Diffusion-limited kinetics of immobilized myosin ATPase.
To test the possibility that ATP diffusion limits the kinetics of myosin ATPase (EC. 3.6.1.3) in situ, myosin was covalently bound to the surface of 2 kinds of films: collagen and Immunodyne. On collagen films, it was bound either with 1-ethyl-3 (3 dimethyl-aminopropyl)carbodiimide (EDC) or with dimethyl-3,3'-dithiobis(propionimidate) (DTP). The apparent Km for K+-ATP rose from 0.26 mM for free myosin in solution to 2-5 mM for covalently bound myosin, and maximum K+-ATPase activity was very low. With the other film, Immunodyne from Pall, the maximum activity of bound myosin was 170 nmol per min per 1.5 cm2 film. The apparent Km for K+-ATP was 2.1 mM when the incubation mixture was vigorously stirred, and the effect of stirring indicated that the kinetics of K+-ATP hydrolysis are limited by external diffusion. The large amount of myosin bound per unit of Immunodyne film surface permitted the study of Mg2+-ATPase activity, although it was 400-500 times less than the K+-ATPase activity. The apparently non-Michaelian kinetics of Mg2+-ATP hydrolysis are attributable to the external diffusion. The apparent Michaelis constant observed at low Mg2+-ATP concentrations rose from 0.27 microM for myosin in solution to 5 microM for myosin bound to Immunodyne film.
Arrio-Dupont
M
M
Physiologie Cellulaire Cardiaque, INSERM U241, Orsay, France.
Béchet
J J
JJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D027682:Cation Transport Proteins; D003094:Collagen; D004058:Diffusion; D004800:Enzymes, Immobilized / Q000378:metabolism; D007700:Kinetics; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits; D012044:Regression Analysis
D027682:Cation Transport Proteins; D004800:Enzymes, Immobilized; D000255:Adenosine Triphosphate; D003094:Collagen; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C022761:potassium transporting ATPase; D009218:Myosins