Allosteric activation of brain hexokinase by magnesium ions and by magnesium-ion–adenosine triphosphate complex

H. S. Bachelard

doi:10.1042/bj1250249

Allosteric activation of brain hexokinase by magnesium ions and by magnesium-ion–adenosine triphosphate complex

Biochemical Journal ◽

10.1042/bj1250249 ◽

1971 ◽

Vol 125 (1) ◽

pp. 249-254 ◽

Cited By ~ 20

Author(s):

H. S. Bachelard

Keyword(s):

Binding Sites ◽

Magnesium Ion ◽

Magnesium Ions ◽

Allosteric Activation ◽

Substrate Saturation ◽

Number Of Binding Sites ◽

High Concentrations ◽

Substrate Binding Sites ◽

Hill Plots ◽

Allosteric Activator

1. Substrate-saturation curves of brain hexokinase for MgATP2− were sigmoidal at sub-saturating concentrations of glucose when the Mg2+/ATP ratio was maintained at 1:1. Under identical conditions, except that Mg2+ was present in excess, hyperbolic curves were observed. 2. The number of binding sites (calculated from Hill plots) is 1.8 at a Mg2+/ATP ratio 1:1, and 1.0 with excess of Mg2+. The apparent Km for MgATP2− is 6.5×10−4m at a Mg2+/ATP ratio 1:1, and 3.5×10−4m with excess of Mg2+. 3. Interdependence between substrate-binding sites was indicated by the effects of varying the concentration of glucose. The sigmoidality and deviation from Michaelis–Menten kinetics at a Mg2+/ATP ratio 1:1 became less pronounced with increasing glucose concentration. Also, although substrate-saturation curves for glucose were hyperbolic when the Mg2+/ATP ratio was 1:1, reciprocal plots were non-linear. These were linear with excess of Mg2+. 4. High concentrations of Mg2+ (Mg2+/ATP ratios above 5:1) were inhibitory. 5. The results are taken to indicate homotropic co-operative binding of MgATP2− and that Mg2+ is an allosteric activator. Possible implications in regulation are discussed.

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Activation of brain hexokinase by magnesium ions and by magnesium ion–adenosine triphosphate complex

Biochemical Journal ◽

10.1042/bj1300063 ◽

1972 ◽

Vol 130 (1) ◽

pp. 63-69 ◽

Cited By ~ 27

Author(s):

Daniel L. Purich ◽

Herbert J. Fromm

Keyword(s):

Simple Model ◽

Complex Formation ◽

Kinetic Data ◽

Alternative Explanation ◽

Metal Substrate ◽

Magnesium Ion ◽

Magnesium Ions ◽

Substrate Complex ◽

The Brain ◽

Hill Plots

1. An alternative explanation for the kinetic data obtained by Bachelard (1971) for the brain hexokinase reaction is presented. 2. Apparently sigmoidal saturation curves for MgATP2− based upon Bachelard's (1971) studies can be corrected to hyperbolic curves by use of a stability constant for MgATP2− complex formation. 3. A number of other effects related to the concentration-dependent stability of the MgATP2− complex and to the presence of the inhibitory free uncomplexed ATP4− concentration are also explained in terms of a non-allosteric role for either Mg2+ or MgATP2− fully consistent with a number of previous reports on this enzyme. 4. A brief discussion of the validity of Hill plots in studies of multisubstrate co-operative enzymes is presented. 5. A simple model is presented that demonstrates how enzymes obeying Michaelis–Menten kinetics can demonstrate sigmoidal velocity responses if the true substrate of the reaction is the metal–substrate complex.

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Phosphofructokinase: structure and control

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1981.0059 ◽

1981 ◽

Vol 293 (1063) ◽

pp. 53-62 ◽

Cited By ~ 139

Keyword(s):

Active Site ◽

Binding Sites ◽

Bacillus Stearothermophilus ◽

Phosphoryl Transfer ◽

Active Conformation ◽

Allosteric Activation ◽

Ligand Binding Sites ◽

And Control ◽

Allosteric Activator ◽

Cooperative Kinetics

Phosphofructokinase from Bacillus stearothermophilus shows cooperative kinetics with respect to the substrate fructose-6-phosphate (F6P), allosteric activation by ADP, and inhibition by phosphoenolpyruvate. The crystal structure of the active conformation of the enzyme has been solved to 2.4 A resolution, and three ligand-binding sites have been located. Two of these form the active site and bind the substrates F6P and ATP. The third site binds both allosteric activator and inhibitor. The complex of the enzyme with F6P and ADP has been partly refined at 2.4 A resolution, and a model of ATP has been built into the active site by using the refined model of ADP and a 6 A resolution map of bound 5'-adenylylimidodiphosphate (AMPPNP). The y-phosphate of ATP is close to the 1-hydroxyl of F6P, in a suitable position for in-line phosphoryl transfer. The binding of the phosphate of F6P involves two arginines from a neighbouring subunit in the tetramer, which suggests that a rearrangement of the subunits could explain the cooperativity of substrate binding. The activator ADP is also bound by residues from two subunits.

