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Journal of Biochemistry Advance Access originally published online on November 21, 2007
Journal of Biochemistry 2008 143(2):217-228; doi:10.1093/jb/mvm211
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© 2007 The Japanese Biochemical Society.

Drosophila Calpain B is Monomeric and Autolyzes Intramolecularly

Min Woo Park and Yasufumi Emori*

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan

*To whom correspondence should be addressed. Tel/Fax: +81-3-5846-4406, E-mail: ss47236{at}mail.ecc.u-tokyo.ac.jp, emori{at}biochem.s.u-tokyo.ac.jp

Received September 12, 2007; Accepted October 28, 2007


  Abstract

Drosophila calpains, Calpain A and Calpain B, show typical calpain domain structures similar to mammalian calpains. However, the small subunit of mammalian calpains, shown to be essential in both genetic and biochemical aspects, is absent in Drosophila calpains and is not required for enzymatic activity. How they compensate for the lack of small subunit is mostly unknown. Here we conducted experiments using recombinant Drosophila Calpain B for further characterization of the enzyme with particular focuses on two issues: possibility of homodimerization and mode of autolysis. The native molecular weight of Calpain B indicates that the active enzyme is primarily monomeric. Co-expression of two recombinant Calpain B proteins each with a unique affinity tag and a subsequent single round of affinity tag purification resulted in isolation of only one recombinant calpain type, suggesting there is no homodimeric interaction. Also the C-termini of Drosophila calpains lack many of the key hydrophobic residues considered to be important in the dimerization of mammalian calpains. Further, initial autolysis of Calpain B seems to occur intramolecularly, which supports the monomeric nature of Drosophila calpains. These results strongly suggest that dimerization is not an essential requirement for Drosophila calpains.

Key Words: autolysis, calpain, Drosophila, intramolecular, monomer

Abbreviations: 2-ME, 2-mercaptoethanol; AP, alkaline phosphatase; DAB, diaminobenzidine tetrahydrochloride; ECL, enhanced chemiluminescence; EDTA, ethylenediamine tetraacetic acid; HRP, horse radish peroxidase; IPTG, isopropyl β-D-1-thiogalactopyranoside; OD, optical density; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethanesulphonylfluoride; PVDF, polyvinylidene difluoride; RT-PCR, reverse transcription polymerase chain reaction; SDS, sodium dodecyl sulphate; TBST, Tris buffered saline containing 0.05% Tween 20


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