INTRACELLULAR-DISTRIBUTION OF HEXOKINASE IN RABBIT BRAIN

Hexokinase in mammalian brain is particulate and usually considered to be bound to the outer mitochondrial membrane. Investigation of rabbit brain mitochondria prepared either by differential centrifugation and discontinuous density gradient centrifugation has provided evidence t hat this particulate fraction also contains endoplasmic vesicles and s ynaptosomes. Solubilization of the bound hexokinase by different combi nations of detergents and metabolites has proved the existence of diff erent hexokinase binding sites. Electron microscopic examination of he xokinase location by immuno-gold labelling techniques confirmed that h exokinase is indeed predominantly bound to mitochondria but that a sig nificant proportion is also bound to non-mitochondrial membranes. Atte mpts to quantify this distribution were unsuccessful since different f igures were obtained using anti-hexokinase IgG affinity purified on im mobilized native or denatured hexokinase. Binding studies of the purif ied rabbit brain mitochondrial hexokinase to rabbit liver mitochondria and microsomes confirmed that in addition to a binding site on mitoch ondria there is another binding site on microsomes. The N-terminal seq uence of hexokinase has been shown to be important for mitochondria bi nding and also for microsome binding. These results suggest that the i ntracellular localization of hexokinase in rabbit brain is not exclusi vely mitochondrial and that the metabolic role of this enzyme should b e reconsidered by including a binding site on the endoplasmic reticulu m.