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Phospholipid Topography Coagulation

10.1055/s-0038-1652952 ◽

1981 ◽

Author(s):

H C Hemker ◽

R F A Zwaal ◽

J Rosing ◽

G van Dieyen ◽

E Bevers

Keyword(s):

Binding Sites ◽

Thrombin Generation ◽

Coagulation Factors ◽

Phosphatidyl Serine ◽

Model Systems ◽

Factor X ◽

Flip Flop ◽

Outer Leaflet ◽

Number Of Binding Sites ◽

High Concentrations

Phospholipids play a role in thrombin generation because the constitute the support on which both the factor X activating enzyme (“tenase”) and the prothrombin activating enzyme (proth rcmbinase) are built.In model systems with pure synthetic phospholipids it can be shown that the binding of the coagulation factors to a phospholipid surface is a function of both phospholipid charge and membrane fluidity. A relatively high molefraction of phosphatidyl serine (PS) is a prerequisite for optimal binding of the vitamin K dependant factors. Precise measurements on the binding constant and the number of binding sites of factor X show that at molefractions of PS between 0.01 and 0.25 aproximately one PS binds per Gla-residue. Above 0.25% of PS vesicle agregation occurs.The normal intact circulating platelet shows a molefraction of PS of ̴ 0.03 in the outer leaf let of its plasma membrane and therefore hardly if at all stimulates thrombinformation.Upon activation by thrombin (2nM) together with collagen, (10μg/ml) the outer leaflet aquires increasing amounts of PS without platelet disruption taking place.This suggests a flip-flop movement of phospholipids across the membrane as part of the activation reaction of platelets. It can be shown that optimal “tenase” supporting phospholipid compositions appear before prothrombine supporting ones. The activation by thrombin and collagen is only partly inhibited by prostacyclin even at high concentrations.

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The Estimation of the Number of Binding Sites for Antibody on Antigen Molecules with Reference to Factor VIII and its Antibody

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647956 ◽

1976 ◽

Vol 35 (02) ◽

pp. 274-288 ◽

Cited By ~ 2

Author(s):

Judith Pool ◽

Rosemary Biggs ◽

R. G Miller

Keyword(s):

Factor Viii ◽

Binding Sites ◽

Theoretical Basis ◽

Human Antibody ◽

Rabbit Antibody ◽

Number Of Binding Sites ◽

Theoretical Considerations

SummaryThe theoretical basis for determining the number of antibody sites on antigen molecules is examined. The theoretical considerations are applied to factor VIII molecules. Examples based on data available at the Oxford Haemophilia Centre are calculated to illustrate the approach. It is concluded that there are few sites on each factor VIII molecule for human antibody. The three antibodies for which reasonable data were available suggest 1–3 sites for human antibody. The data for rabbit antibody suggest 5–6 sites per factor VIII molecule.

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Abnormal Platelet Serotonin Uptake and Binding Sites in Myeloproliferative Disorders

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661099 ◽

1984 ◽

Vol 51 (03) ◽

pp. 349-353 ◽

Cited By ~ 9

Author(s):

C Caranobe ◽

P Sié ◽

F Fernandez ◽

J Pris ◽

S Moatti ◽

...

Keyword(s):

Binding Sites ◽

High Capacity ◽

Specific Inhibitor ◽

Myeloproliferative Disorders ◽

Normal Subjects ◽

Binding Data ◽

Full Explanation ◽

Number Of Binding Sites ◽

5 Ht Uptake ◽

Kinetics Of

SummaryA simultaneous investigation of the kinetics of serotonin (5 HT) uptake and of binding sites was carried out in the platelets of normal subjects and of 10 patients affected with various types of myeloproliferative disorders (MD). The 5 HT uptake was analysed according to the Lineweaver-Burk and the Eadie-Hofstee methods. With the two methods, the patient’s platelets exhibited a dramatic reduction of the Vi max and of the Km; in some patients the Eadie-Hofstee analysis revealed that a passive diffusion phenomenon is superimposed on the active 5 HT uptake at least for the higher concentration used. The binding data were analysed with the Scatchard method. Two classes of binding sites (high affinity - low capacity, low affinity - high capacity) were found in normal subjects and patients. Pharmacological studies with imipramine, a specific inhibitor of 5 HT uptake, suggested that both the sites are involved in 5 HT uptake. The number of both binding sites was significantly decreased in patient’s platelets while the affinity constants of these binding sites were not significantly reduced in comparison with those of the control subjects. No correlations were found between Vi max, Km and the number of binding sites. These results suggest that a reduction in the number of platelet membrane acceptors for 5 HT commonly occurs in myeloproliferative disorders but does not provide a full explanation of the uptake defect.

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Functional Involvement of Thrombospondin in Platelet Aggregation Induced by Low Versus High Concentrations of Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661464 ◽

1986 ◽

Vol 55 (01) ◽

pp. 136-142 ◽

Cited By ~ 4

Author(s):

K J Kao ◽

David M Shaut ◽

Paul A Klein

Keyword(s):

Platelet Aggregation ◽

Binding Sites ◽

Inhibitory Effect ◽

Activated Platelets ◽

Low Concentrations ◽

Platelet Binding ◽

Functional Involvement ◽

Thrombin Activation ◽

Platelet Aggregates ◽

High Concentrations

SummaryThrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (≤0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (≥60 μg/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (≥60 μg/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin. In conclusion, our findings demonstrate that TSP is functionally important for platelet aggregation induced by low (≤0.05 U/ml) but not high (≥0.1 U/ml) concentrations of thrombin. High concentrations of exogenous TSP may univalently saturate all its platelet binding sites consequently interfering with TSP-crosslinking of thrombin-activated platelets.

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