LEUCINE-SPECIFIC BINDING OF PHOTOREACTIVE LEU(7)-MAP TO A HIGH-MOLECULAR-WEIGHT PROTEIN ON THE PLASMA-MEMBRANE OF THE ISOLATED RAT HEPATOCYTE

Leu(8)-MAP (Multiple Antigen Peptide) is an effective inhibitor of macroautophagy and proteolysis in the isolated rat hepatocyte, having an apparent K-m (0.1 mM) equaling leucine. Since it is not transported into the cytosolic compartment, it very likely mediates its effect through a plasma membrane site. In an attempt to identify the site we photoreacted intact cells with a biologically active, iodinatable azide derivative of Leu(7)-MAP. A approximate to 340,000 M(r) protein whose labeling was protected 83% with 20 mM Leu was found in plasma membrane fractions when electrophoresed in 7.5-20% gradient gels under nonreducing conditions; addition of 20 mM dithiothreitol generated smaller m.w. products, possibly subunits, of consistent size. No specific labeling was observed with photoreactive derivatives of Ile(7)-MAP or Val(7)-MAP. (C) 1994 Academic Press, Inc.