Separate effects of Mg2+, MgATP, and ATP4- on the kinetic mechanism for insulin receptor tyrosine kinase.

The separate effects of the equilibrium species Mg2+, MgATP substrate, and ATP4- on the reaction catalyzed by insulin receptor tyrosine kinase were examined. The separated kinetic constants show that the K0.5 value for Mg2+ decreased from 23 to 0.43 mM and the Hill coefficient for Mg2+ (hMg2+) decreased from 1.43 to 0.668 when the concentration of ATPT (MgATP + ATP4-) was increased from 50 to 1000 microM. The apparent Ki for ATP4- increased from 0.20 to 136 microM and the Hill coefficient for ATP4- (hATP4-) decreased from 1.41 to 0.82 as the concentration of total ATP (ATPT) increased. These findings suggest that the [ATP4-]/[Mg2+] ratio modulates the shift from positive to negative cooperativity. It was also shown that the apparent affinity of the kinase for MgATP increased as the concentration of free Mg2+ increased and that the apparent affinity of the kinase for free Mg2+ increased as the concentration of MgATP substrate increased. Thus, Mg2+ and MgATP interact with the kinase in a mutually inclusive manner which leads to an increase in the ratio of the enzyme (E) rate-limiting species, [Mg-E-MgATP]/[E-MgATP]. Free ATP4- not only acts as a competitive inhibitor of the substrate but also decreases the relative concentration of Mg-E-MgATP. ATPT-dependent activation of the kinase is, therefore, a result of MgATP's increasing the affinity of the kinase for Mg2+, thereby leading to saturation of the enzyme with Mg2+ at lower concentrations of the divalent metal. This results in an increase in the [Mg-E-MgATP]/[E-MgATP] ratio, and therefore decreases saturation of the kinase with ATP4- inhibitor, not only at the active site but also at a kinetically distinct regulatory site. This kinetic relationship allows not only for the mutually inclusive interaction between Mg2+ and MgATP, but also for the mutually exclusive interaction toward ATP4-, hence indicating that the effect of Mg2+ will be to form an enzyme complex (Mg-E) which will have a higher affinity for MgATP substrate and a lower affinity for ATP4- than E alone. The role of the equilibrium concentrations of Mg-E,E, and ATP-E on the activation of insulin receptor tyrosine kinase is discussed which may account, at least in part, for modulation of cooperativity and the metal-dependent increase in turnover (VM).

Role of divalent metals in the kinetic mechanism of insulin receptor tyrosine kinase.

The kinetic and thermodynamic interrelationships of peptide substrate (Val5-angiotensin 11), metal-ATP, and divalent metal cations with rat liver insulin receptor tyrosine kinase (IRTK) were investigated. Results of the initial rate studies with varying peptide and MnATP substrates indicates that the kinetic mechanism for IRTK is of the sequential type and therefore rules out a ping pong Bi Bi pathway. Hence, peptide substrate and metal-ATP bind to the kinase prior to the release of products. MnADP was a linear competitive inhibitor of MnATP and a noncompetitive inhibitor of peptide substrate. A synthetic tyrosine-containing pentapeptide, Glu-Glu-Phe-Tyr-Phe (EEFYF), was a linear competitive inhibitor of peptide substrate and a noncompetitive inhibitor of MnATP. Accordingly, the data show that phosphorylation of peptide substrate occurs via a rapid random equilibrium Bi Bi mechanism in which the kinase has the potential to react initially with either of the two substrates. In contrast, divalent metal cations and metal-ATP were found to interact with the kinase in a mutually inclusive manner, with metal binding to the kinase prior to MnATP. It was also found that divalent metals increase the affinity of the kinase for metal-ATP but do not affect the affinity of IRTK for metal-ADP product. Hence, divalent metals, during the reaction of association of enzyme with one of its substrates to form the binary complex, increase the relative concentration of E-ATP complex versus E-peptide complex, thus introducing a thermodynamic-dependent ordering for the interaction of substrates with the enzyme. To investigate the thermodynamics of this system, we assumed that under initial conditions the kinetic data we obtained reflected the association constants of reactants with the enzyme.

