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Enzyme kinetics is an essential topic in undergraduate Biochemistry courses. A laboratory work that covers the principal basic concepts of enzyme kinetics in steady state is presented. The alkaline phosphatase catalyzed reaction of phenyl-phosphate hydrolysis was studied as a model. The laboratory experience was designed to reinforce the concepts of initial velocity dependence on substrate and enzyme concentration, and to highlight the importance of the accurate determination of initial reaction rate. The laboratory work consists in two parts, in which students first determine the enzyme concentration and the time to be used in the following session to obtain the kinetic parameters (K[subscript M] and V[subscript max]) by non-lineal fitting of the Michaelis-Menten equation to the initial velocity dependence with substrate concentration results. The experimental methodology is robust, the cost per student is low and the equipment and reagents used are of easy access.

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca2+-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca2+ in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca2+ concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μm, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of LaIII (inducing accumulation of phosphoenzyme in the E1P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E1P-phosphorylated intermediate of the PMCA. [ABSTRACT FROM AUTHOR]

This study examined how the-quaternary organic ammonium ion, benzyltriethylamine (BTEA), binds to the Na,K-ATPase to produce membrane potential (VM)-dependent inhibition and tested the prediction that such a VM-dependent inhibitor would display electrogenic binding kinetics. BTEA competitively inhibited K+ activation of Na,K-ATPase activity and steady-state 86Rb+ occlusion. The initial rate of 86Rb+ occlusion was decreased by BTEA to a similar degree whether it was added to the enzyme prior to or simultaneously with Rb+, a demonstration that BTEA inhibits the Na,K-ATPase without being occluded. Several BTEA structural analogues reversibly inhibited Na,K-pump current, but none blocked current in a VM-dependent manner except BTEA and its para-nitro derivative, pNBTEA. Under conditions that promoted electroneutral K+-K+ exchange by the Na,K-ATPase, step changes in VM elicited pNBTEA-activated ouabain-sensitive transient currents that had similarities to those produced with the K+ congener, TI+. pNBTEA- and TI+-dependent transient currents both displayed saturation of charge moved at extreme negative and positive VM, equivalence of charge moved during and after step changes in VM, and similar apparent valence. The fate constant (ktot) for TI+-dependent transient current asymptotically approached a minimum value at positive VM. In contrast, ktot for pNBTEA-dependent transient current was a "U"-shaped function of VM with a minimum value near 0 mV. Homology models of the Na,K-ATPase alpha subunit suggested that quaternary amines can bind to two extracellularly accessible sites, one of them located at K+ binding sites positioned between transmembrane helices 4, 5, and 6. Altogether, these data revealed important information about electrogenic ion binding reactions of the Na,K-ATPase that are not directly measurable during ion transport by this enzyme. [ABSTRACT FROM AUTHOR]

Occlusion of K+ in the Na+/K+-ATPase can be achieved under two conditions: during hydrolysis of ATP, in media with Na+ and Mg2+, after the K+-stimulated dephosphorylation of E2P (physiological route) or spontaneously, after binding of K+ to the enzyme (direct route). We investigated the sidedness of spontaneous occlusion and deocclusion of Rb+ in an unsided, purified preparation of Na+/K+-ATPase. Our studies were based on two propositions: (i) in the absence of ATP, deocclusion of K+ and its congeners is a sequential process where two ions are released according to a single file mechanism, both in the absence and in the presence of Mg2+ plus inorganic orthophosphate (Pi), and (ii) in the presence of Mg2+ plus Pi, exchange of K+ would take place through sites exposed to the extracellular surface of the membrane. The experiments included a double incubation sequence where one of the two Rb+ ions was labeled as 86Rb+. We found that, when the enzyme is in the E2 conformation, the first Rb+ that entered the enzyme in media without Mg2+ and Pi was the last to leave after addition of Mg2+ plus Pi, and vice-versa. This indicates that spontaneous exchange of Rb+ between E2(Rb2) and the medium takes place when the transport sites are exposed to the extracellular surface of the membrane. Our results open the question if occlusion and deocclusion via the direct route participates in any significant degree in the transport of K+ during the ATPase activity. [ABSTRACT FROM AUTHOR]

We used suspensions of partially purified Na+/K+-ATPase from pig kidney to compare the effects of Rb+, as a K+ congener, on the time course and on the equilibrium values of eosin fluorescence and of Rb+ occlusion. Both sets of data were collected under identical conditions in the same enzyme preparations. The incubation media lacked ATP so that all changes led to an equilibrium distribution between enzyme conformers with and without bound eosin and with and without bound or occluded Rb+. Results showed that as Rb+ concentration was increased, the equilibrium value of fluorescence decreased and occlusion increased along rectangular hyperbolas with similar half-maximal values. The time courses of attainment of equilibrium showed an initial phase which was so quick as to fall below the time resolution of our rapid-mixing apparatus. This phase was followed by the sum of at least two exponential functions of time. In the case of fluorescence the fast exponential term accounted for a larger fraction of the time course than in the case of occlusion. Comparison between experimental and simulated results suggests that fluorescence changes express a process that is coupled to Rb+ occlusion but that is completed before occlusion reaches equilibrium. [ABSTRACT FROM AUTHOR]

We used partially purified Na+/K+ATPase from pig kidney to study dephosphorylation, occlusion, and ATPase activity in the same enzyme preparation and in media of identical composition containing 10 μM ATP and different concentrations of Rb+, used as a K+ congener. The experiments were performed using a rapid-mixing apparatus with a time resolution of 3.5 ms. The main findings were as follows. (i) At sufficiently low Rb+ concentration the initial rate of dephosphorylation was higher than that of occlusion, (ii) as [Rb+] tended to zero the slope of the time course of occlusion but not that of the time course of dephosphorylation approached zero and, (iii) as Rb+ concentration increased, ATPase activity first increased and, after passing through a maximum, tended to a value that was lower than that observed in media without Rb+. None of these results is compatible with the currently held idea that binding of a single Rb+ to the E2P conformer of the ATPase does not modify the rate of dephosphorylation and strongly suggest that a single Rb+ does promote dephosphorylation through a mechanism that is not stoichiometrically coupled to Rb+ occlusion. If this mechanism is included in the currently accepted scheme for ATP hydrolysis by the Na+/K+ATPase, a reasonable prediction of the experimental results is obtained. [ABSTRACT FROM AUTHOR]

Na + ,K + -ATPase is an integral membrane protein which couples ATP hydrolysis to the transport of three Na + out and two K + into the cell. The aim of this work is to characterize the effect of K + , ATP, and Mg 2+ (essential activator) on the Na + ,K + -ATPase thermal stability. Under all conditions tested, thermal inactivation of the enzyme is concomitant with a structural change involving the ATP binding site and membrane-associated regions. Both ligands exert a clear stabilizing effect due to both enthalpic and entropic contributions. Competition experiments between ATP and K + showed that, when ATP is present, the inactivation rate coefficient exhibits a biphasic dependence on K + concentration. At low [K + ], destabilization of the enzyme is observed, while stabilization occurred at larger cation concentrations. This is not expected for a simple competition between the enzyme and two ligands that individually protect the enzyme. A model that includes enzyme species with none, one, or two K + and/or one molecule of ATP bound explains the experimental data. We concluded that, despite both ligands stabilizing the enzyme, the species with one K + and one ATP simultaneously bound is unstable.