Early Phosphate Burst of Heart Myosins. Phylogenic Variations

Abstract

The phosphate burst of cardiac myosins from different animal species was studied by a rapid quench-flow technique. The rate constant of the phosphate burst varied from one species to another. Moreover it was lower for cardiac myosins than for skeletal myosin. The phosphate burst rate correlated with the ATPase in the steady state and with the muscle-shortening speed.By contrast, the amplitude of the phosphate burst did not vary significantly. Its value was 0.8-0.9 per myosin site for each of the myosins studied. Deviations from this stoichiometry were related to the mode of preparation of the myosin and to denaturation by a high pH.As shown several years ago by Barany [l], the shortening speed of skeletal muscles (expressed in muscle lengths per s) correlates with Ca2+-activated and actin-Mg2+-activated myosin ATPase in the steady state. Later, a similar relationship was observed in the heart [2]. The heart has advantages for such a study since it is homogeneous well characterized muscle and also because it is now well established that its shortening speed is inversely proportional to its weight, i.e. the smaller the heart, the faster it shortens [3].ATP hydrolysis by myosin in the steady-state is slow; by contrast, the transient initial phase is very rapid and proceeds in the same speed range as that of the contractile process. This is the main reason why the transient-state steps are of considerable interest and were studied in detail in the usual model, i.e. rabbit white skeletal muscle myosin [4-111. Transient kinetic evidence indicates that at least two conformations occur (see stars in the equation below), bringing about the cleavage of ATP. The products of the hydrolysis remain bound to the myosin. The following equation [9,12] concerning ATP hydrolysis is now generally accepted:

Myosin isoenzymes in normal and hypertrophied human ventricular myocardium.

“…53, No. 1, July 1983 found by others on normal skeletal (Taylor and Weeds, 1977) or cardiac (Van Thiem et al, 1978;Lompre et al, 1979) muscles, clearly indicated that our preparations were neither aggregated nor oxidized, and thus could be considered as functionally intact.…”

“…Determination of early phosphate burst size was carried out manually, as described by Taylor and Weeds (1977) and Van Thiem et al (1978), under stoichiometric conditions; 5 fiM myosin (= 10 ^M active site) and 10 fiM [7-32 P] ATP were mixed on a Vortex mixer in the presence of 0.5 M KC1, 50 mM Tris-HCl, 5 mu MgCl 2 , and 0.1 mM dithiothreitol (pH 7.4, 22°C) and at several time intervals, aliquots were withdrawn and rapidly mixed with equal volumes of quenching solution ( 1 M HC1 and 0.35 M KH2PO4). The amount of 32 P released was determined after filtration on an activated charcoal/cellulose column.…”

“…Klotz et al (1975) observed that the Ca ++activated ATPase of human myosin was not modified if the delay between death and the beginning of the myosin preparation was less than 24 hours. The size of the early burst of phosphate at the very beginning of ATP hydrolysis by myosin (Taylor and Weeds, 1977) appeared later to be a very good test for evaluating the functional status of cardiac myosin (Van Thiem et al, 1978). We have thus used this technique to assess the condition of six of our myosins, and Figure 6 shows the typical time-course of such a determination.…”

ATPase activity of the cross-linked complex between cardiac myosin subfragment 1 and actin in several models of chronic overloading. A new approach to the biochemistry of contractility.

“…In addition, it is a very minor component representing, in any type of heart, 3% of free plus cross-linked myosin SI after 3 minutes condensation and 16% after 20 minutes. Assuming a composition of one myosin SI and three actins for C2, 3 this complex would only slightly modify the calculations. The yield in Cl was 25-26% after 3 minutes condensation and 40-46% after 20 minutes.…”

“…3 This relation is one of the basics of muscle physiology and pathology since, in response to chronic overload in some animal species such as rats, it has been shown that the drop in V m ax correlates with a diminution in myosin ATPase and to a corresponding isomyosin shift from the fast isoform V, to the slow isoform V 3 . reagent 1 -ethyl-3 -(3 -dimethylaminopropy l)-carbodiimide (EDC).…”