title: Dicyclohexylcarbodiimide interaction with the voltage-dependent anion channel from sarcoplasmic reticulum.
abstract: In a recent study [Shoshan-Barmatz, V., Orr, I., Weil, S., Meyer, H., Varsanyi, M. & Heilmeyer, L. M. G. (1996) FEBS Lett. 386, 205-210] we have demonstrated the presence of the voltage-dependent anion channel (VDAC) in skeletal muscle sarcoplasmic reticulum (SR) as supported here by co-localization of VDAC and (Ca2+ + Mg2+)ATPase in the SR using double-immunogold labeling. The interaction of the carboxyl-modifying reagent dicyclohexylcarbodiimide with the SR-VDAC is characterized by labeling with [14C]dicyclohexylcarbodiimide and by dicyclohexylcarbodiimide modification of the reconstituted-purified VDAC channel activity. In both SR and mitochondrial membranes, [14C]dicyclohexylcarbodiimide most specifically labeled a 35-kDa protein, identified as VDAC by specific anti-VDAC Ig. Labeling of the SR-VDAC was about twofold higher than that of the mitochondrial VDAC, which could result either form higher labeling of the SR protein or from relatively higher amounts of VDAC/mg total protein in the SR membranes. [14C]Dicyclohexylcarbodiimide labeling of the SR, but not the mitochondrial VDAC, was biphasic with respect to time and concentration of [14C]dicyclohexylcarbodiimide. Partial digestion of [14C]dicyclohexylcarbodiimide-labeled SR-VDAC with chymotrypsin yielded five proteolytic fragments which were recognized by the anti-VDAC Ig, and the dicyclohexylcarbodiimide-binding site was localized in the 19-kDa fragment. VDAC was purified from SR and mitochondrial membranes by spermine-agarose column. The interaction of dicyclohexylcarbodiimide with functional carboxyl residue(s) in the purified VDAC is demonstrated by recording its channel activity, following its reconstitution into planar lipid bilayer (PLB). Dicyclohexylcarbodiimide inhibited the channel activity in a voltage-dependent manner, requiring incubation with dicyclohexylcarbodiimide at high (negative or positive) potentials. Dicyclohexylcarbodiimide slowed down the transition from the high-conducting to a long-lived low-conducting states of the channel (approximately 20% of its maximal conductance), by stabilizing the intermediate states. Similar results were also obtained with purified-reconstituted mitochondrial VDAC. Hydrophilic carboxyl reagents [[1-ethyl-3-(3-dimethylamino)propyl] carbodiimide, N-ethyl-phenylisoxazolium-3'-sulfonate] neither modified the channel activity nor prevented [14C]dicyclohexylcarbodiimide labeling. These results indicate that dicyclohexylcarbodiimide interacts with a carboxyl group located in a hydrophobic region of the protein which is involved in the channel gating.
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: I
authors.initials: I
authors.lastname: Shafir
authors.forename: W
authors.initials: W
authors.lastname: Feng
authors.forename: V
authors.initials: V
authors.lastname: Shoshan-Barmatz
mesh_terms: D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000032:analysis; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism* / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007473:Ion Channels / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D016253:Microscopy, Immunoelectron; D008930:Mitochondria, Liver / Q000378:metabolism* / Q000648:ultrastructure; D008931:Mitochondria, Muscle / Q000378:metabolism* / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D018272:Porins; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D050994:Voltage-Dependent Anion Channels
chemical_list: D007473:Ion Channels; D008565:Membrane Proteins; D010446:Peptide Fragments; D018272:Porins; D050994:Voltage-Dependent Anion Channels; D004024:Dicyclohexylcarbodiimide; D017301:Ca(2+) Mg(2+)-ATPase