Mechanism of the phosphorylation-dependent regulation of smooth muscle heavy meromyosin.
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.
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D005895:Gizzard, Avian / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D009130:Muscle, Smooth / Q000201:enzymology*; D015879:Myosin Subfragments / Q000378:metabolism*; D009391:Nephelometry and Turbidimetry; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D013997:Time Factors; D014422:Turkeys; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D015879:Myosin Subfragments; D010710:Phosphates; D014639:Vanadium; D014638:Vanadates; D000244:Adenosine Diphosphate; D017301:Ca(2+) Mg(2+)-ATPase