Cheung C W, Cohen N S, Raijman L
Department of Biochemistry, University of Southern California School of Medicine, Los Angeles 90033.
J Biol Chem. 1989 Mar 5;264(7):4038-44.
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.
利用α-毒素使分离的大鼠肝细胞对低分子量化合物具有通透性，观察到尿素循环中的酶优先利用内源性生成的中间产物。通透细胞以琥珀酸盐作为呼吸底物，从添加的氯化铵、[14C]碳酸氢盐、鸟氨酸和天冬氨酸合成[14C]尿素；在所有底物饱和的情况下，每min/mg细胞干重形成约4 nmol尿素。尿素通常占总（氨 + 鸟氨酸）依赖性计数的约40 - 50%，精氨酸占不到10%，瓜氨酸占约30%。精氨琥珀酸裂解酶和精氨酸酶之间精氨酸的紧密通道化表现为，向孵育体系中添加200倍过量的未标记精氨酸，并未降低尿素中计数的百分比，也未增加精氨酸中计数的百分比，尽管添加的大量精氨酸在细胞内被水解。通过类似观察证明了精氨琥珀酸在其合成酶和裂解酶之间的通道化；200倍过量添加的未标记精氨琥珀酸仅使尿素中计数的百分比降低了25%。还显示了瓜氨酸从其在线粒体基质中由鸟氨酸转氨甲酰酶合成的位点到细胞质空间中的精氨琥珀酸合成酶的通道化。这些结果强烈表明，尿素循环的三种“可溶性”细胞质酶围绕线粒体聚集，并在细胞内进行空间组织，使得中间产物能够在它们之间有效转移。
