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The elementary steps of the cross-bridge cycle in which troponin C (TnC) was partially extracted were investigated by sinusoidal analysis in rabbit psoas muscle fibers. The effects of MgATP and phosphate on the rate constants of exponential processes were studied at 200 mM ionic strength, pCa 4.20, pH 7.00, and at 20 degrees C. The results were analyzed with the following cross-bridge scheme: [formula: see text] where A is actin, M is myosin, S is MgATP, D is MgADP, and P is phosphate (Pi). When TnC was extracted so that the average remaining tension was 11% (range 8-15%), K1 (MgATP association constant) increased to 7x, k2 (rate constant of cross-bridge detachment) increased to 1.55x, k-2 (reversal of detachment) decreased to 0.27x, and K2 (= k2/k-2: equilibrium constant of cross-bridge detachment) increased to 6.6x, k4 (rate constant of force generation) decreased to 0.4x, k-4 (reversal of force generation) increased to 2x, K4 (= k4/k-4) decreased to 0.17x, and K5 (Pi association constant) did not change. The activation factor alpha, which represents the fraction of cross-bridges participating in the cycling, decreased from 1 to 0.14 with TnC extraction. The fact that K1 increased with TnC extraction implies that the condition of the thin filament modifies the contour of the substrate binding site on the myosin head and is consistent with the Fenn effect. The fact that alpha decreased to 0.14 is consistent with the steric blocking mechanism (recruitment hypothesis) and indicates that some of the cross-bridges disappear from the active cycling pool. The fact that the equilibrium constants changed is consistent with the cooperative activation mechanism (graded activation hypothesis) among thin-filament regulatory units that consist of troponin (TnC, Tnl, TnT), tropomyosin, and seven actin molecules, and possibly include cross-bridges.

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The electrochemically-driven and peroxidase-mediated oxidations of 5-hydroxytryptophan (5-HTPP) in acidic solution have been studied. Both oxidation processes yield a complex mixture of identical products. Under a limited set of experimental conditions, the first voltammetric oxidation peak of 5-HTPP at a pyrolytic graphite electrode exhibits linear diffusion control. Based upon the peak characteristics it may be implied that the initial step in the electro-oxidation is a reversible one-electron abstraction to give a radical cation that deprotonates to a neutral radical, 5-HTPP'. Attack by 5-HTPP on this radical leads to three sets of diastereomeric dimers. 5-HTPP" can also be further oxidized (le) to a quinone imine that is attacked by water and then oxidized to give tryptophan-4,5-dione. Additional chemical/electrochemical reactions generate other products including one fully characterized trimer. The electrochemical process appears to exactly mimic the enzymatic reaction.There is considerable interest in understanding the oxidation chemistry and biochemistry of various indoleamines found naturally in the central nervous system because such chemistry might provide some insights into the etiology of certain mental illnesses (1, 2). We have recently initiated a program to study the electrochemical oxidation of such indoleamines with the expectation that the information obtained might provide a basis for understanding the enzyme-mediated oxidations of these compounds. Recently, we reported preliminary results concerning the electrochemical oxidation of 5-hydroxytryptophan (5-HTPP) in aqueous acidic solution at a pyrolytic graphite electrode (PGE) (2). Some of the products were identified and included several dimeric compounds and, as a result, it seemed likely that radical intermediates might be involved. However, because of strong adsorption of 5-HTPP at the PGE, voltammetric techniques could not be employed to probe mechanistic aspects of the electrochemically driven reaction. Furthermore, not all major oxidation products were isolated and identified.In this report a more complete description of the products and mechanisms of the electrochemical oxidation of 5-HTPP is presented. In addition, the electrochemically driven and peroxidase-mediated oxidations of 5-HTPP are compared.