Role of divalent metals in the activation and regulation of insulin receptor tyrosine kinase.

Multiple equilibrium equations were solved to separate the individual effects of ionic divalent metals, free nucleotides and their chelated species on insulin receptor tyrosine kinase (IRTK). Basal IRTK is activated by divalent metal cations when present in excess of that required for substrate formation, indicating the presence of a divalent cation-dependent regulatory site on the kinase. The activatory order for basal activity was Mn2+ greater than Co2+ greater than Mg2+ and Ca2+ = 0. The insulin-dependent activation of IRTK was minimal in the absence of excess free divalent metal, even when the concentration of MnATP or MgATP substrate present exceeded the apparent Km of the kinase. The activatory order for insulin-dependent activation of IRTK changed to Mg2+ greater than Mn2+ and Co2+ = 0. The titration of the MnCl2 saturation response at several concentrations of MgCl2 revealed that the insulin-dependent response of IRTK increases as a function of increasing MgCl2, while basal activity was unaffected. This enhancement of the responsiveness to insulin in the presence of both cations was not due to differing affinities of the kinase for substrate, as evidenced by nearly identical apparent Km values for MnATP and MgATP. The Mg2+-dependent increase in the response of the kinase to insulin may be due to Mg2+ inducing a stronger coupling between receptor and kinase than that observed with Mn2+ alone. The plotting of the effect of several concentrations of free divalent metals on substrate saturation curves revealed that an increase in either of the reactants increased the affinity of the insulin-activated kinase for the other respective reactant. Accordingly, free divalent metal and metal-ATP substrate interact with IRTK in a mutually inclusive manner. CaCl2 saturation curves in the presence of constant MnCl2 and increasing MgCl2 showed that the affinity of IRTK for Ca2+ decreases and the affinity for CaATP increased with increasing Mg2+. Our data suggests that IRTK contains three sites for interaction with divalent metal cations: a MeATP (active) site, a regulatory site, and a metal-dependent site acting to couple the receptor with the kinase.

A coupling mechanism to inter-relate regulatory with haem-haem interactions of haemoglobin.

The Me2+ (Zn2+ or Mg2+)-dependent increase in the oxygenation of Haemoglobin (Hb) is examined theoretically using an overview in scale of the interface of the alpha 1 beta 2 dimer of Hb. A model was developed showing that 2,3 DPG and Me2+ would maintain a pH-modulated mutually exclusive relationship as ligands of Hb because they both share a common binding His-beta (2)143. A symmetric relationship applies to alpha 2 beta 1. One atom of metal, within each dimer, would compete in turn with each of the haem groups to sequentially dislocate both proximal histidines during the transition from deoxyHb to oxyHb. Hence, the pH-modulated reaction path of the multiple equilibrium of Hb would integrate the interactions of either one 2,3 DPG with four histidines for the increase of hindrance effects, or the interactions of two Me2+ with four haem groups to increase their affinity for O2. The model predicts that increases in pO2 would increase the affinity of Hb for Me2+ and therefore for O2, simultaneously decreasing its affinity for 2,3 DPG and protons.

Stimulation of the hexose monophosphate pathway in the human erythrocyte by Mn2+: evidence for a Mn2+-dependent NADPH peroxidase activity.

In human RBC hemolysates, Mn2+ was found to stimulate the HMP as determined by the release of 14CO2 from [1-14C]glucose, providing activities of 125, 200, and 300% of basal at Mn2+ concentrations of 1, 10, and 100 mM, respectively. To explore the possibility that this stimulatory effect upon the HMP is a result of redox recycling of NADPH, RBC hemolysates were used to study NADPH oxidation. Mn2+, alone or in combination with a free radical-generating system, did not enhance the ability of hemolysates to oxidize NADPH. However, hemolysates + 10 mM H2O2 brought about a 10-fold increase in NADPH oxidation (0.51 +/- 0.05 nmole/min to 5.67 +/- 0.84 nmole/min) and the addition of 10 mM Mn2+ to this system increased the rate of oxidation to 34.10 +/- 2.97 nmole/min. Boiled hemolysates, either in the presence or absence of Mn2+, had some residual catalytic activity.

