Subcellular distribution of imidazoline-guanidinium-receptive sites in human and rabbit liver. Major localization to the mitochondrial outer membrane

Abstract

Imidazoline-guanidinium-receptive site (IGRS) is a membrane protein that, even if recognized by a series of imidazoline and guanidinium alpha 2-adrenergic compounds, is insensitive to catecholamine and physically distinct from alpha 2 receptors (Parini, A., Coupry, I., Graham, R. M., Uzielli, I., Atlas, D., and Lanier, S. M. (1989) J. Biol. Chem. 264, 11874-11878). In the present report, we defined the subcellular localization of IGRS by performing binding studies with the imidazoline radioligand [3H]idazoxan. Binding studies on subcellular fractions of homogenates from human and rabbit liver showed a significant increase in [3H]idazoxan binding in a membrane fraction enriched in cytochrome oxidase activity, a specific marker for mitochondria. The enrichment in [3H]idazoxan binding sites correlates closely with cytochrome oxidase activity in the nuclear, mitochondrial, plasma membrane, microsomal, and soluble fractions (r = 0.966, p less than 0.002) but not with the specific markers for other cell compartments, suggesting a major localization of IGRS in mitochondria. Separation of inner and outer mitochondrial membranes by digitonin treatment showed that [3H]idazoxan binding correlates positively with monoamine oxidase (r = 0.960) and negatively with cytochrome oxidase (r = -0.950) activities. In addition, in highly purified preparations of outer mitochondrial membranes obtained by hypotonic shock, [3H]idazoxan binding activity was 12.5-fold enriched with respect to intact mitochondria. Taken together, these data show, for the first time, that IGRS in human and rabbit liver are mainly associated with the outer mitochondrial membranes. This demonstration of the major mitochondrial localization of IGRS will facilitate the characterization of its functional activity in liver.