Purification and characterization of arginine kinase from the mantle muscle of the squid, Symplectoteuthis oualaniensis: Role of the phosphagen/phosphagen kinase system in a highly aerobic muscle

Abstract

Arginine kinase (EC 2.7.3.3) from squid mantle muscle was purified to homogeneity as judged by isoelectric focusing and electrophoresis (sodium dodecyl sulfate, starch). The enzyme is a monomer of molecular weight 55,000 ± 6000 and occurs in mantle muscle in a single electrophoretic form with a specific activity of 310 units/g wet wt at 25 °C. The pH optima are 8.2 and 6.9 in the arginine phosphate-synthesizing and ATP-synthesizing directions, respectively. The divalent cation requirement of the enzyme is satisfied by Mg 2+ or Mn 2+ but added Ca 2+, Zn 2+, Cu 2+, and Fe 2+ are inhibitory. Monovalent ions ( NH 4 +, K +, Na +, Cl −) inhibit the enzyme. Michaelis constants for ATP and arginine (both measured at pH 7.6) and for ADP and arginine phosphate (pH 7.2) are 0.8, 1.0, 0.2, and 3.5 m m, respectively, the K m 's for any one substrate being independent of the concentration of its cosubstrate. Of over 40 metabolites tested for in vitro effects on the purified enzyme, only NADH, within its physiological range, affected enzyme activity. NADH is a noncompetitive inhibitor with a K i of 0.15 m m. Like other enzymes of energy metabolism in squid mantle muscle, arginine kinase is tightly regulated, its activity being closely integrated with that of glycolysis and the Krebs cycle by NADH control. A theory is proposed, based on the data obtained, that the arginine kinase/arginine phosphate system in this highly aerobic muscle serves to reversibly regulate glycolytic flux and that its role in “energy storage” is very limited.