the two subunits o ( and j3 of phenylalanyl-tRNA synthetase were determined by the method of Weber and Osborn [20].…”

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Phenylalanyl‐tRNA Synthetase from Yeast

Berther¹,

Mayer²,

Dutler³

1974

European Journal of Biochemistry

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It has been shown that the initial rates, w, of the formation of aminoacyl-tRNA, catalyzed by phenylalanine-tRNA synthetase, do not depend on the concentration of the substrates ATP and phenylalanine in a simple way. In contrast a normal Michaelis-Menten behaviour has been observed for the substrate tRNA. The reciprocal plots of w versus v/[S] for ATP and phenylalanine are curved and only approach linearity at low and high concentrations of the varied substrate. From these limiting slopes two Km and two V values corresponding to low and high concentrations of ATP and phenylalanine have been determined. Since the reciprocal plots for tRNA are linear, only one Km and Vvalue has been obtained for this substrate. In order to show that this kinetic behaviour is an intrinsic property of the enzyme and not an artifact, a new procedure for the purification of the enzyme has been devised and the purity and stability of the preparation carefully checked. In addition the influence of ammonium ions and of spermine on the initial rates has been measured to verify that the non-linearity of the reciprocal plots is not the result of the substrate acting as a base. Inhibition experiments with tRNA,, (a modified tRNA obtained from native tRNA by periodate oxidation of the 2': 3'-diol in the 3'-terminal ribose to the corresponding dialdehyde) at low and high phenylalanine concentrations have shown that it is also not a consequence of two fundamentally different reaction mechanisms being followed at the two concentration ranges. On the basis of this and other evidence it is proposed that two mutually interacting sites are engaged, of which one or both are operative, depending on the concentration of the substrates. Studies of pyrophosphate inhibition at low tRNA concentration have provided evidence that the aminoacylation of tRNA proceeds via two catalytic steps in which aminoacyl-AMP occurs as an intermediate. At high tRNA concentrations it has been found that this substrate is adding prior to the dissociation of pyrophosphate.

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Quaternary Structure and Enzymic Properties of Beef Liver Glutamate Dehydrogenase

Cited by 14 publications

References 33 publications

Studies of Glutamate Dehydrogenase

Studies of Glutamate Dehydrogenase

Kinetic studies on the mechanism of the action of ADP on the glutamate dehydrogenase reaction

Phenylalanyl‐tRNA Synthetase from Yeast

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