Identification of a single binding protein for endothelin-1 and endothelin-3 in bovine cerebellum membranes.
Competition binding experiments of [125I]endothelin-3 ([125I]ET-3) to bovine cerebellum membrane preparations in the presence of either ET-3 or ET-1 have indicated the presence of a single class of high affinity binding sites for these two peptides in the brain. Cross-linking of [125I]ET-3 to cerebellum membrane preparations with dissuccinimidyl suberate (DSS) resulted in the labeling of two bands with apparent mol wt of 52 and 30 kDa. Under these conditions the labeling intensities of these two bands were similar. However, addition of 5 mM EDTA to the protease inhibitor mixture during membrane preparations as well as the binding and cross-linking reaction increased the labeling of the 52-kDa protein while reducing the labeling of the 30-kDa protein. Peptide map comparisons of the 52- and 30-kDa protein bands using Staphylococcus aureus V8 protease and papain revealed that the 30-kDa band is a proteolytic degradation product of the 52-kDa protein. These results suggest that the 52-kDa protein represents the specific binding protein of ET-3, and thus, the apparent mol wt of the ET receptor is 50 kDa, subtracting the mol wt of the iodinated ET. Since cross-linking of [125I]ET-1 to cerebellum membrane preparations revealed the same two bands of 52 and 30 kDa, peptide mapping of the 52-kDa proteins, cross-linked with either [125I]ET-1 or ET-3, was conducted. Under these experimental conditions, identical peptide fragments were generated by both Staphylococcus aureus V8 protease and papain. These results suggest that ET-1 and ET-3 bind to a common brain binding protein with an apparent mol wt of 50 kDa.
Schvartz
I
I
Division of Endocrinology, Glaxo Research Laboratories, Research Triangle Park, North Carolina 27709.
Ittoop
O
O
Hazum
E
E
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016232:Endothelins / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010449:Peptide Mapping; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017466:Receptors, Endothelin
D016232:Endothelins; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin