Localization of the Glycerol‐Phosphate Dehydrogenase in the Outer Phase of the Mitochondrial Inner Membrane

Evidence for the localization of the mitochondrial glycerol‐phosphate dehydrogenase in the outer phase of the inner membrane is presented. Glycerol‐phosphate does not permeate through the inner membrane to the matrix space in a number of mitochondria which actively oxidize glycerol‐phosphate. The impermeability of the inner membrane to ferricyanide is demonstrated and this forms a basis for elucidating membrane localization of carriers. In mitochondria from various tissues ferricyanide can interact directly with glycerolphosphate dehydrogenase but not with succinate dehydrogenase. The oxidation of glycerol‐phosphate, in contrast to that of succinate, is insensitive to substrate depletion brought about by uncouplers. The results are interpreted to show that both the substrate and the acceptor‐site of glycerol‐phosphate dehydrogenase are exposed to the outer side of the inner membrane. Thus, for the operation of the glycerol‐phosphate dehydrogenase, mitochondria do not require translocases for glycerol‐phosphate and dihydroxyacetone‐phosphate.