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An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms
A S Rovner1, Y Freyzon, K M Trybus
1Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington 05405-0068, USA.
Abstract:
Smooth muscle myosin isoforms of the heavy chain and the essential light chain have been hypothesized to contribute to the different shortening velocities of phasic and tonic smooth muscles, and to their different affinities for MgADP. We used the baculovirus/insect cell system to express homogeneous heavy meromyosin molecules differing only in seven amino acid insert (QGPSFSY) in the motor domain near the active site, or in the type of essential light chain isoform. Myosin from tonic rabbit uterine smooth muscle lacks the heavy chain insert, while myosin from phasic chicken gizzard contains it. The properties of a mutant uterine heavy meromyosin with added insert, and a mutant gizzard heavy meromyosin with the insert deleted, were compared with their wild type progenitors. Phosphorylated heavy meromyosins with the insert have a twofold higher enzymatic activity and in vitro motility han heavy meromyosins without the insert. These functional properties were not altered by the essential light chain isoforms. The altered motility caused by the insert implies that it modulates the rate of ADP release, the molecular step believed to limit shortening velocity. The insert may thus account in part for both the lower sensitivity to MgADP and the higher shortening velocity of phasic compared to tonic smooth muscles.
Insights
Smooth muscle myosin heavy chain isoforms, specifically a seven amino acid insert, significantly impact muscle function. This insert enhances enzymatic activity and motility, explaining differences in phasic and tonic smooth muscle characteristics.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Smooth muscle myosin isoforms are hypothesized to influence muscle shortening velocity and MgADP affinity.
- Differences between phasic and tonic smooth muscles suggest underlying molecular variations in myosin.
Purpose of the Study:
- To investigate the role of a specific seven amino acid insert in the smooth muscle myosin heavy chain motor domain.
- To determine if this insert or essential light chain isoforms are responsible for functional differences between phasic and tonic smooth muscles.
Main Methods:
- Utilized the baculovirus/insect cell system to express homogeneous heavy meromyosin (HMM) molecules.
- Created mutant HMMs by adding or deleting the seven amino acid insert (QGPSFSY) and compared their properties to wild-type progenitors.
- Assessed enzymatic activity and in vitro motility of phosphorylated HMMs.
Main Results:
- Phosphorylated HMMs with the insert exhibited twofold higher enzymatic activity and in vitro motility compared to those without.
- Essential light chain isoforms did not alter these functional properties.
- The insert was shown to modulate the rate of ADP release, a key step limiting shortening velocity.
Conclusions:
- The seven amino acid insert in smooth muscle myosin heavy chain significantly enhances enzymatic activity and motility.
- This insert likely contributes to the higher shortening velocity and lower MgADP sensitivity observed in phasic smooth muscles compared to tonic smooth muscles.
- Essential light chain isoforms do not appear to play a role in these specific functional differences.