Effects of phosphorylation, MgATP, and ionic strength on the rates of papain degradation of heavy and light chains of smooth muscle heavy meromyosin at the S1-S2 junction.

H Suzuki,Y Kondo,A D Carlos,J C Seidel

doi:10.1016/s0021-9258(18)38065-7

H Suzuki, Y Kondo + Show 2 more

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https://doi.org/10.1016/s0021-9258(18)38065-7

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Abstract

The effects of ionic strength, MgATP, and phosphorylation on the degradation rates of heavy meromyosin (HMM) by papain have been compared to their effects on the sedimentation coefficient (s20,w) to determine the relationship of the degradation rate to the equilibrium between the flexed and the extended forms (Suzuki, H., Stafford, W. F., Slayter, H. S., and Seidel, J. C. (1985) J. Biol. Chem. 260, 14810-14817). At 0.025 M NaCl, where HMM is predominantly in the flexed form, MgATP, Mg-adenylyl imidodiphosphate or MgADP reduce kH by 80-90%. MgATP exerts its optimal effect at this ionic strength, where at least 70% of HMM is flexed in the presence or absence of MgATP, suggesting that nucleotides reduce kH by decreasing the proteolytic susceptibility of the flexed form. At 0.5 M NaCl, where HMM is in the extended form, MgATP has no effect on kH. At low ionic strengths phosphorylation decreases kH but increases it in the presence of MgATP. Plots of kH against s20,w determined at various ionic strengths are linear, the data for phosphorylated and dephosphorylated HMM falling on the same line. Thus, raising the ionic strength or phosphorylating the 20-kDa light chain appears to alter kH by increasing the fraction of HMM in the extended form. The degradation rate of the 20-kDa light chain (kL) of dephosphorylated HMM responds to changes in ionic strength in essentially the same way as does kH, suggesting that the response of kL to changes in ionic strength can also be attributed to conversion of HMM to the extended form. However, kL for phosphorylated HMM measured in the presence of MgATP exhibits very little dependence on ionic strength.

Highlights

From the Department of Muscle Research, Boston BiomedicaRl esearch Institute, Boston, Massachusetts 02114 and the Departments of Biological Chemistry and Neurology, Harvard Medical School, Boston,Massachusetts 02115
MgATP exerts its optimal effect at this ionic strength, where at least 70% ofHMM is flexed in thepresence or absence of MgATP, suggestwhile phosphorylation or raising the ionic strength favors the extended form [1,2,3,4,5,6]
The ATPase activityin theabsence of actin and thsedimentation velocity of mixtures of the two forms are determined largely by the fraction of HMM in each form [10]. This provides a molecular basis on which to relate the changes in proteolytic degradation rates to thereorientation of HMM heads that is reflected in changes in the sedimentation velocity

Summary

MATERIALS ANDMETHODS

Myosin from chicken gizzardwas prepared by the method of Ebashi [15],and phosphorylated according to Aiba et al [16].Phosphorylated gizzard HMM was obtained by digesting phosphorylated myosin, 810 mg/ml, with a-chymotrypsin, 0.04 mg/ml, in a solution containing 0.5 M NaC1, 1mM EDTA, 1mM dithiothreitol, and 20 mM Tris-HC1, pH 7.5, for 8 min a t 10 "C [17]. The digestion was stopped by addition of iodoacetic acid to a finalconcentration of 2 mM, and the digested myosin was dialyzed againsta solution containing 0.025 M NaC1,O.l mM EDTA, 0.1 mM dithiothreitol, and 5 mM MOPS, pH 7.0, and centrifuged at 20,000 X g for 30 min to remove insoluble myosin rod and undigested myosin. This procedure yielded S1 containing at least 90% of the 20-kDa light chain intact, as indicated bygel electrophoresis in sodium dodecyl sulfate, and at least 90% of the light chain was phosphorylated as indicated bygel electrophoresis in 6 M urea. Sedimentation velocity was measured in aBeckman Model-E analytical ultracentrifuge a t 20 "C as described previously [10]

RESULTS

Digestion of Smooth Muscle Heavy Meromyosin with Papain

Dephosphorylated None

DISCUSSION