Molecular basis of interactions between mitochondrial proteins and hydroxyapatite in the presence of Triton X-100, as revealed by proteomic and recombinant techniques.

Hydroxyapatite chromatography is a very important step in the purification of voltage-dependent anion channels (VDACs) and several members of solute carrier family 25 (Slc25) from isolated mitochondria. In the presence of Triton X-100, VDACs and Slc25 members present a peculiar property, i.e., a lack of interaction with hydroxyapatite, resulting in their presence in the flow-through fraction of hydroxyapatite chromatography. This property has allowed selective isolation of VDACs and Slc25 members from a mixture of total mitochondrial proteins. However, the reason why only these few proteins are selectively obtained in the presence of Triton X-100 from the flow-though fraction of hydroxyapatite chromatography has not yet been adequately understood. In this study, when we examined the protein species in the flow-through fractions by proteomic analysis, VDAC isoforms, Slc25 members, and some other membrane proteins were identified. All the mitochondrial proteins had in common high hydrophobicity over their entire protein sequences. When the proteins were fused to soluble proteins, the fused proteins showed affinity for hydroxyapatite even in the presence of Triton X-100. Based on these results, we discussed the molecular basis of the interactions between proteins and hydroxyapatite in the presence of Triton X-100.

Specific formation of trypsin-resistant micelles on a hydrophobic peptide observed with Triton X-100 but not with octylglucoside.

The manner of interaction of the coat peptide of the Pf3 phage (Pf3 peptide) with lipid bilayers has been extensively studied. Presently, we designed a derivative of the Pf3 peptide, referred to as the DDRK peptide, and subjected it to trypsin digestion to understand its physicochemical properties. In the presence of Triton X-100 used for solubilization of the peptide, digestion of DDRK with trypsin caused specific cleavage at the lysine (Lys) residue in its N-terminal region but not at other Lys residues or at the arginine residue. As the N-terminal region of the DDRK peptide is relatively hydrophilic, but its remaining region is hydrophobic, this hydrophobic region of the peptide would be expected to be coated by Triton micelles. Thus, we propose that the presence of such micelles protected against cleavage there, leading to selective cleavage by trypsin of the DDRK peptide at its hydrophilic Lys residue in the N-terminal part of the molecule. However, such a protective effect on the DDRK peptide against trypsin digestion was not observed with octylglucoside. The observed results are important for better understanding of the manner of interaction between detergents and hydrophobic peptides.

Substitution of certain amino acids in a short peptide causes a significant difference in their immunoreactivities with antibodies against different epitopes: evidence for possible folding of the peptide on a nitrocellulose or PVDF membrane.

Substitution of amino acids in a peptide caused remarkable differences in its immunoreactivities with antibodies against 3 epitopes in the immobilized peptide. The observed differences in immunoreactivities among the peptides were not due to the differences in efficiencies of their transfer onto nitrocellulose or PVDF membranes. Rather, possible folding of the peptide on the membrane was considered to be the reason for their distinct immunoreactivities with the antibodies.