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Journal of Biochemistry Advance Access originally published online on August 30, 2007
Journal of Biochemistry 2007 142(4):501-506; doi:10.1093/jb/mvm154
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© 2007 The Japanese Biochemical Society.

Recombinant Sea Urchin Flagellar Adenylate Kinase

Masashi Kinukawa* and Victor D. Vacquier

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

*To whom correspondence should be addressed. Tel: 858-534-2146, Fax: 858-534-7313, E-mail: makinukawa{at}ucsd.edu

Received June 29, 2007; Accepted July 19, 2007


  Abstract

Adenylate kinase (AK) is localized in sea urchin sperm flagella and embryonic cilia. To investigate sea urchin Strongylocentrotus purpuratus AK (SpAK) enzymatic characteristics, the full-length recombinant protein of 130 kDa (SpAKr) and each of its three catalytic domains were expressed in Escherichia 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 µmol ATP formed per minute per milligram of 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

Abbreviations: AK, adenylate kinase; SpAK, sea urchin Strongylocentrotus purpuratus AK; 130 kDa SpAK, S. purpuratus AK of 130 kDa relative molecular mass; SpAKr, the recombinant full-length 130 kDa SpAK protein expressed in Escherichia coli; PKA, cyclic AMP-dependent protein kinase; ALP, calf intestinal alkaline phosphatase; DTT, dithiothreitol; PMSF, phenylmethylsulfonylfluoride; Ap5A, P1,P5-di(adenosine-5')-pentaphosphate


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