Ohta Sho, Nishikawa Atsushi, Imamura Kiichi
Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1, Ridai-cho, Okayama 700-0005, Japan.
Comp Biochem Physiol B Biochem Mol Biol. 2003 Jun;135(2):397-405. doi: 10.1016/s1096-4959(03)00095-2.
A cDNA clone encoding pyruvate kinase (PK) was isolated from a skeletal muscle cDNA library of globefish (Fugu rubripes), which is a kind of lower vertebrate. The full-length cDNA of globefish skeletal muscle pyruvate kinase (FM-PK) is approximately 2 kb and encodes a protein comprising 530 amino acids. The FM-PK gene is spanning approximately 4.8 kb and consists of 11 exons. FM-PK mRNA was detected in muscle and heart using Northern blots. The recombinant FM-PK (rFM-PK) was expressed in a baculovirus-insect cell system and purified using ion-exchange chromatography. The purified rFM-PK was shown to exist a 230 kDa homotetramer composed of 57 kDa subunits. Gel filtration showed 230000 as the tetramer of the subunit. The apparent K(m) (or S(0.5)) and the Hill coefficient for phosphoenolpyruvate (PEP) and ADP are 0.14 mM, 1.3 and 0.30 mM 0.98 at pH 7.4, respectively, when the enzyme is saturated with the second substrate. The rFM-PK is strongly activated by fructose-1,6-bisphosphate, the apparent K(m) for PEP changes to 0.059 mM and the Hill coefficient to 1.1. ATP, which is the product of the enzyme reaction, inhibits activity. This is the first report to show the full-length cDNA and amino acid sequence of PK for a species of fish.
从一种低等脊椎动物——河豚（红鳍东方鲀）的骨骼肌cDNA文库中分离出一个编码丙酮酸激酶（PK）的cDNA克隆。河豚骨骼肌丙酮酸激酶（FM-PK）的全长cDNA约为2 kb，编码一个由530个氨基酸组成的蛋白质。FM-PK基因跨度约为4.8 kb，由11个外显子组成。使用Northern印迹法在肌肉和心脏中检测到了FM-PK mRNA。重组FM-PK（rFM-PK）在杆状病毒-昆虫细胞系统中表达，并通过离子交换色谱法进行纯化。纯化后的rFM-PK显示为一种由57 kDa亚基组成的230 kDa同型四聚体。凝胶过滤显示该亚基的四聚体分子量为230000。当酶被第二种底物饱和时，在pH 7.4条件下，磷酸烯醇丙酮酸（PEP）和ADP的表观K(m)（或S(0.5)）以及希尔系数分别为0.14 mM、1.3和0.30 mM、0.98。rFM-PK被1,6-二磷酸果糖强烈激活，PEP的表观K(m)变为0.059 mM，希尔系数变为1.1。该酶反应的产物ATP抑制其活性。这是首次报道一种鱼类PK的全长cDNA和氨基酸序列。
