Purification, characterization, and hydrodynamic properties of arginine kinase from gulf shrimp (Penaeus aztecus)

Arginine kinase from the tail muscle of the Gulf shrimp (Penaeus aztecus) was purified to apparent homogeneity, using a rapid, high-yield method. The protein exhibits a molecular weight of 40 kDa according to the methods of gel filtration and gel electrophoresis in sodium dodecyl sulfate, also indicating that arginine kinase from shrimp is a monomer. The amino acid content of arginine kinase from shrimp is similar to arginine kinases from several species and to creatine kinase from rabbit muscle. Arginine kinase derivatized at the reactive sulfhydryl with 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid exhibits significant changes in fluorescence anisotropy only in the presence of the guanidino substrate and the so-called "dead-end complex" containing arginine + MgADP. Several compounds structurally similar to arginine, e.g., ornithine do not interact with arginine kinase, suggesting a narrow specificity for substrate binding. The most suitable description of the decay of the fluorescence of arginine kinase derivatized with 5-[(((acetyl)-amino)ethyl)amino]naphthalene-1-sulfonate (AEDANS-AK), from among discrete and distributed models, is a triple exponential discrete decay. The presence of the dead-end complex only marginally increases the rate of decay, but significantly shifts the magnitude of the preexponentials (amplitudes) between the two major decay components. One interpretation of these results is that multiple conformational isomers may occur, in which the relative concentrations are dependent upon the presence of the dead-end complex. Measurement of the time-dependent anisotropy decay of AEDANS-AK reveals a two-term decay law with rotational correlation times of 0.88 and 15.2 ns. The slower component is close to the theoretical value of 16.7 ns for an isotropic rotator of the molecular mass of arginine kinase. This finding suggests that the overall conformation of arginine kinase may differ considerably from the prolate ellipsoidal subunits of the functionally analogous creatine kinase.