Characterization and partial purification of the VDAC-channel-modulating protein from calf liver mitochondria.
The mitochondrial channel, VDAC, mediates metabolic flux across the mitochondrial outer membrane. When reconstituted into planar phospholipid membranes, VDAC is voltage-dependent, existing in multiple conformational states with different selectivities and permeabilities. At low membrane potentials, these channels are in the open state and are anion-selective. VDAC channels switch to lower-conductive closed states at high membrane potentials. The VDAC modulator, a soluble mitochondrial protein, has been demonstrated to dramatically increase the voltage dependence of VDAC channels and induce the channels to enter closed states even at low membrane potentials. We have isolated and partially purified this modulating protein and the activity is associated with a 54 kDa protein on SDS-PAGE. Under native reduced conditions the activity eluted around 100 kDa from a gel filtration column. As little as 200 ng/ml of the partially purified protein was sufficient to modulate reconstituted VDAC channels. This protein had a pI of 5.1. A second activity with a pI of 4.8 was far more potent, making VDAC-channel-containing membranes virtually non-conductive in some experiments. The effects of both modulator activities could be completely reversed by the addition of pronase. Simple perfusion of the chamber did not reverse the effect of the modulator on VDAC. By controlling the gating of VDAC channels, the VDAC modulator could play an important role in regulating cellular metabolism.
Liu
M Y
MY
Department of Zoology, University of Maryland, College Park 20742.
Torgrimson
A
A
Colombini
M
M
D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D007473:Ion Channels / Q000302:isolation & purification* / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D011402:Pronase / Q000494:pharmacology; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism
D002413:Cations, Divalent; D007473:Ion Channels; D011506:Proteins; D011402:Pronase