Graduation Year


Document Type




Degree Granting Department


Major Professor

Steven H. Grossman, Ph.D.


Phosphotransferase, Enzyme characterization, Purification, Echinoderm, Quaternary structure


Arginine kinase catalyzes the reversible phosphorylation of arginine using ATP. This phosphotransferase is found throughout invertebrate species; whereas a homologous enzyme, creatine kinase, is found in both vertebrate and invertebrate species. Arginine kinases are found as monomers of 40 kDa or 80 kDa and dimers of 80 kDa while creatine kinases are found as dimers of 80 kDa, monomers of 150 kDa, or octamers of 320 kDa. The significance or advantage of the dimeric state or various quaternary structures is still not understood for this family of enzymes. Here, arginine kinase from Isostychopus badonotus muscle was purified to homogeneity and analyzed for physical, kinetic, and immunological characteristics. The results indicate that arginine kinase from the sea cucumber, I. badonotus, is a dimer with a molecular weight of 87 kDa that displays physical and kinetic characteristics similar to other arginine kinases regardless of their weight or subunit composition.

However, immunological cross-reactivity using I. badonotus polyclonal antibodies shows that dimeric arginine kinase from the sea cucumber can react with dimeric arginine and creatine kinases but not with monomeric arginine or creatine kinases. Comparable results are seen with polyclonal antibodies raised against purified monomeric arginine kinase from the American cockroach, Periplaneta americana. Monomeric arginine kinase from the cockroach reacted with monomeric arginine kinases but not with dimeric arginine or creatine kinases or monomeric creatine kinases. Arginine kinase from the sea cucumber and the cockroach is substantially inhibited by the anion nitrate which mimics the transferable phosphoryl group in the assumed rapid equilibrium, random addition reaction. Here, nitrate has been shown to inhibit both the initial velocity and percent of product formed from arginine kinase in I. badonotus and P. americana.

Difference spectra for each enzyme in the presence of varying components of the transition state analog suggest that nitrate has an effect on the enzyme itself and inhibits through a mechanism beyond that of stabilization of the dead-end complex. Further characterization of the dimeric state in these enzymes on a structural level included the elucidation of the protein sequence from the American cockroach and a comparison with dimeric arginine and creatine kinases.