Isolation and some biochemical properties of porcine pancreas mitochondria

Morphologically and functionally intact mitochondria were isolated from the porcine pancreas using a conventional, differential centrifugation method. The homogenate of the porcine pancreas made in a medium containing 0.25 M sucrose, 10 mM Tris-Cl, pH 7.4, 0.5% bovine serum albumin and 1 mM EDTA, in the presence or in the absence of dibucaine and trypsin inhibitor, was centrifuged for 10 min at 700xg. The supernatant was centrifuged for 10 min at 7000xg, and the pellet was washed three times. Trypsin activity of mitochondria isolated in the absence of dibucaine and trypsin inhibitor was as low as that of mitochondria isolated in the presence of dibucaine and trypsin inhibitor, and a major part of the activity remained inactivated. Phospholipase A2 activity of the former mitochondria was as low as that of the latter, and remained unchanged up to 8-10 hr at 4 degrees C. The presence of bovine serum albumin and EDTA in the respiration medium was absolutely required to obtain good respiratory controls of those mitochondria. These data suggest that well-coupled mitochondria can be obtained from the pancreas by a conventional isolation procedure without activating the major part of trypsin.