TY - JOUR
T1 - Identification of an endosome-specific antigen
AU - Pitt, Alan
AU - Schwartz, Alan L.
N1 - Funding Information:
These studies were supported in part by NIH. We would like to thank Rob Fallon and Cliff Harding for critically reading the manuscript and Willem Stoorvogel for assisting in the DAB quench experiments.
PY - 1991/5
Y1 - 1991/5
N2 - We have used successive density gradient centrifugation with vesicles prepared from a human hepatoma Hep G2 post nuclear supernatant to obtain a highly enriched preparation of early endosomes. A monoclonal antibody (8E4) raised against this early endosome preparation recognizes a single polypeptide highly enriched in light vesicle membranes. The antigen has a molecular weight of 195 kDa by SDS-PAGE in the presence or absence of a reducing agent. Western blot analysis shows that the 8E4 antigen is detectable only in light vesicle membranes and not among heavy membranes, whole cytosol, or nuclear pellet proteins. The 8E4 antigen appears to be an integral membrane protein as it is precipitated by Triton X-114. The distribution of the 8E4 antigen in a Nycodenz density gradient fractionation of light vesicle membranes is identical to the distribution of 125I-ASOR-labeled early endosomes but distinct from the distribution of the plasma membrane enzyme, alkaline phosphodiesterase. In addition, incubation of cells with a horseradish peroxidase-transferrin conjugate followed by 3, 3′-diaminobenzidine cytochemistry specifically quenches 8E4 antigen detection by protein dot blot analysis. These data strongly suggest that the 8E4 antigen is an integral membrane protein primarily located in endocytic vesicles.
AB - We have used successive density gradient centrifugation with vesicles prepared from a human hepatoma Hep G2 post nuclear supernatant to obtain a highly enriched preparation of early endosomes. A monoclonal antibody (8E4) raised against this early endosome preparation recognizes a single polypeptide highly enriched in light vesicle membranes. The antigen has a molecular weight of 195 kDa by SDS-PAGE in the presence or absence of a reducing agent. Western blot analysis shows that the 8E4 antigen is detectable only in light vesicle membranes and not among heavy membranes, whole cytosol, or nuclear pellet proteins. The 8E4 antigen appears to be an integral membrane protein as it is precipitated by Triton X-114. The distribution of the 8E4 antigen in a Nycodenz density gradient fractionation of light vesicle membranes is identical to the distribution of 125I-ASOR-labeled early endosomes but distinct from the distribution of the plasma membrane enzyme, alkaline phosphodiesterase. In addition, incubation of cells with a horseradish peroxidase-transferrin conjugate followed by 3, 3′-diaminobenzidine cytochemistry specifically quenches 8E4 antigen detection by protein dot blot analysis. These data strongly suggest that the 8E4 antigen is an integral membrane protein primarily located in endocytic vesicles.
UR - http://www.scopus.com/inward/record.url?scp=0026166443&partnerID=8YFLogxK
U2 - 10.1016/0014-4827(91)90141-G
DO - 10.1016/0014-4827(91)90141-G
M3 - Article
C2 - 1849826
AN - SCOPUS:0026166443
VL - 194
SP - 128
EP - 134
JO - Experimental Cell Research
JF - Experimental Cell Research
SN - 0014-4827
IS - 1
ER -