Heat treatment could affect the biochemical properties of caldesmon.
Smooth muscle caldesmon (CaD) exhibits apparent heat stability. A widely used purification procedure of CaD involves extensive heat treatment (Bretscher, A. (1984) J. Biol. Chem. 259, 12873-12880). CaD thus purified co-sediments with actin, inhibits actomyosin ATPase activity, and interacts with Ca2+/calmodulin, similarly to the unheated protein. On the other hand, heat-treated CaD binds to actin filaments in a tether-like fashion, whereas lengthwise binding dominates in vivo (Mabuchi, K., Lin, J. J.-C., and Wang, C.-L. A. (1993) J. Muscle Res. Cell Motil. 14, 54-64), suggesting that differences do exist between heat-purified CaD and the native protein. We have isolated, without heat treatment, full-length recombinant chicken gizzard CaD overexpressed in insect cells (High-FiveTM) using a baculovirus expression system. We found that such unheated CaD interacts with calmodulin 10 times stronger than does the heated CaD; its inhibitory action on actomyosin ATPase is reversed by a much lesser amount of calmodulin. Moreover, electron microscopic examination indicated that actin binding at the N-terminal region is more frequent in the unheated CaD, resulting in more lengthwise binding. These findings point to the fact that CaD is not entirely heat-stable; the C-terminal CaM-binding regions and the N-terminal actin-binding region are possibly affected by heat treatment.
Zhuang
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Muscle Research Group, Boston Biomedical Research Institute, Boston, Massachusetts 02114, USA. Wang@bbri.harvard.edu
Mabuchi
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K
Wang
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CA
D000199:Actins / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D002645:Chickens; D006358:Hot Temperature; D008854:Microscopy, Electron; D009218:Myosins / Q000037:antagonists & inhibitors; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D011994:Recombinant Proteins; D009218:Myosins