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Stimulation of adenosine triphosphatase activity of sarcoplasmic reticulum by adenylyl methylene diphosphate
Abstract:
The effects of adenylyl methylene diphosphate (AMD), a non-hydrolyzable ATP analogue, were examined in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Ca2+-dependent APTase activity measured at 5 degrees C and pH 7.0 in 5.2 micrometer [gamma-32P]ATP and in the absence of added alkali metal salts was stimulated by added AMD. The steady state level of phosphoenzyme, however, was not decreased greatly by added AMP under these conditions. The hydrolysis of the phosphoenzyme formed at the steady state in the absence of added alkali metal salts was accelerated by added AMD to an extent that can account for the stimulation of the ATPase activity. At 5 degrees C and pH 7.0 the maximum stimulation of phosphoenzyme hydrolysis by AMD and the Km value for this ATP analogue were 4.3-fold and 40 micrometer, respectively. These results provide further support for our previous conclusion (Shigekawa, M., Dougherty, J.P. and Katz, A.M. (1978) J.Biol. Chem. 253, 1442--1450) that 2 classes of ATP site exist in the calcium pump ATPase in the absence of added alkali metal salts, one being the catalytic site and the other being the regulation site which activates the activity of the catalytic site.
Insights
Adenylyl methylene diphosphate (AMD) stimulates calcium pump ATPase activity in rabbit skeletal muscle sarcoplasmic reticulum. This stimulation is due to AMD accelerating phosphoenzyme hydrolysis, supporting the existence of two ATP sites.
Area of Science:
- Biochemistry
- Muscle Physiology
- Enzymology
Background:
- Sarcoplasmic reticulum vesicles are crucial for muscle contraction.
- Calcium pump ATPase activity is vital for muscle relaxation.
- Understanding ATP hydrolysis mechanisms is key to muscle function.
Purpose of the Study:
- To investigate the effects of adenylyl methylene diphosphate (AMD), a non-hydrolyzable ATP analogue, on calcium pump ATPase activity.
- To elucidate the role of AMD in the hydrolysis of phosphoenzyme intermediates.
- To provide further evidence for distinct ATP binding sites in the calcium pump ATPase.
Main Methods:
- Isolation of sarcoplasmic reticulum vesicles from rabbit skeletal muscle.
- Measurement of Ca2+-dependent ATPase activity using [gamma-32P]ATP.
- Analysis of phosphoenzyme formation and hydrolysis kinetics in the presence of AMD.
Main Results:
- AMD significantly stimulated Ca2+-dependent ATPase activity.
- AMD accelerated the hydrolysis of the steady-state phosphoenzyme.
- Maximum stimulation of phosphoenzyme hydrolysis by AMD was 4.3-fold with a Km of 40 micromolar.
Conclusions:
- AMD's stimulation of ATPase activity is attributed to enhanced phosphoenzyme hydrolysis.
- These findings support the model of two classes of ATP sites in the calcium pump ATPase.
- A regulatory site activated by ATP binding influences the catalytic site's activity in the absence of alkali metal salts.