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A key step in the initiation of apoptosis is the release from the mitochondrial intermembrane space of cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO, Omi/HtrA2, apoptosis-inducing factor (AIF), and endonuclease G (EndoG). Discrepancies have arisen, however, as to whether all these proteins are released in different systems. Our results suggest that failure to observe cytochrome c release may be due to the use of different buffers because after permeabilization by caspase-8 cleaved human Bid (tBid), cytochrome c dissociation from mitochondria was highly dependent on ionic strength and required 50-80 mM KCl, NaCl, or LiCl. In addition, mitochondria isolated from apoptotic cells using low ionic strength buffer bound a greater proportion of endogenous cytochrome c. In contrast to cytochrome c, Smac/DIABLO and Omi/HtrA2 were released independent of ionic strength, and AIF and EndoG behaved as if they are exposed to the intermembrane space but tethered to or within the inner membrane. AIF and EndoG were also not released by active caspases, which suggests their involvement in apoptosis may be limited. In summary, whereas tBid permeabilizes the outer membrane to cytochrome c, Smac/DIABLO, and Omi/HtrA2, the release of cytochrome c during apoptosis will be underestimated unless sufficient ionic strength is maintained to overcome the electrostatic association of cytochrome c with membranes. [ABSTRACT FROM AUTHOR]

CEL-I is a C-type lectin, purified from the sea cucumber Cucumaria echinata, that shows a high specificity for N-acetylgalactosamine (GalNAc). We determined the crystal structures of CEL-I and its complex with GalNAc at 2.0 and 1.7 Å resolution, respectively. CEL-I forms a disulfide-linked homodimer and contains two intramolecular disulfide bonds, although it lacks one intramolecular disulfide bond that is widely conserved among various C-type carbohydrate recognition domains (CRDs). Although the sequence similarity of CEL-I with other C-type CRDs is low, the overall folding of CEL-I was quite similar to those of other C-type CRDs. The structure of the complex with GalNAc revealed that the basic recognition mode of GalNAc was very similar to that for the GalNAc-binding mutant of the mannosebinding protein. However, the acetamido group of GalNAc appeared to be recognized more strongly by the combination of hydrogen bonds to Arg115 and van der Waals interaction with Gln70. Mutational analyses, in which Gln70 and/or Arg115 were replaced by alanine, confirmed that these residues contributed to GalNAc recognition in a cooperative manner. [ABSTRACT FROM AUTHOR]