scholarly journals Kinetic studies of dogfish liver glutamate dehydrogenase

1979 ◽  
Vol 177 (2) ◽  
pp. 449-459 ◽  
Author(s):  
A H Electricwala ◽  
F M Dickinson
Keyword(s):  
Glutamate Dehydrogenase ◽  
Kinetic Data ◽  
Reductive Amination ◽  
Initial Rate ◽  
Kinetic Studies ◽  
Bovine Liver ◽  
Binding Studies ◽  
Degree Of Protection ◽  
Binding Sequence ◽  
Kinetic Features

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621–631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver–Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5′-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.

scholarly journals Kinetic studies of glutamate dehydrogenase. The reductive amination of 2-oxoglutarate

1970 ◽  
Vol 118 (3) ◽  
pp. 409-419 ◽  
Author(s):  
P. C. Engel ◽  
K. Dalziel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Reductive Amination ◽  
Initial Rate ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Random Order ◽  
Order Type ◽  
Reverse Reaction ◽  
Enzyme Subunits ◽  
High Concentrations

1. Kinetic studies of the reductive amination of 2-oxoglutarate catalysed by glutamate dehydrogenase with NADH and NADPH as coenzyme were made at pH7.0 and pH 8.0. The concentrations of both substrates and coenzymes were simultaneously varied over wide ranges. Lineweaver–Burk plots with respect to each substrate and coenzyme were linear, except that with high concentrations of 2-oxoglutarate or coenzyme inhibition occurred. There was no evidence of the negative homotropic interactions between the enzyme subunits that were revealed in previous kinetic studies of the reverse reaction. 2. The initial-rate results are shown to be inconsistent with any of the six possible compulsory-order mechanisms for this three-substrate reaction, and it is concluded that a random-order mechanism is the most likely one. On the basis of this mechanism, the dissociation constants of all the binary, ternary and quaternary complexes of the enzyme and substrates are calculated from initial-rate parameters. 3. The results are discussed in relation to those of earlier workers who concluded that the mechanism is of the compulsory-order type.

scholarly journals The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides

1972 ◽  
Vol 126 (4) ◽  
pp. 975-984 ◽  
Author(s):  
K. Dalziel ◽  
R. R. Egan
Keyword(s):  
Glutamate Dehydrogenase ◽  
Dissociation Constants ◽  
Equilibrium Dialysis ◽  
Kinetic Studies ◽  
Binding Studies ◽  
Purine Nucleotides ◽  
Active Centre ◽  
Sodium Phosphate Buffer ◽  
Low Concentrations ◽  
High Concentrations

1. The binding of NAD+ and NADP+ to glutamate dehydrogenase has been studied in sodium phosphate buffer, pH7.0, by equilibrium dialysis. Approximate values for the dissociation constants are 0.47 and 2.5mm respectively. For NAD+ the value agrees with that estimated from initial-rate results. 2. In the presence of the substrate analogue glutarate both coenzymes are bound more firmly, and there is one active centre per enzyme subunit. The binding results cannot be described in terms of independent and identical active centres, and binding is stronger at low coenzyme concentrations than at high concentrations. Either the six subunits of the oligomer are not identical or there are negative interactions between them in the binding of coenzymes in ternary complexes with glutarate. The latter explanation is favoured. 3. The binding studies support the conclusions drawn from earlier kinetic studies of the glutamate reaction. 4. ADP and GTP respectively decrease and increase the affinity of the enzyme for NAD+ and NADP+, in both the presence and absence of glutarate. The negative binding interactions in the presence of glutarate are abolished by ADP, which decreases the affinity for the coenzymes at low concentrations of the latter. 5. In the presence of glutarate, GTP and NAD+ or NADP+, the association of enzyme oligomers is prevented, and the solubility of the enzyme is decreased; the complex of enzyme and ligands readily crystallizes. 6. The results are discussed in relation to earlier kinetic studies.

scholarly journals Binding Studies of a Spin-Labelled Oxidized Coenzyme to Bovine-Liver Glutamate Dehydrogenase

1977 ◽  
Vol 72 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Alt ZANTEMA ◽  
Wolfgang E. TROMMER ◽  
Herbert WENZEL ◽  
George T. ROBILLARD
Keyword(s):  
Glutamate Dehydrogenase ◽  
Bovine Liver ◽  
Binding Studies

scholarly journals Kinetic studies of bovine liver carbamoyl phosphate synthetase

1974 ◽  
Vol 141 (3) ◽  
pp. 807-816 ◽  
Author(s):  
Keith R. F. Elliott ◽  
Keith F. Tipton
Keyword(s):  
Initial Rate ◽  
Kinetic Studies ◽  
Bovine Liver ◽  
Kinetic Mechanism ◽  
Rate Equations ◽  
British Library ◽  
Rate Data ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Lending Library

