Structural Differences in the Crossbridge Head of Temperature‐Associated Myosin Subfragment‐1 Isoforms from Carp Fast Skeletal Muscle

Abstract

We determined the primary structures of the three acclimation-temperature-associated isoforms of myosin subfragment-1 heavy chain from fast skeletal muscle of thermally acclimated carp. These isoforms were cloned by extending %regions of cDNAs that encode the rod part of myosin heavy chain specifically expressed in 10°C-and 30°C-acclimated carp, together with the region that encodes an intermediate Biol. 200,. These three isoforms generally resembled each other in primary structure, showing 94.8, 90.9, and 92% similarity between the 10°C-and intermediate-type, between the 10°C-and 30"C-type, and between the intermediate-and 30°C-type myosin heavy chains, respectively. However, isoform-specific differences were clearly observed between the 10°C-and 30°C-type heavy chains in the first 60 amino acid residues from the N-terminus, where the intermediate-type showed an intermediate feature in its sequence compared to the 10°C-and 30°C-type isoforms. Other striking differences were observed in two surface loops between the 10°C-and 30°C-type isoform. Five amino acid residues out of sixteen were different in loop 1 near the ATP-binding pocket, and six out of twenty were different in loop 2 on the actin-binding site. The loops connecting /3-sheets that are known to surround the ATP-binding pocket were highly conserved in primary structure for the three types. In northern blot analysis, the accumulated mRNA levels of the 10°C-and intermediate-type isoforms were significantly higher in carp acclimated to 10°C and 20°C than carp acclimated to 30"C, whereas the level of the 30°C-type isoform was significantly higher in carp acclimated to 30°C than those acclimated to 10°C and 20°C.Keywords: carp ; Cyprinus carpio ; fast skeletal muscle; myosin subfragment-I ; temperature acclimation.Water temperature is one of the most important physical factors for poikilotherms, since their body temperature closely parallels the temperature of the environment. Eurythermal fish such as carp and goldfish experience a wide range of water temperature throughout the year. However, they maintain constant physiological and biochemical rate processes, which compensate for fluctuation of environmental temperatures. Fry and Hart (1948) found that goldfish change cruising speed following temperature acclimation. Biochemical evidence for such physiological temperature-acclimation-inducing changes was obtained by Johnston et al. (1975) who showed that goldfish experience changes in myofibrillar Mg" adenosine 5'-triphosphatase (ATPase) activity after acclimation to warm and cold temperatures. At a given experimental temperature, goldfish acclimated to low temperature exhibit higher ATPase activity than fish acclimated to warm temperature. Later, Heap et al. (1985) obCorrespondence to S. Watabe,

Divergent evolution of the myosin heavy chain gene family in fish and tetrapods: evidence from comparative genomic analysis

“…Evolutionary pressure to maintain a normal physiology including locomotion and speed at various temperatures thus allowed teleosts to acquire a larger MYH repertoire than homoeothermic tetrapods. In fact, we found that common carp (7,22,19,55,56), grass carp (49), and medaka (33) express several types of fast skeletal MYHs in response to temperature change. In this regard, it is worth noting that eurythermal temperate fish such as common carp (27) and medaka (33) have more MYHs than torafugu, at least in cluster C, further substantiating the fact that the number of MYHs is related to a particular lineage or species-specific physiological needs.…”

“…Interestingly, common carp express several types of fast skeletal MYHs encoding isoforms with different ATPase and motor activities following temperature acclimation (7,19,22,55,56). It has recently been found that grass carp (Ctenopharyngodon idellus) undergoes similar changes in temperature-dependent expressions of fast skeletal MYHs (49), although grass carp is a diploid fish compared with tetraploid common carp (39).…”

Molecular cloning and mRNA expression analysis of carp embryonic, slow and cardiac myosin heavy chain isoforms

“…The deduced amino acid sequences obtained in this study contained part of a myosin rod region corresponding to amino acid residues 1792-1924 from the N terminus of MYH F10 (BAA22067; Hirayama and Watabe, 1997). These sequences together with those for the corresponding regions in the fast and cardiac/slow type MYHs reported from human Homo sapiens, zebrafish Danio rerio and torafugu pufferfish Takifugu rubripes were used for phylogenetic analysis following paired alignment of the sequences with CLUSTAL W (Thompson et al, 1994).…”

“…We have shown that changes in the expression of MYH isoforms play a key role in the plasticity of myofibrillar ATPase activity and contractile properties with temperature acclimation (Hwang et al, 1990;Watabe et al, 1992Watabe et al, , 1995Guo et al, 1994). Three distinct MYH DNAs were cloned from the fast myotomal muscle of carp acclimated to either 10°C or 30°C for a minimum of 6·weeks (Imai et al, 1997;Hirayama and Watabe, 1997). The nomenclature used for these clones was based on the acclimation temperature at which the corresponding mRNAs were most strongly expressed.…”

Temperature and the expression of seven muscle-specific protein genes during embryogenesis in the Atlantic codGadus morhuaL.

“…The MyHC cDNA was a partial sequence (the full coding cDNA is >5000·bp in carp; Hirayama and Watabe, 1997). The isolated clone came from the 3′ end and spanned 779·bp of coding region and 89·bp of untranslated region (UTR).…”