Origin of the Ammonia Found in Protein‐Free Extracts of Rat‐Liver Mitochondria and Rat Hepatocytes

Abstract

Protein-free extracts of isolated rat liver mitochondria contain 5.17 .+-. .+-. 0.19 nmol ammonia/mg protein. The ammonia found in the protein-free extracts does not originate from lysosomes contaminating the mitochondrial preparation. When isolated mitochondria are incubated with ornithine, 14CO2 and a source of ATP, a small amount of citrulline is formed. This amount is stoichiometrically equivalent to the ammonia that disappears from the extramitochondrial space, whereas the amount of ammonia found in the protein-free extracts of the mitochondria remains unchanged. Similar results were obtained when the reductive amination of 2-oxoglutarate was used as an ammonia-consuming reaction. When isolated mitochondria are incubated under conditions such that the glutamate dehydrogenase (EC 1.4.1.3) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) reactions reach equilibrium, the thermodynamically active concentration of ammonia is not equal to the concentration measured in the protein-free extracts. About 80% of the ammonia found in protein-free extracts of rat-liver mitochondria is derived from a component or components with a MW .gtoreq. 50,000. Protein-free extracts of isolated rat-liver cells contain considerable amounts of ammonia. After digitonin fraction, this ammonia is found in the protein-free extract of the particulate fraction. The ammonia found in protein-free extracts of rat-liver tissue is probably derived from a component or components in the mitochondria and is released during deproteinization.