Isolation of mitochondrial porin from Neurospora crassa

Mitochondrial porin forms channels in the outer mitochondrial membrane which allows the passage of molecules with diameters up to -20 A [l-6]. We have described the purification of this protein [7] from purified outer membranes by differential extraction with detergents and anionic exchange chromatography. The purified porin displayed an app. Mr of 31 000 as judged by polyacrylamide gel electrophoresis. The isolated protein could be inserted into artificial lipid bilayers in an asymmetric fashion producing voltage-dependent channels for anions and cations [3,4,7]. Mitochondrial porin bears a number of similarities to the porins of the outer membrane of Gram-negative bacteria [8,9]. A detailed investigation of this interesting protein is hampered by the fact that only small quantities can be prepared when the isolation procedure starts with purified outer mitochondrial membrane. Therefore we have developed a method for a rapid isolation which gives pure, active porin, in high yield. This procedure entails chromatography of detergent solubilized mitochondrial protein on hydroxyapatite and celite. The latter procedure was introduced for the isolation of the phosphate carrier from animal mitochondria [lo]. The porin purified by this procedure shows a single band upon SDS gel electrophoresis (M, = 31 000). Upon isoelectric focusing it displays two bands with PI-values of 7.7 and 7.8. It is functionally active after insertion into lipid bilayers.