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Journal of Biochemistry Advance Access published online on August 30, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm154
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© 2007 The Japanese Biochemical Society

Recombinant Sea Urchin Flagellar Adenylate Kinase

Masashi Kinukawa{ddagger} and Victor D. Vacquier

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093-0202 USA

{ddagger} Corresponding author: Dr. Kinukawa, Masashi: E-mail, makinukawa{at}ucsd.edu, Tel. 858-534-2146, Fax 858-534-7313

Received June 29, 2007; Accepted July 19, 2007


  Abstract

Adenylate kinase (SpAK) is localized in sea urchin sperm flagella and embryonic cilia. To investigate SpAK enzymatic characteristics, the full-length recombinant protein of 130-kDa (SpAKr) and each of its three catalytic domains were expressed in E. coli. Although the full-length SpAK had high enzymatic activity, each of the three catalytic domains had no activity. The Km for ATP synthesis from ADP was 0.23 mM and the Vmax was 4.51 µmole ATP formed per min per mg protein. The specific AK inhibitor, Ap5A, blocks SpAKr enzymatic activity with an IC50 of 0.53 µM. The pH optimum for SpAKr is 8.1, as compared to 7.7 for the natural SpAK. Calcium inhibits SpAKr activity in a dose-dependent manner. Although SpAKr has three cAMP-dependent protein kinase phosphorylation sites, and can be phosphorylated in vitro, the enzymatic kinetics after phosphorylation are not significantly altered. SpAK and Chlamydomonas flagellar AKs are the only AKs with three catalytic sites. Further study of the SpAKr will aid in understanding the active site of this interesting and important ATP synthase.

Key Words: adenylate kinase, protein expression, ATP synthesis, cell motility, axoneme, protein phosphorylation


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