A through study of initial-rate data has been made on carbamoyl phosphate synthetase from bovine liver. On the basis of the results the order of substrate binding to the enzyme is ATPMg followed by HCO3−, ATPMg and NH4+. A model for the enzymic mechanism is proposed, and the rate equations describing it are presented. Details of the derivation of the initial-rate equation for the kinetic mechanism proposed have been deposited as Supplementary Publication SUP 50032 (6 pages) at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

scholarly journals Kinetic studies of glutamate dehydrogenase with glutamate and norvaline as substrates. Coenzyme activation and negative homotropic interactions in allosteric enzymes

1969 ◽  
Vol 115 (4) ◽  
pp. 621-631 ◽  
Author(s):  
P. C. Engel ◽  
K. Dalziel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Phosphate Buffer ◽  
Initial Rate ◽  
Kinetic Studies ◽  
Glutamate Concentration ◽  
Enzyme Subunits ◽  
New Type ◽  
Michaelis Constants ◽  
Allosteric Enzymes ◽  
Absence Of Evidence

1. Kinetic studies of glutamate dehydrogenase were made with wide concentration ranges of the coenzymes NAD+ and NADP+ and the substrates glutamate and norvaline. Initial-rate parameters were evaluated. 2. Deviations from Michaelis–Menten behaviour towards higher activity were observed with increasing concentrations of either coenzyme with glutamate as substrate, but not with norvaline as substrate. 3. In phosphate buffer, pH7·0, Lineweaver–Burk plots with either coenzyme as variable and a constant, large glutamate concentration showed three or four linear regions of different slope with relatively sharp discontinuities. Maximum rates obtained by extrapolation and Michaelis constants for the coenzymes increased in steps with increase of coenzyme concentration. 4. In the absence of evidence of heterogeneity of the enzyme and coenzyme preparations, the results are interpreted in terms of negative homotropic interactions between the enzyme subunits. It is suggested that sharp discontinuities in Lineweaver–Burk plots or reciprocal binding plots may be characteristic of this new type of interaction, which can be explained in terms of an Adair–Koshland model, but not by the model of Monod, Wyman & Changeux.

scholarly journals The kinetic mechanism of ox liver glutamate dehydrogenase in the presence of the allosteric effector ADP. The oxidative deamination of L-glutamate

1984 ◽  
Vol 223 (1) ◽  
pp. 161-168 ◽  
Author(s):  
D P Hornby ◽  
M J Aitchison ◽  
P C Engel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Initial Rate ◽  
Potassium Phosphate ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Major Effect ◽  
Oxidative Deamination ◽  
Allosteric Effector ◽  
Steady State Kinetic ◽  
State Kinetic

In steady-state kinetic studies of ox liver glutamate dehydrogenase in 0.11 M-potassium phosphate buffer, pH7, at 25 degrees C, the concentration of ADP was varied from 0.5 to 1000 microM. Inhibition was observed except when the concentrations of both glutamate and coenzyme were high, when activation was seen. With NAD+ or NADP+ as coenzyme, 200 microM-ADP was sufficient to saturate the enzyme with respect to the major effect of this nucleotide. In the presence of 210 microM-ADP, widely varied concentrations of coenzyme give linear Lineweaver-Burk plots, in marked contrast with results obtained previously for kinetics without ADP. This has allowed evaluation for the reaction with NAD+, NADP+ and acetylpyridine-adenine dinucleotide (315 microM-ADP in the last case) of all four initial rate parameters, i.e. the phi coefficients in the equation: (Formula: see text) where A is coenzyme and B is glutamate. The relative constancy of phi B and of phi AB/phi A with the different coenzymes point to a compulsory-order mechanism with glutamate as the leading substrate. This conclusion, though unexpected, agrees well with various previous observations on the binding of oxidized coenzyme.

Factor V from Human Plasma

1961 ◽  
Vol 05 (01) ◽  
pp. 001-020
Author(s):  
Douglas M. Surgenor ◽  
Nancy A. Wilson ◽  
Anne S. Henry
Keyword(s):  
Human Serum ◽  
Human Plasma ◽  
Kinetic Data ◽  
Human Factor ◽  
Kinetic Studies ◽  
Partial Purification ◽  
Factor V ◽  
Two Stage ◽  
Plasma Factor ◽  
Tissue Thromboplastin

SummaryA method is described for the partial purification of a human plasma factor which accelerates the conversion of prothrombin to thrombin in the presence of tissue thromboplastin. This factor may be dried from the frozen state, and may be kept in stable dry form for long periods of time. The quantitative assay of this activity is done in a classical two-stage prothrombin system using tissue thromboplastin and calcium. From its properties, it is concluded that this activity corresponds to factor V, labile factor and plasma Ac-globulin.Chemical and kinetic studies reveal that human factor V is active in plasma and is destroyed by thrombin. Human serum has little or no factor V activity.These results thus fail to support the postulated activation of factor V during clotting. All of the kinetic data are consistent with an enzymatic role for factor V in the formation of tissue prothrombin activator (thromboplastin).

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