PubMed · 7326531
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C A Lawrence. 1981. Ion-selective electrodes.. https://pubmed.ncbi.nlm.nih.gov/7326531/
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The release of fibrinopeptides A and B by the slow and fast forms of thrombin was studied over the temperature range from 5 to 45 degrees C and the salt concentration range from 100 to 800 mM. The sequential mechanism for the release of fibrinopeptides originally proposed by Shafer was found to be obeyed under all conditions examined. The origin of preferential binding of fibrinogen and fibrin I to the fast form of thrombin in the transition state is in the second-order rate constant for association, k(l). In the case of fibrinogen, the values of k(l) for interaction with the fast and slow forms at 25 degrees C are 19 +/- 4 and 2.5 +/- 0.3 microM(-1) s(-1), with an activation energy of about 10 kcal/mol in both forms. In the case of fibrin I, the analogous values of k(l) are 9.1 +/- 0.7 and 2.5 +/- 0.2 microM(-1) s(-1), and the activation energy is about 4.5 kcal/mol in both forms. The mechanism of recognition of fibrinogen and fibrin I by thrombin entails a diffusion-controlled step with a small energy barrier. Analysis of the temperature dependence of the coupling free energy for allosteric switching indicates that the preferential interaction of fibrinogen and fibrin I with the fast form of thrombin in the transition state is entropy-driven, signaling a contribution of the hydrophobic effect to the slow-->fast transition. The salt dependence of the release of fibrinopeptides shows a constant coefficient Gamma(salt) = d ln(k(cat)/K(m))/d ln [salt] in the concentration range examined. Interestingly, the value of Gamma(salt) is independent of the salt used (NaCl, ChCl, or NaF) and is -1.5 +/- 0.1 for fibrinopeptide A and -2.5 +/- 0.1 for fibrinopeptide B. Hence, Gamma(salt) reflects predominantly the electrostatic contribution to the formation of the transition state, with a larger contribution seen in the interaction of thrombin with fibrin I. It is concluded that the interaction of thrombin with fibrinogen and fibrin I, leading to the release of fibrinopeptides A and B, is driven by electrostatic forces that presumably favor the correct preorientation of the enzyme and the substrate to form a productive complex in the transition state. This electrostatic-steering effect, also reported for thrombin-hirudin interaction, leads to a diffusion-controlled encounter with a very small energy barrier. Once the complex is formed, the enzyme switches to the fast form as a result of entropic factors presumably linked to water release from a more extended surface of recognition. While the release of fibrinopeptides as a function of salt concentration was being studied, an important observation was made on the role of Cl- in the formation of the fibrin clot. This anion drastically and specifically reduces the thickness of fibrin fibers, as judged by the 10-fold decrease in the equilibrium turbidity of clots developed in NaCl as compared to the turbidity of clots developed in NaF. Hence, the transition from a "coarse" to a "fine" clot induced by an increase in ionic strength as first described by Ferry is, instead, due to the specific binding of Cl- to intermediates in the ensuing polymerization. In fact, no change in the clotting curve is observed when the ionic strength is changed with NaF.
Possible regulation of fast Na+ channels by tyrosine kinase was examined in human uterine smooth muscle cell line, using whole-cell voltage clamp (at a holding potential of - 90 mV). Bath application of genistein, an inhibitor tyrosine kinase, decreased the fast Na+ current (INa(f)) dose-dependently. The maximal inhibition of INa(f) was 98%, and the concentration for half-maximal inhibition (IC50) was 9 microM. The effect of genistein was rapidly reversible. Daidzein, an inactive analog of genistein, had a similar inhibitory effect on INa(f). These results suggest that the fast Na+ channels in uterine sarcoma cells may be directly blocked by genistein and daidzein, i.e., their effect may be independent of tyrosine kinase inhibition.
The known high-resolution structure and dynamics characterization of the lipid bilayer-bound polypeptide gramicidin A provides a unique opportunity to study structure-function and dynamics-function correlations in a model membrane protein. In particular, the indoles have a variety of very important functional roles in this cation channel that will undoubtedly be recognized in membrane proteins. That indoles and phenols are oriented at the hydrophobic-hydrophilic interface of lipid bilayers is already well-recognized in membrane proteins. The most buried indole of the gramicidin channel, Trp9, is shown by 15N solid state NMR to be exposed to the hydrophilic surface through hydrogen exchange. Here the importance of the indole dipole moments is described for cation conductance. Preparation of samples with high concentrations of Na+ is shown by high-resolution orientational constraints derived from 2H NMR to have no structural effect on the indole side chain conformations. These dipoles stabilize cations in the binding sites near the channel entrance and substantially reduce the potential energy barrier at the bilayer center. This latter finding conclusively documents that the rate-limiting step in cation conductance by this channel involves the barrier at the bilayer center. Furthermore, dynamics of the indole rings cause significant fluctuations in the energy of stabilization at the binding site that may result in a rapid mechanism for gating the channel.