Leucine-specific binding of photoreactive Leu7-MAP to a high molecular weight protein on the plasma membrane of the isolated rat hepatocyte.
Leu8-MAP (Multiple Antigen Peptide) is an effective inhibitor of macroautophagy and proteolysis in the isolated rat hepatocyte, having an apparent Km (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 Leu7-MAP. A approximately 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 Ile7-MAP or Val7-MAP.
Mortimore
G E
GE
Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey 17033.
Wert
J J
JJ
Miotto
G
G
Venerando
R
R
Kadowaki
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D007700:Kinetics; D007930:Leucine; D008099:Liver / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew
D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D010455:Peptides; C089108:multiple antigen peptide, Leu(7)-; D007930:Leucine