Rat liver aspartate aminotransferase (AAT) (EC 18.104.22.168) exists in two isozymic forms, cytosolic (c-AAT) and mitochondrial (m-AAT). The previous study (Kamimoto, Y. et al., Hepatology an accompanying paper in this issue) demonstrated that these isozymes were cleared from blood at different half-lives via adsorptive endocytosis by sinusoidal liver cells. To understand the cellular mechanism for the differential uptake of the isozymes, we have further studied in vivo uptake of 125I-labeled AAT isozymes by sinusoidal cells. The results indicated that the uptake of the isozymes occurred via a typical endocytotic pathway: the initial binding, internalization and subsequent degradation in the lysosomes. Quantitation of the isozymes bound to the cell surface prior to internalization either by binding at 4°C or by a combined use of anti-AAT antibody and 125I-protein A at 37°C revealed that the differential plasma clearance of AAT isozymes could be attributable to the isozymic difference in capacity of surface membranes to bind the isozymes. The uptake of 125I-c-AAT was inhibited by unlabeled c-AAT more significantly than by m-AAT, but not by other ligands known to be taken up by sinusoidal cells via receptor-mediated pathways. Similarly, the uptake of 125I-m-AAT was inhibited predominantly by unlabeled m-AAT. Similar ligand specificity was also demonstrated in the binding study at 4°C. The binding of 125I-m-AAT and c-AAT followed saturation kinetics with an apparent Kd of 1.3 × 10−6M and 1.7 × 10−6M, respectively.
These findings indicate that differential uptake of AAT isozymes results from the presence on the surface membrane of two binding sites with a definite specificity for each isozyme.