A mechanistic mathematical model for the catalytic action of glutathione peroxidase.

Glutathione peroxidase (GPx) is a well-known seleno-enzyme that protects cells from oxidative stress (e.g., lipid peroxidation and oxidation of other cellular proteins and macromolecules), by catalyzing the reduction of harmful peroxides (e.g., hydrogen peroxide: H2O2) with reduced glutathione (GSH). However, the catalytic mechanism of GPx kinetics is not well characterized in terms of a mathematical model. We developed here a mechanistic mathematical model of GPx kinetics by considering a unified catalytic scheme and estimated the unknown model parameters based on different experimental data from the literature on the kinetics of the enzyme. The model predictions are consistent with the consensus that GPx operates via a ping-pong mechanism. The unified catalytic scheme proposed here for GPx kinetics clarifies various anomalies, such as what are the individual steps in the catalytic scheme by estimating their associated rate constant values and a plausible rationale for the contradicting experimental results. The developed model presents a unique opportunity to understand the effects of pH and product GSSG on the GPx activity under both physiological and pathophysiological conditions. Although model parameters related to the product GSSG were not identifiable due to lack of product-inhibition data, the preliminary model simulations with the assumed range of parameters show that the inhibition by the product GSSG is negligible, consistent with what is known in the literature. In addition, the model is able to simulate the bi-modal behavior of the GPx activity with respect to pH with the pH-range for maximal GPx activity decreasing significantly as the GSH levels decrease and H2O2 levels increase (characteristics of oxidative stress). The model provides a key component for an integrated model of H2O2 balance under normal and oxidative stress conditions.

Mitochondrial inner membrane electrophysiology assessed by rhodamine-123 transport and fluorescence.

Rhodamine-123 is widely used to make dynamic measurements of mitochondrial membrane potential both in vitro and in situ. Yet data interpretation is difficult due to a lack of quantitative understanding of how membrane potential and measured fluorescence are related. To develop such understanding, a model for dye transport across the mitochondrial inner membrane and partition into the membrane was developed. The model accounts for experimentally measured dye self-quenching and was integrated into a model of mitochondrial electrophysiology to estimate transients in mitochondrial membrane potential from kinetic fluorescence measurements. Our analysis indicates that (i) R123 fluorescence peaks at concentrations near 50 microM due to self-quenching; (ii) measured fluorescence intensity and membrane potential are related by a non-linear calibration curve sensitive to certain experimental details, including total concentration of dye and mitochondria in suspensions; and (iii) the time courses of membrane potential and electron transport fluxes following a perturbation (i.e. addition of ADP) significantly differ from observed transients in fluorescence intensity. These findings are consistent with the model predictions that mitochondria display a characteristic time of response to changes in substrate concentration of less than 0.1 s, corresponding to the time scale over which the rate of ATP synthesis changes to meet changes in ADP concentration.

Comparison of hyperkalemic cardioplegia with altered [CaCl2] and [MgCl2] on [Ca2+]i transients and function after warm global ischemia in isolated hearts.

AIM: [MgCl(2)] and [CaCl(2)] may modify the cardioprotective effects of hyperkalemic cardioplegia (CP). We changed [MgCl(2)] and [CaCl(2)] in a CP solution to examine their effects on [Ca(2+)]i transients and cardiac function before and after global normothermic ischemia. METHODS: After stabilization and loading of indo 1-AM in Kreb's solution (KR), each heart was perfused with either KR or 1 of 4 CP solutions before 37 degrees C, 30 min ischemia followed by reperfusion with KR. The KR solution contained, in mM, 4.5 KCl, 2.4 MgCl(2) and 2.5 CaCl(2); the CP solutions had in addition to 18 KCl: CP 1 (control CP): 2.4 MgCl(2), 2.5 CaCl(2); CP 2: 7.2 MgCl(2), 2.5 CaCl(2); CP 3, 7.2 MgCl(2), 1.25 CaCl(2); CP 4: 2.4 MgCl(2), 1.25 CaCl(2). RESULTS: In the KR group [Ca(2+)]i markedly increased on early reperfusion while functional return (LVP, dLVP/dt((max and min))) was much reduced; each CP group led to reduced [Ca(2+)]i loading and improved function. The rates of cytosolic Ca(2+) fluxes (d[Ca(2+)]/dt(max) and d[Ca(2+)]/dt(min)) increased significantly compared to baseline in the KR group, but were mostly suppressed in the CP groups, and d[Ca(2+)]/dt(min) was lower after CP 4 compared to CP 1 on reperfusion. At 60 min reperfusion, LVP area to [Ca(2+)] area and cardiac efficiency to phasic [Ca(2+)] relationships were shifted after KR, but not after CP 1-4. With similar functional recovery, [Ca(2+)] transient and [Ca(2+)] area were significantly lower after CP 4 than after CP 1. CONCLUSION: Increasing [MgCl(2)] (CP 2 and 3) did not improve cardiac function or reduce Ca(2+) transients on reperfusion better than the other CP groups, but reducing [CaCl(2)] (CP 3 and 4) was more effective in reducing [Ca(2+)] transients on reperfusion after global ischemia.