Taurocyamine Kinase from Body‐Wall Musculature of the Lugworm Arenicola marina

Abstract

ATP: taurocyamine phosphotransferase was extracted from body‐wall musculature of the lugworm Arenicola marina and purified by sequential ammonium sulfate precipitation, Sephadex G‐25, DEAE‐cellulose and hydroxylapatite column chromatography to polyacrylamide discelectrophoretic homogeneity. The enzyme was enriched 860‐fold to a specific activity of 1200–1300 μmol×min−1× mg protein−1. For direct measurement of the enzyme activity of forward as well as of reverse reaction one spectrophotometrical assay was evolved. The purified enzyme has a molecular weight of approximately 60000 (assayed by means of sucrose‐density‐gradient centrifugation and by gel‐sieve chromatography on a Sephadex G‐100 column). Dodecylsulfate/polyacrylamide gel electrophoresis revealed three bands: at 22000, 13000–14000, and 11000. Temperature optimum of the taurocyamine kinase activity was found to be 33°C. The pH optimum of the reaction velocity of the forward reaction is at pH 8.0, that of the reverse reaction at pH 7.2. K m values for the substrates of the forward reaction are 1.0 × 10−4 M for taurocyamine and 3.3 × 10−3 M for ATP, and for those of the reverse reaction 8.3 × 10−4 M for phosphotaurocyamine and 1.2 × 10−3 M for ADP. V value of the forward reaction is 240 μmol×min−1× mg protein−1, and that of the reverse 1340 μmol×min−1× mg protein−1. Phosphocreatine and phosphoarginine are inactive. To become active the taurocyamine kinase needs Mg2+ (10 mM). Isoelectric focusing of the purified enzyme showed a pattern of about 8 bands between pH 6.1 and 7.8, the main band lying at pH 7.3. Within the cells of the body‐wall musculature 92–94% of the taurocyamine kinase activity were found to be in the cytosol, the mitochondria, in all probability, have 6–8% of the total activity. Copyright © 1979, Wiley Blackwell. All rights reserved