Cyclic nucleotide metabolism in the human erythrocyte.

Erythrocytes, which show little or no guanylate or adenylate cyclase activity, take up cyclic nucleotides from blood. Studies were done by incubating human erythrocytes in isotonic medium with the dibutyryl derivatives of cAMP and cGMP and in a hypotonic medium in which the cells are partially hemolyzed and, therefore, freely permeable to cAMP and cGMP. At cAMP and cGMP concentrations of 50 microM and above, the amount of 14CO2 generated from 1-14C-glucose was decreased significantly. This effect was suppressed by 4.6 mM theophylline. Inosine and ribose, which are catabolites of cAMP and cGMP also decreased formation of 14CO2 from 1-14C-glucose. Accordingly, it is postulated that in the absence of theophylline, the activity of phosphodiesterase resulted in AMP and GMP formation. These mononucleotides enter into the purine salvage pathways to form ribose phosphate. Additionally, the production of lactate and 2,3-diphosphoglycerate (2,3-DPG) was measured in human erythrocytes after incubation with the dibutyryl derivatives of cAMP (bt2-cAMP) and cGMP (bt2-cGMP). At a concentration of 0.1 microM, bta2-cGMP inhibits lactate production at 60 min (p less than 0.01). Slight increases in 2,3-DPG were not statistically significant. Catabolism of cyclic nucleotides may prevent diffusion equilibria allowing for the erythrocyte's continuous uptake of cyclic nucleotides and providing a detoxification mechanism that could compensate for conditions in which elevations of these substances are observed.

Cyclic GMP in the human erythrocyte. Intracellular levels and transport in normal subjects and chronic hemodialysis patients.

We have studied the intracellular levels of cyclic GMP in the red blood cells of chronic hemodialysis patients before and after dialysis treatment and in normal subjects. Cyclic GMP is present at levels (mean +/- 1SD) of 19.6 +/- 4.7 and 18.4 +/- 5.8 nM/L before and after treatment, respectively. The levels in normal subjects are 3.6 +/- 0.9. The uptake of cyclic GMP was also studied and the data demonstrate that the red blood cell is slightly permeable to cyclic GMP, suggesting that cyclic GMP uptake could substitute for guanylate cyclase activity in the human erythrocyte.

A model for the regulation of brain adenylate cyclase by ionic equilibria.

Multiple-equilibrium equations were solved to investigate the individual and separate effects of Mg2+, Mn2+, Ca2+, ATP4-, and their complexes on the kinetics of brain adenylate cyclase. The effects of divalent metals and/or ATP4- (in excess of their participation in complex formation) were determined and, from the corresponding apparent affinity values, the following kinetic constants were obtained: Km(MgATP) = 1.0 mM, Ki(ATP4-) = 0.27 mM, Km(MnATP) = 0.07 mM, and Ki(CaATP) = 0.015 mM. MgATP, MnATP, ATP4-, and CaATP were shown to compete for the active site of the enzyme. Hence, it is proposed that endogenous metabolites with a strong ligand activity for divalent metals, such as citrate and some amino acids, become integrated into a metabolite feedback control of the enzyme through the release of ATP4- from MgATP. Ca2+ fluxes may participate in the endogenous regulation of adenylate cyclase by modifying the level of CaATP. The free divalent metals show an order of affinity K0.5(Ca2+) = 0.02 mM, K0.5(Mn2+) = 3.8 mM, K0.5(Mg2+) - 4.7 mM, and an order of activity Mn2+ greater than Mg2+ greater than Ca2+. The data indicate that Mn2+ and Mg2+ ions may compete for a regulatory site distinct from the active site and increase Vm without changing Km(MgATP), Km(MnATP), or Ki(ATP4-). The interactions of ATP4- and CaATP, which act as competitive inhibitors of the reaction of the enzyme with the substrates MgATP and MnATP, and Mg2+ and Mn2+, which act as activators of the enzyme in the absence of hormones, are shown to follow the random rapid equilibrium BiBi group-transfer mechanism of Cleland with the stipulation that neither Mg2+ nor Mn2+, in excess of their respective participation in substrate formation, are obligatorily required for basal activity. ATP4- and CaATP are involved in dead-end inhibition. For MgCl2 saturation curves at constant total ATP concentration, the computer-generated curves based on the RARE BiBi model predict a change in the Hill cooperativity h from a basal value of 2.6, when Mg2+ is not obligatorily required, to 4.0 when the addition of hormones or neurotransmitters induces an obligatory requirement for Mg2+.

