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Mammalian skeletal muscle myosin light chain kinases. A comparison by antiserum cross-reactivity
Abstract:
Purified myosin light chain kinases from skeletal muscle are reported to be significantly smaller (Mr = 75,000-90,000) than the kinases purified from smooth muscle (Mr = 130,000-155,000). It has been suggested that the smaller kinases from striated muscle are proteolytic fragments of a larger enzyme which is homologous, if not identical, to myosin light chain kinase from smooth muscle. Therefore, we have used an antiserum to rabbit skeletal muscle myosin light chain kinase and Western blot analysis to compare the subunit molecular weight of the kinase in skeletal muscle extracts of several mammalian species. In rabbit skeletal muscle, the antiserum only recognized a polypeptide of Mr = 87,000, with no indication that this polypeptide was a proteolyzed fragment of a larger protein. The apparent molecular weights observed in different animal species were 75,000 (mouse), 83,000 (guinea pig), 82,000 (rat), 87,000 (rabbit), 100,000 (dog), and 108,000 (steer). The molecular weight of myosin light chain kinase was constant within an animal species, regardless of skeletal muscle fiber type. The antiserum inhibited the catalytic activity of skeletal muscle myosin light chain kinase. Similar antibody dilution curves for inhibition of myosin light chain kinase activity in extracts were observed for all animal species (rabbit, rat, mouse, guinea pig, dog, cat, steer, and chicken) and different fibers (slow twitch oxidative, fast twitch oxidative glycolytic, and fast twitch glycolytic) tested. The antiserum did not inhibit the activity of rabbit smooth muscle myosin light chain kinase. These results suggest that there may be at least two classes of muscle myosin light chain kinase represented in skeletal and smooth muscles, respectively.
Insights
Skeletal muscle myosin light chain kinase is not a smaller fragment of a larger enzyme. This study reveals distinct myosin light chain kinase classes in skeletal versus smooth muscles across species.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Myosin light chain kinases (MLCKs) purified from skeletal muscle are smaller than those from smooth muscle.
- It has been hypothesized that skeletal muscle MLCKs are proteolytic fragments of a larger enzyme homologous to smooth muscle MLCK.
Purpose of the Study:
- To investigate the molecular weight and immunological properties of skeletal muscle myosin light chain kinase (MLCK) across various mammalian species.
- To determine if skeletal muscle MLCK is a proteolytic fragment of a larger enzyme.
Main Methods:
- Western blot analysis using an antiserum against rabbit skeletal muscle MLCK.
- Comparison of subunit molecular weights in skeletal muscle extracts from different species.
- Enzyme inhibition assays using the antiserum on MLCK activity.
Main Results:
- The antiserum recognized a single polypeptide of Mr = 87,000 in rabbit skeletal muscle, with no evidence of proteolysis.
- Apparent molecular weights of skeletal muscle MLCK varied across species (75,000–108,000 Da) but were constant within a species and independent of muscle fiber type.
- The antiserum inhibited skeletal muscle MLCK activity across all tested species and fiber types but did not inhibit rabbit smooth muscle MLCK.
Conclusions:
- Skeletal muscle myosin light chain kinase (MLCK) is likely not a proteolytic fragment of a larger enzyme.
- Evidence suggests at least two distinct classes of muscle MLCK: one in skeletal muscle and another in smooth muscle.