Abstract
Humans express five distinct myosin isoforms in the sarcomeres of adult striated muscle (fast IIa, IId, the slow/cardiac isoform I/β, the cardiac specific isoform α, and the specialized extraocular muscle isoform). An additional isoform, IIb, is present in the genome but is not normally expressed in healthy human muscles. Muscle fibers expressing each isoform have distinct characteristics including shortening velocity. Defining the properties of the isoforms in detail has been limited by the availability of pure samples of the individual proteins. Here we study purified recombinant human myosin motor domains expressed in mouse C2C12 muscle cells. The results of kinetic analysis show that among the closely related adult skeletal isoforms, the affinity of ADP for actin·myosin (K(AD)) is the characteristic that most readily distinguishes the isoforms. The three fast muscle myosins have K(AD) values of 118, 80, and 55 μM for IId, IIa, and IIb, respectively, which follows the speed in motility assays from fastest to slowest. Extraocular muscle is unusually fast with a far weaker K(AD) = 352 μM. Sequence comparisons and homology modeling of the structures identify a few key areas of sequence that may define the differences between the isoforms, including a region of the upper 50-kDa domain important in signaling between the nucleotide pocket and the actin-binding site.
Highlights
Characterization of individual muscle myosin isoforms has been limited by the availability of pure samples of isoforms
The Affinity of extraocular isoform (EO) for ATP Is Weaker than IIa, IIb, and IId in the Presence of Actin—ATP binding to actin1⁄7S1 was determined for each myosin isoform using rapid mixing in a stopped flow apparatus
We report the characterization of the human IIb isoform, which is not normally expressed in healthy human muscle
Summary
Characterization of individual muscle myosin isoforms has been limited by the availability of pure samples of isoforms. Conclusion: The superfast extraocular myosin is kinetically distinct from the fast skeletal IIa, IId, and IIb isoforms. Humans express five distinct myosin isoforms in the sarcomeres of adult striated muscle (fast IIa, IId, the slow/cardiac isoform I/, the cardiac specific isoform ␣, and the specialized extraocular muscle isoform). The motor domain of the MyHC imparts the primary contractile character to the muscle fiber, whereas the ELC and RLC play modulating roles. Fast/type II fibers in adult skeletal muscles express a mixture of the MyHC isoforms IIa and IId and, in a few specific muscles, the extraocular isoform (EO). MyHC-EO is thought to be a very fast isoform because of its expression only in uniquely rapid twitch fibers in the extraocular muscles and laryngeal muscles [7,8,9].
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