Kinetic characterization of brush border myosin-I ATPase

Abstract

Brush border myosin-I (BBM-I) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells. We used stopped-flow kinetic analysis to measure the rate and equilibrium constants for several steps in the BBM-I ATPase cycle. We determined the rates for ATP binding to BBM-I and brush border actomyosin-I (actoBBM-I), the rate of actoBBM-I dissociation by ATP, and the rates for the steps in ADP dissociation from actoBBM-I. The rate and equilibrium constants for several of the steps in the actoBBM-I ATPase are significantly different from those of other members of the myosin superfamily. Most notably, dissociation of the actoBBM-I complex by ATP and release of ADP from actoBBM-I are both very slow. The slow rates of these steps may play a role in lengthening the time spent in force-generating states and in limiting the maximal rate of BBM-I motility. In addition, release of ADP from the actoBBM-I complex occurs in at least two steps. This study provides evidence for a member of the myosin superfamily with markedly divergent kinetic behavior.

Figure 1

Rate of mantATP binding to BBM-I (•) and actoBBM-I ▪. Each point is the average of 1–5 transients from two protein preparations. The solid lines are the best linear fits through the data points. K₁k₂ = 0.95 ± 0.04 μM⁻¹⋅s⁻¹ for BBM-I. K′₁k′₂ = 0.45 ± 0.04 μM⁻¹⋅s⁻¹ for actoBBM-I.

Figure 2

(Upper) Fluorescence titration of 0.5 μM pyrene-actin-phalloidin with BBM-I. Maximum quenching of fluorescence by BBM-I is plotted as 1.0. The solid line is a fit as described in the text: 1/K₇ < 0.01 μM. (Lower) Rate of ATP-induced dissociation of BBM-I from pyrene-actin-phalloidin as a function of nucleotide concentration. Each data point is the average of 1–5 transients from three protein preparations. The solid line is a fit to Eq. 5 as described in the text: K′₁ = 1.6 × 10³ ± 300 M⁻¹ and k′₂ = 200 ± 19 s⁻¹.

Figure 3

Dependence of the rate of 1.2 mM ATP binding to a 0.5 μM complex of pyrene-actin-phalloidin-BBM-I on the concentration of ADP. (Upper) The observed first-order rate constants at each ADP concentration were obtained by fitting the stopped-flow data to a single exponential. The solid line is a fit to Eq. 7 as described in the text: K′₆ = 1.0 μM. (Lower) Experimental transients at each ADP concentration were fit to two exponential rates. The rate of the slow component (k_obs1, ▪) is defined by k′₅, and the rate of the fast component (k_obs2, •) is defined by k′₆ and the ADP concentration. The solid line through the circles is a fit to Eq. 8 as described in the text.

Figure 4

Stopped-flow fluorescence transients showing the dissociation of 0.5 μM BBM-I from pyrene-actin-phalloidin by 1.2 mM ATP in the presence of 12.5 μM ADP. Solid lines show single- and double-exponential fits to the data. Residuals obtained by subtracting the experimental data from the single-exponential fit (Middle) and the double-exponential fit (Bottom) are shown.

Figure 5

Stopped-flow fluorescence transients showing the dissociation of 0.5 μM BBM-I from 0.5 μM pyrene-actin-phalloidin by 1.2 mM ATP in the presence of 12.5, 50, 150, and 300 μM ADP (top to bottom, respectively). Solid lines show simulated transients to Eq. 6 calculated as described in the text.