Evolution of phosphagen kinase. Isolation, characterization and cDNA-derived amino acid sequence of two-domain arginine kinase from the sea anemone Anthopleura japonicus.
Evolution of phosphagen kinase. Isolation, characterization and cDNA-derived amino acid sequence of two-domain arginine kinase from the sea anemone Anthopleura japonicus.
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DOI：
10.1042/bj3280301
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发表时间：
1997-11
期刊：
影响因子：
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通讯作者：
Tomohiko Suzuki;Yoshitada Kawasaki;T. Furukohri
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文献类型：
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作者：
Tomohiko Suzuki;Yoshitada Kawasaki;T. Furukohri
采用Ultrogel AcA 34凝胶过滤、DEAE-32柱层析、Cosmogel-SP柱洗脱等方法，从原始海葵Anthopleura sp.体壁肌肉中分离出精氨酸激酶（AK）。用SDS/PAGE测定的变性分子量为80 kDa，是通常AK亚基的两倍，表明这种AK具有不寻常的双结构域结构。天然形式在Superose 12柱上洗脱，保留时间与兔同型二聚肌酸激酶相同，表明花侧壳AK是80 kDa的单体。在pH7.9 -9.1的范围内，分离的酶对于正向反应给出100-120微摩尔Pi/min/mg蛋白质的比活性。该酶被Ca 2+完全激活，因为它与Mg 2+。测定了花侧耳属AK的715个氨基酸的cDNA序列。内部胰蛋白酶肽的化学测序支持序列的有效性。在Ala-364密码子的第二和第三个核苷酸之间存在一个686 bp的桥内含子，它将花侧耳属AK的两个结构域分开。这是第一个被测序的双结构域AK。Anthopleura AK与已知的无脊椎动物AK显示出48-54%的氨基酸序列同一性，并且还显示出与海洋蠕虫糖胞胺激酶和兔肌酸激酶的较低但显著的相似性（39-46%）。
Arginine kinase (AK) was isolated from the body wall muscle of the primitive sea anemone Anthopleura japonicus by Ultrogel AcA34 gel filtration, DEAE-32 chromatography and elution on a Cosmogel-SP column. The denatured molecular mass as determined with SDS/PAGE was 80 kDa, twice that of the usual AK subunit, indicating that this AK has an unusual two-domain structure. The native form was eluted on a Superose 12 column with the same retention time as that of rabbit homodimeric creatine kinase, indicating that Anthopleura AK is a monomer of 80 kDa. The isolated enzyme gave a specific activity of 100-120 micromol of Pi/min per mg of protein in the pH range 7.9-9.1 for the forward reaction. The enzyme is fully activated by Ca2+, as it is with Mg2+. The cDNA-derived amino acid sequence of 715 residues of Anthopleura AK was determined. The validity of the sequence was supported by chemical sequencing of internal tryptic peptides. A bridge intron of 686 bp, which separates the two domains of Anthopleura AK, is present between the second and third nucleotide in the codon of Ala-364. This is the first two-domain AK to be sequenced. Anthopleura AK shows 48-54% amino acid sequence identity with known invertebrate AKs, and also shows a lower, but significant, similarity (39-46%) to marine worm glycocyamine kinase and rabbit creatine kinase.