Effect of phosphorylation on the interaction and functional properties of rabbit striated muscle alpha alpha-tropomyosin.
Phosphorylated rabbit cardiac alpha alpha-tropomyosin has been prepared either enzymatically (Montgomery, K., and Mak, A.S. (1984) J. Biol. Chem. 259, 5555-5560) or by fractionation of the phosphorylated and nonphosphorylated forms on a Mono Q column in 9 M urea, 50 mM Tris, pH 8.0. Although the phosphorylated and nonphosphorylated forms showed no difference in their F-actin binding properties, the phosphorylated protein had substantially higher viscosities at low ionic strengths, indicating a greater propensity for head-to-tail interaction. Similar measurements showed the strengthening of this interaction by whole troponin to be substantially reduced by phosphorylation even though the binding of whole troponin and troponin T to tropomyosin was demonstrated by affinity chromatography to be, if anything, strengthened by phosphorylation. In a reconstituted actin (4 microM) plus myosin subfragment 1 ATPase assay (50 mM ionic strength), significantly higher activities over a range (1 to 8 microM) of subfragment 1 concentrations were observed with phosphorylated tropomyosin compared with the nonphosphorylated protein. In the fully reconstituted system with troponin, there was no significant difference in the inhibition of ATPase in the absence of Ca2+. However, in its presence, the activities were appreciably increased with the phosphorylated tropomyosin compared to those with the nonphosphorylated form. These differences were eliminated by treatment of the phosphorylated tropomyosin with alkaline phosphatase. This is the first demonstration of an effect of phosphorylation on the functional properties of tropomyosin.
Heeley
D H
DH
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Watson
M H
MH
Mak
A S
AS
Dubord
P
P
Smillie
L B
LB
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002846:Chromatography, Affinity; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D009994:Osmolar Concentration; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D014335:Tropomyosin / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D014783:Viscosity
D000199:Actins; D014335:Tropomyosin; D000251:Adenosine Triphosphatases