Molecular Biological Studies on Functional Properties of Fish Myosin with Reference to Carp Fast Skeletal Muscle

参考鲤鱼快骨骼肌鱼肌球蛋白功能特性的分子生物学研究

• 批准号: 07456095
• 负责人: WATABE Shugo
• 金额: $ 4.03万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07456095/
• 关键词: carp; fast skeletal muscle; myosin; myosin heavy chain; light meromyosin; cDNA cloning; temperature acclimation; primary structure; cDNAクローリニング; cDNA; PCR

Carp, Cyprinus carpio (0.5-0.8kg in body weight), were acclimated to either 10 or 30ﾟC for a minimum of 5 weeks, and their dorsal fast skeletal muscles were used for isolation of light meromyosin (LMM), C-terminal one third of myosin heavy chain. The microsequence analysis after SDS-PAGE revealed that the LMM component from the 10ﾟC-acclimated carp contained an N-terminal amino acid sequence different from that of the 30ﾟC-acclimated carp. DNA fragments encoding an N-terminal region of LMM were amplified by PCR or reverse transcription-PCR,demonstrating that the two acclimated groups further contained several amino acids substituted.cDNA clones encoding LMM were isolated from cDNA libraries constructed from fast skeletal muscles of carp acclimated to 10 and 30ﾟC,using the above-mentioned DNA as a probe. All clones covered at least the full length of LMM.cDNAs were identified which were the predominant types expressed in 10ﾟC-and 30ﾟC-acclimated fish, as well as an intermediate type present at all acclimation temperature. Northern blot analysis using probes ofthree kinds of DNA fragments from the 3'untranslated region of carp acclimated to 10,20 and 30ﾟC further confirmed the presence of acclimation temperature-specific isoforms. In addition, it was found that mRNA levels of three isoforms were altered in an acclimation temperature-dependent manner. The deduced amino acid sequence identity of LMM isoforms were 95.6% between the 10 and 30ﾟC types and 97.8% between the 10ﾟC and intermediate types. When these were compared with those of homoiotherms, the 30ﾟC-acclimated type was more similar to those of homoiotherms than was the 10ﾟC-acclimated type. These results suggest that the 30ﾟC type of LMM is more thermostable than that of 10ﾟC type.

鲤鱼（Cyprinus carpio）（体重0.5-0.8kg）在10 ℃或30 ℃下驯化5周后，取其背侧快肌分离肌球蛋白重链C端1/3的轻肌球蛋白（LMM）。SDS-PAGE后的微序列分析显示，来自10 ℃驯化的鲤鱼的LMM组分含有与30 ℃驯化的鲤鱼不同的N-末端氨基酸序列。用PCR或RT-PCR扩增出编码LMM N端的DNA片段，证明这两个驯化组还含有几个氨基酸的替代，以上述DNA为探针，从10 ℃和30 ℃驯化的鲤鱼快速骨骼肌cDNA文库中分离出编码LMM的cDNA克隆。所有克隆至少覆盖LMM的全长。cDNA被鉴定为在10 ℃和30 ℃驯化的鱼中表达的主要类型，以及在所有驯化温度下存在的中间类型。以10 ℃、20 ℃和30 ℃驯化的鲤鱼3 '端非翻译区的3种DNA片段为探针进行北方印迹分析，进一步证实了驯化温度特异性异构体的存在。此外，有人发现，三个异构体的mRNA水平的变化在驯化温度依赖性的方式。LMM 10 ~ 30个亚型的氨基酸同源性为95.6%，10 ~ 30个亚型的氨基酸同源性为97.8%。当这些与恒温动物的那些比较时，30 ℃-驯化型比10 ℃-驯化型更类似于恒温动物的那些。这些结果表明，30 ℃型LMM比10 ℃型LMM更耐热。

项目成果

Kiyoshi Kikuchi et al.: "The warm temperature acclimation-related 65-kDa protein,Wap65,in goldfish and its gene expression." The Journal of Biological Chemistry. 270. 17087-17092 (1995)

Kiyoshi Kikuchi 等人：“金鱼中与温暖温度驯化相关的 65-kDa 蛋白 Wap65 及其基因表达。”

Shugo Watabe, Jun-ichi Imai, Misako Nakaya, Yasushi Hirayama, Yoh Okamoto, Haruhiko Masaki, Takeshi Uozumi, Ikuo Hirono, and Takashi Aoki: "Temperature acclimation induces light meromyosin isoforms with different primary structures in carp fast skeletal m

Shugo Watabe、Jun-ichi Imai、Misako Nakaya、Yasushi Hirayama、Yoh Okamoto、Haruhiko Masaki、Takeshi Uozumi、Ikuo Hirono 和 Takashi Aoki：“温度适应在鲤鱼快速骨骼肌中诱导具有不同初级结构的轻肌球蛋白亚型

Kiyoshi Kikuchi, Michiaki Yamashita, Shugo Watabe, and Katsumi Aida: "The warm temperature acclimation-related 65-kDa protein, Wap65, in goldfish and its gene expression" J.Biol.Chem.270(29). 17087-17092 (1995)

Kiyoshi Kikuchi、Michiaki Yamashita、Shugo Watabe 和 Katsumi Aida：“金鱼中与温暖温度驯化相关的 65-kDa 蛋白 Wap65 及其基因表达”J.Biol.Chem.270(29)。

Shin-ya Saito, Shugo Watabe, Hiroshi Ozaki, Hideo Kigoshi, Kiyohuki Yamada, Nobuhiro Fusetani, and Hideaki Karaki: "Novel actin depolymerizing macrolide aplyronine A" J.Biochem.120. 552-555 (1996)

Shin-ya Saito、Shugo Watabe、Hiroshi Ozaki、Hideo Kigoshi、Kiyohuki Yamada、Nobuhiro Fusetani 和 Hideaki Karaki：“新型肌动蛋白解聚大环内酯 aplyronine A”J.Biochem.120。

S.Chaen: "Lower activation energy for sliding of F-action on a less themostable isoform of carp myosin." J.Biochem.120. 788-791 (1996)

S.Chaen：“在不太稳定的鲤鱼肌球蛋白亚型上滑动 F 作用的活化能较低。”