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Reaction of two heads of gizzard myosin with ATP
Abstract:
The reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, MPADP, formed was measured from the Pi-burst size over a wide range of ATP concentrations. At low concentrations of ATP, the Pi-burst size was 0.5 mol/mol myosin head, and the apparent Kd value was about 0.15 microM. However, at high ATP concentrations, the Pi burst size increased from 0.5 to 0.75 mol/mol myosin head with an observed Kd value of 15 microM. The binding of nucleotides to gizzard myosin during the ATPase reaction was directly measured by a centrifugation method. Myosin bound 0.5 mol of nucleotides (ATP and ADP) with high affinity (Kd congruent to 1 microM) and 0.35 mol of nucleotides with low affinity (Kd = 24 microM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms MPADP with high affinity for ATP while the other forms MPADP and MATP with low affinity for ATP. We studied the correlation between the formation of MPADP and the dissociation of actomyosin. The amount of Pi-burst size was not affected by the existence of F-actin, and when 0.5 mol of ATP per mol of myosin head was added to actomyosin (1 mg/ml F-actin, 5 microM myosin at 0 degrees C) most (93%) of the added ATP was hydrolyzed in the Pi-burst phase. All gizzard actomyosin dissociated when 1 mol of ATP per mol myosin head was added to actomyosin.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Smooth muscle myosin has two nucleotide binding sites with different affinities for ATP, influencing its ATPase reaction and actomyosin dissociation. This study reveals distinct binding characteristics crucial for muscle contraction mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Smooth muscle myosin is a key motor protein involved in muscle contraction.
- Understanding the kinetics of ATP hydrolysis and nucleotide binding is essential for elucidating myosin function.
Purpose of the Study:
- To investigate the reaction intermediates formed by smooth muscle myosin heads.
- To characterize the nucleotide binding sites and their affinities for ATP and ADP.
- To correlate myosin intermediate formation with actomyosin dissociation.
Main Methods:
- Measurement of myosin-phosphate-ADP (MPADP) complex formation via Pi-burst size at varying ATP concentrations.
- Direct measurement of nucleotide binding to gizzard myosin using centrifugation.
- Study of actomyosin dissociation in response to ATP addition.
Main Results:
- Gizzard myosin exhibits two types of nucleotide binding sites with distinct affinities (Kd ≈ 1 µM and Kd = 24 µM for ATP).
- Pi-burst size varied with ATP concentration, indicating different intermediate formations (0.5 to 0.75 mol/mol myosin head).
- Actomyosin dissociation was observed upon addition of 1 mol ATP per mol myosin, with 93% ATP hydrolysis during the Pi-burst phase.
Conclusions:
- Gizzard myosin possesses two distinct nucleotide binding sites, affecting MPADP and M-ATP formation.
- These differential binding properties are critical for the ATPase cycle and actomyosin dynamics.
- The findings provide insights into the molecular mechanisms of smooth muscle contraction.