Several lines of evidence are presented that an entity is present in mitochondria which converts lysine and carbamylphosphate to homocitrulline, distinct from the ornithine transcarbamylase (OTC): lack of inhibition by lysine of OTC with ornithine as substrate at the pH of the mitochondrial matrix, a K(m) for lysine of 6.3 mmol/l in the crude mitochondrial extract, versus a K(m) of 55.3 mmol/l after partial purification of OTC, decrease of the specific activity of OTC with lysine as substrate and increase of the specific activity with ornithine as substrate after heat denaturation, and separation of the activities for ornithine and lysine after isoelectric focusing. This would explain the excretion of homocitrulline in the HHH-syndrome, saccharopinuria, citrullinemia and hyperlysinemia. The fact that homocitrulline excretion is not observed in OTC deficiency suggests that the activity responsible for homocitrulline excretion is closely related to OTC.

Differential digitonin extraction of rat liver mitochondria and of mitochondria of livers of affected and unaffected male sparse fur mice released a lysine transcarbamylase activity from the mitochondria at a digitonin to protein ratio in between that for myokinase and glutamate dehydrogenase, but at a slightly lower ratio than the ornithine transcarbamylase activity. Homocitrulline formation by isolated rat liver mitochondria is independent of the uptake of lysine by mitochondria as evidenced by the insensitivity of homocitrulline formation to changes in the matrix pH, in contrast to citrulline formation from ornithine. Highperformance liquid chromatography separates the lysine transcarbamylase activity from the ornithine transcarbamylase activity. It is concluded that the lysine transcarbamylase activity is localized outside the inner mitochondrial membrane.