Hepatic Clearance of Rat Liver Aspartate Aminotransferase Isozymes: Evidence for Endocytotic Uptake Via Different Binding Sites on Sinusoidal Liver Cells

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Abstract
Rat liver aspartate aminotransferase (AAT) (EC 2.6.1.1) 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 × 10-6 M and 1.7 × 10-6 M, 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.

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