Mechanism of the phosphorylation-dependent regulation of smooth muscle heavy meromyosin.

J R Sellers

doi:10.1016/s0021-9258(17)36331-7

Abstract

Phosphorylation of smooth muscle heavy meromyosin (HMM) has been shown to result in about a 25-fold increase in the steady-state Vmax of the actin-activated MgATPase activity from 0.07 s-1 for unphosphorylated HMM to 1.9 s-1 for phosphorylated HMM. The steady-state MgATPase activity of unphosphorylated HMM in the absence of actin is 0.004 s-1. The true extent of regulation might be even larger since the actin activation of the MgATPase activity of unphosphorylated HMM (from 0.004 to 0.07 s-1 at Vmax) could be arising from a small fraction of modified HMM molecules which are no longer regulated and that truly regulated unphosphorylated HMM molecules are not activated by actin. To test this idea, a "limited turnover" experiment was used to measure the reassociation rate of acto-unphosphorylated HMM following addition of a 2-4-fold molar excess of ATP. The reassociation rate was very slow and was not significantly increased by raising the actin concentration from 10 to 75 microM or by addition of trace phosphorylated HMM. The rate constant was estimated to be about 0.002 s-1, which is in good agreement with the rate of product (both Pi and ADP) release estimated from unphosphorylated HMM alone measured by a gel filtration technique. These two experiments suggest that the rate of product release from unphosphorylated HMM may not be significantly affected by actin and that perhaps the true extent of regulation of HMM by phosphorylation is much greater than that determined by steady-state methods. It also suggests that phosphorylation may operate by increasing the forward rate constant for product release by approximately 1000-fold.

Highlights

SCHEME1 ing addition of a 2-4-fold molar excess of ATP
0.002 s-l, which is in good agreement with therate of lation does not operateby preventing the binding of unphosproduct release estimated from un- phorylated heavy meromyosin (HMM) .ATPor HMM .ADP
The final experimentalconditions were 20 mM KC1,lO mM imidazole, 1.8mM MgClz, 0.1 mM EGTA, 1mM dithiothreitol, 15 p~ HMM, 30 p~ radioactively labeled ATP, and 1.0 mM unlabeled ATP at25 “C.Experimentally, HMM was added to initiate the reaction in thepresence of labeled ATP followed by addition of 1mM unlabeled ATP at 10 s

Summary

THEJOURNAL OF BIOLOGICACHLEMISTRY

Vol 260,No 29,Issue of December 15,pp. 1P5r8i1n5t1-e9d1855i8n19l?.S.A. Mechanism of the Phosphorylation-dependentRegulation of Smooth Muscle Heavy Meromyosin*. Rate of product release from unphosphorylated HMM was very rapid and was not affected by phosphorylation (3, may not be significantly affected by actin and that 9) These data suggested that the kinetic step most likely to perhaps the true xtent of regulation of H” by phos- be regulated was the step(s) in which phosphate was released phorylation is much greater than that determined by (k+a).A separate study found somewhat different values for steady-state methods. “single turnover” experiments show that actin does not significantly activate the majority of the unphosphorylated smooth muscle HMM molecules, indicating thatthetrue extent of regulation by phosphorylation is much greater than the 25-fold suggested by steady-state experiments [3] and is perhaps nearly 1000-fold.A preliminary account of these data has been reported [14]

MATERIALS AND METHODS

Regulation of SmoothMHMeeruaosvmcylyeosin

RESULTS

These molecules wouldact as scavengers to hydrolyze the free

DISCUSSION