Evolution of phosphagen kinase. Isolation, characterization and cDNA-derived amino acid sequence of two-domain arginine kinase from the sea anemone Anthopleura japonicus.
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.
Suzuki
T
T
Department of Biology, Faculty of Science, Kochi University, Kochi 780, Japan.
Kawasaki
Y
Y
Furukohri
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D018076:DNA, Complementary / Q000302:isolation & purification* / Q000378:metabolism; D019143:Evolution, Molecular; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012615:Sea Anemones / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D001122:Arginine Kinase; D008274:Magnesium; D002118:Calcium