Calcium-induced exposure of a hydrophobic surface of mouse ALG-2, which is a member of the penta-EF-hand protein family.
ALG-2 is a 22 kDa EF-hand type Ca2+-binding protein associated with lymphocyte apoptosis. Comparison of the primary structure of ALG-2 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-2 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-2 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-PEF 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-2 exposed a hydrophobic surface in a Ca2+-concentration dependent manner, the half-maximal effect occurring at approximately 6 microM. Mg2+ was not effective for the conformational change. On Western blotting, ALG-2 was detected in particulate fractions from cultured mammalian cells, suggesting the association of the protein with macromolecules in the cells.
Maki
M
M
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan. mmaki@agr.nagoya-u.ac.jp
Yamaguchi
K
K
Kitaura
Y
Y
Satoh
H
H
Hitomi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D019556:COS Cells / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics; D011232:Chemical Precipitation; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000378:metabolism; D017830:Octoxynol / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D013347:Subcellular Fractions
D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D009282:Naphthalenesulfonates; C490815:Pdcd6 protein, mouse; D011994:Recombinant Proteins; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D017830:Octoxynol; D002118:Calcium