ISOLATION AND CHARACTERIZATION OF THE MITOCHONDRIAL CHANNEL, VDAC, FROM THE INSECT HELIOTHIS-VIRESCENS

A 31 kDa voltage-dependent anion-selective channel (VDAC) protein was purified from the insect Heliothis virescens (tobacco budworm, denoted TBW) using an alkali extraction and filtration procedure and was char acterized by SDS-PAGE, amino acid sequencing, biophysical properties a nd immunocytochemistry. The N-terminal sequence has highest identity w ith VDACs from mammals (50-66%) followed by plants (34-41%) and lower eukaryotes (30-34%). Reconstitution in planar phospholipid membranes y ielded properties typical of VDACs from other organisms including a si ngle-channel conductance of 4.1 nS (in 1 M KCl), closure in response t o positive and negative transmembrane voltage, and a reversal potentia l of 11.8 mV indicating anion selectivity in the open state. A polyclo nal antiserum (R19) raised against gel-purified 31 kDa protein specifi cally labelled mitochondria and mitochondrial outer membranes in TBW f light muscle by light and electron microscope immunocytochemistry. (C) 1997 Elsevier Science B.V.