Channeling of urea cycle intermediates in situ in permeabilized hepatocytes

Preferential use of endogenously generated intermediates by the enzymes of the urea cycle was observed using isolated rat hepatocytes made permeable to low molecular weight compounds with alpha-toxin. The permeabilized cells synthesized 14C]urea from added NH4Cl, 14C]HCO3-, ornithine, and aspartate, using succinate as a respiratory substrate; with all substrates saturating, about 4 nmol of urea were formed per min/mg dry weight of cells. Urea usually accounted for about 40-50% of the total (NH3 + ornithine)-dependent counts, arginine for less than 10%, and citrulline for about 30%. Very tight channeling of arginine between argininosuccinate lyase and arginase was shown by the fact that the addition of a 200-fold excess of unlabeled arginine to the incubations did not decrease the percentage of counts found in urea or increase that found in arginine, even though a substantial amount of the added arginine was hydrolyzed inside the cells. The channeling of argininosuccinate between its synthetase and lyase was demonstrated by similar observations; unlabeled argininosuccinate added in 200-fold excess decreased the percentage of counts in urea by only 25%. Channeling of citrulline from its site of synthesis by ornithine transcarbamylase in the mitochondrial matrix to argininosuccinate synthetase in the cytoplasmic space was also shown. These results strongly suggest that the three "soluble" cytoplasmic enzymes of the urea cycle are grouped around the mitochondria and are spatially organized within the cell in such a way that intermediates can be efficiently transferred between them.