Effects of noradrenaline on the activation and the stability of brain adenylate cyclase.

At constant 1 mM-ATP, the Mg2+-saturation curves for adenylate cyclase (EC 4.6.1.1) particulate preparations obtained from corpus striatum and cortex tissues of rat brain show that addition of 0.1 mM-noradrenaline increases the apparent Vmax. for Mg2+ by 300% in corpus striatum particles, and by 280% in cortex particles. At 10 mM-MgCl2, the addition of 0.1 mM-noradrenaline increased by 800% the adenylate cyclase activity of corpus striatum particles. At all Mg2+ concentrations, the addition of 0.3 mM-CaCl2 suppressed the noradrenaline-induced stimulation of adenylate cyclase of corpus striatum particles, and even resulted in a strong inhibition of the activating effect of Mg2+ itself on adenylate cyclase of corpus striatum particles, and even resulted in a strong inhibition of the activating effect of Mg2+ itself on adenylate cyclase activity of cortex particles. The addition of noradrenaline during a 3 h preincubation of particle preparations of brain cortex at 38 degrees C decreased by more than 4-fold the half-life of the decay of adenylate cyclase activity. The addition of MgATP protected against noradrenaline-induced inactivation.

Hormonal control of fat cells adenylate cyclase.

Fat cells were preincubated for 2 h in the presence and absence of growth hormone (GH) and Dexamethasone (Dex) before the addition of increasing concentrations of either epinephrine, theophylline or glucagon and final incubation of the cells for an additional 5 minutes. GH and Dex increased by 85%, 28% and 72%, respectively, the cAMP levels reached in the sole presence of 10(-5)M epinephrine, 10(-2)M theophylline or 5 X 10(-5)M glucagon. An adenylate cyclase particulate preparation shows that epinephrine decreases Km from 2mM to 0.6 MM and increases Vmax and the strength of interaction value (n) from 0.91 to 1.75.

Hypothesis on the role of liganded states of proteins in energy transducing systems.

In energy transducing systems the direction of energy transfer is proposed to be maintained by the synchronized turnovers of the conformational change of one protein coupling up to affect another. Catalysis by those systems implies, therefore, that under new space restrictions the groups of the transducing enzyme increase and decrease reactivity between themselves, with activatory and/or inhibitory ligands (H+, H2O, metals, etc.) and with the electron shells of the reactant molecules. The exergonic reaction-dependent turnover of the forms of the enzyme within the transition complexes would be maintained, therefore, under asymmetric phase angles of conformational-dependent reactivity that would effectively restrict the microscopic reversibility of transducing systems. Some well known reactions, such as hemoglobins Bohr effect, can be used to illustrate that microscopic (molecular) interactions subject to thermodynamic equilibria laws may similarly paricipate as driving forces in energy transducing sytems. This would allow the thermodynamic description of the role of proton translocation as that of a modificatory force of the structural parameters of proteins. Similarly, the relationship between the liganded states of hemoglobin and its change in conformation has been used to develop an illustrative model relating changes in oxido-reduction of electron carriers to induced-fit effects leading to a sequence of ATPase forms in transition complexes which become stabilized as high energy intermediates under the constraints imposed by the membrane of energy transducing organelles.