Biochemical kinetics of porcine cardiac subfragment-1. II. Pre-steady-state studies of the initial phosphate burst.

Abstract

The actin dependence of the rate and magnitude of the initial phosphate burst was measured using both quench-How and stopped-flow kinetic techniques. These studies revealed that even at high actin concentrations the magnitude of the phosphate burst was a significant fraction of the magnitude that exists in the absence of actin. Furthermore, it was shown that the rate of the burst rises rapidly as a function of the actin concentration. Detailed modeling with the four-state model revealed that if the predicted V^ is constrained to be approximately equal to the extrapolated value (double reciprocal plot) and if the apparent dissociation constant of subfragment-1 to actin divided by the apparent activation constant of the actin-activated myosin ATPase activity (K^^^/K ArPmK ) is constrained to be considerably different from one, then the model is unable to simultaneously account for the ATPase activity and the rate and magnitude of the initial inorganic phosphate burst. (Circulation Research 1989;65:515-525) R ecent studies on the steady-state properties of porcine cardiac subfragment-1 (S-l) 1 have revealed that the biochemical kinetics of cardiac S-l are very similar to the kinetics of rabbit skeletal S-l.2 It was shown that ^bindmgj the apparent dissociation constant of S-l to actin, and /CATP»K> the apparent activation constant of the actin-activated myosin ATPase activity, of porcine cardiac S-l were, within experimental error, equal to those of skeletal S-l and that the ratio of these constants (i.e., Ktinding/iCATp^) for the cardiac proteins is approximately 5 : 1. The only significant difference between skeletal and cardiac S-l was that the extrapolated V^, of the double reciprocal plot 3 was approximately 2/sec for cardiac S-l and 4-5/sec for skeletal S-l at 15° C and low ionic strength (0.013 M). Furthermore, it was shown that cross-linked actoS-1, prepared using the zero-length cross-linking agent l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), possesses a significant ATPase activity. 4 ' 5 This lack of significant inhibition of the ATPase activity of cardiac actoS-1 at saturating actin con- centrations was reported previously for skeletal S-l, and detailed kinetic modeling revealed that a "nondissociating pathway" for ATP hydrolysis was required to account for the data. 5 It was then concluded that the four-state model was the minimal model that could account for the data (see Figure I).1 Because the steady-state kinetics of cardiac S-l are almost identical to rabbit skeletal S-l, except for a significantly different V^, it becomes interesting to pursue the kinetic mechanism of this difference. The important question is whether a four-state model can adequately account for the cardiac data or whether a more complex model (e.g., a six-state model) needs to be postulated. In the current work, we have expanded our prior kinetic studies of cardiac S-l to include presteady-state measurements of the rate and magnitude of the initial phosphate burst, both in the presence and absence of actin. As wa...

Kinetic Analysis of the Slow Skeletal Myosin MHC-1 Isoform from Bovine Masseter Muscle

“…However, the quench flow data reported here show that k +3  +  k - 3 defines the medium phase observed in the stopped flow measurements and the maximum observed rate constant of the medium phase is k +3  +  k -3  = 19 s −1 , whereas the fast phase represents k +2 . The ATP hydrolysis step of BM S1 is much slower than that of fast skeletal S1 from rabbit ( k +3  +  k -3  = 131 s −1 ) but it is similar to the slow skeletal myosin II, MHC-II-1 from rabbit soleus (22 s −1 ) 9 and pig MHC-II-1 (24.5 s −1 ) 24 . It may therefore be a general feature of slow myosin II isoforms.…”

Oxygen exchange kinetics of procine cardiac acto-subfragment 1

“…In the present paper, we studied the amount of 180 exchange which occurs with porcine cardiac S-l. We used porcine cardiac S-l because it shows the same difference between /fATPase and Kbinding that is shown by skeletal muscle S-l. Of course, some variability does exist in various preparations of cardiac S-l. At 15 °C and low ionic strength, the Vmax for cardiac S-l has been consistently in the range of 1.7-2.2 s"1 with about 20 different protein preparations. AfATPasc has also been found to be consistently in the range of 3-6 µ while different preparations gave values for Afbinding in the range of 20-25 µ using airfuge techniques and 30-40 µ with light-scattering methods (Stein & White, 1987;Stein et al, 1989). When determined on the same protein preparation, the ratio of Aibinding to AfATPase has always been at least 4.…”

“…In fitting our data to the four-state model, we first at- tempted to fit the ATPase data. The rate constants k5 and fc_5 are generally determined from the fluorescence enhancement rate in the absence of actin (i.e., k5 + k_5 = 24 s"1) and the fact that the equilibrium constant K5 (ks/k-5) is generally found to be in the range of 2-3 using quench-flow techniques, and has been assumed to be 2 in the present case (Stein & White, 1987; Stein et al, 1989). As we discussed in an earlier paper, the difference noted between AfATPase and Aibinding can only be explained by the four-state model if k6 is rate limiting and kw is relatively fast (Stein et al, 1981).…”