Calcium-induced exposure of a hydrophobic surface of mouse ALG-2, which is a member of the penta-EF-hand protein family

ALG-8 is a 22 kDa EF-hand type Ca2+-binding protein associated with lymphocyte apoptosis, Comparison of the primary structure of ALG-8 with those of EF-hand type proteins revealed that it belongs to the penta-EF-hand (PEF) protein family including the small subunit of calpain, We established a convenient method for the purification of the recombinant mouse ALG-8 expressed in Escherichia coli, The recombinant protein was first pelleted from a lysate in the absence of a Ca2+-chelator, and then extracted with buffer containing EDTA/EGTA followed by purification by conventional column chromatographies, Estimation of the molecular mass by gel filtration suggested that the recombinant ALG-8 occurred as a monomeric form, Ca2+-dependent precipitation was blocked by inclusion of non-ionic detergent Triton X-100, suggesting hydrophobic self-aggregation at high concentrations of the protein. The N-terminal deletion mutant lacking the hydrophobic non-PEE region was found to be more soluble than the wild type in the presence of Ca2+, Analysis using a fluorescent hydrophobicity probe indicated that ALG-g exposed a hydrophobic surface in a Ca2+-concentration dependent manner, the half-maximal effect occurring at approximately 6 mu M. Mg2+ was not effective for the conformational change. On Western blotting, ALG-8 was detected in particulate fractions from cultured mammalian cells, suggesting the association of the protein with macromolecules in the cells.