[55] Isolation of carbodiimide-binding proteins from mitochondria and Escherichia coli

N,N'-Dicyclohexylcarbodiimide (DCCD) is a potent inhibitor of the proton-translocating ATPase systems found in prokaryotes and eukaryotes. When membrane preparations from beef heart mitochondria, yeast mitochondria, and Escherichia coli are treated under controlled conditions with the radioactive carbodiimides [14C] DCCD and [14C] EDC or the spin-labeled NCCD, these carbodiimides bind covalently to the membrane. It has been shown that DCCD binds to a specific protein. When beef heart submitochondrial particles (SMP), E. coli membrane preparations, or detergent-dispersed inhibitor-sensitive ATPase preparations are treated with low titers of [14C] DCCD and then dissolved in sodium dodecyl sulfate (SDS) and subjected to electrophoresis on SDS-containing gels, the major labeled component in all cases comigrates with a protein that has an apparent molecular weight of 10,000 daltons. However, membrane phospholipids migrate to this region of the polyacrylamide gel, and DCCD has been shown to react with phospholipids. That DCCD is binding to a protein and not to a membrane phospholipid is suggested by two additional observations. Thin-layer chromatography of purified DCCD-binding materials separates all detectable phosphate-containing compounds from the [14C] DCCD-binding components; digestion of purified [14C] DCCD-binding fraction into a water-soluble form. The ATPase activity of these mutants is less sensitive to inhibition by DCCD, and the resistance in the mutants correlates with a lower level of labeling of the 10,000-dalton protein by [14C] DCCD.