A simple and rapid preparation of fully phosphorylated and fully dephosphorylated skeletal muscle myosin. Application to the preparation of a phosphorylated LC2-modified artificial isozyme.
Fast skeletal myosin LC2 is phosphorylated on ser-15 by a specific myosin light chain kinase (MLCK) in the presence of Ca2+ and calmodulin, and dephosphorylated by a muscle phosphate in the presence of Mg2+. Fully dephosphorylated myosin is obtained by dialysis of muscle crude extract (0.06 M NaCl, 0.01 M Tris-HCl, pH 7.5, 50 microM EGTA); fully phosphorylated myosin is obtained by addition of Ca2+ (0.2 mM), Mg2+ (10 mM) and ATP (3 mM) and 5 min incubation at 28 degrees C. The following reaction characteristics were noted. The crude extract is a very efficient phosphorylating complex and can be diluted to phosphorylate or dephosphorylate purified myosin. Phosphorylation and dephosphorylation appear monophasic, showing no evidence of negative cooperativity in this particular type of myosin and medium. Phosphorylation is 24 times slower in the presence of 0.45 M KCl, 5 mM pyrophosphate. Thiophosphorylated myosin is slowly dephosphorylated by phosphatase. At the crude myosin stage the dephosphorylation reaction is efficiently inhibited (at 0-4 degrees C) by the presence of 70 mM NaF. Myosin-[(T)-LC2'] (a myosin species in which LC2 has been selectively modified by trypsin) is an interesting species refractory to phosphorylation. The myosin-[(T)-LC2'] isozyme can be obtained fully phosphorylated by phosphorylation of myosin followed by limited tryptic proteolysis as described earlier. Urea-PAGE as used separates LC2, phosphoryl-LC2, LC2' and phosphoryl-LC2' effectively and in this order. Through this procedure the (de)-phosphorylating complex is ipso facto specific to the myosin species considered; the method avoids lengthy preparations of purified proteins and is easy, rapid and efficient.
D000818:Animals; D011756:Diphosphates; D007527:Isoenzymes / Q000302:isolation & purification; D007700:Kinetics; D009132:Muscles / Q000032:analysis; D009219:Myosin-Light-Chain Kinase; D009218:Myosins / Q000302:isolation & purification*; D009994:Osmolar Concentration; D010766:Phosphorylation; D011817:Rabbits; D012969:Sodium Fluoride
D011756:Diphosphates; D007527:Isoenzymes; D012969:Sodium Fluoride; D009219:Myosin-Light-Chain Kinase; D009218:Myosins