Solubilization of the receptors for avian pancreatic polypeptide in chicken, canine, and pig brains.
When n-octyl-beta-D-glucoside was used in several detergents to extract active avian pancreatic polypeptide (APP) receptors, a specific binding of [125I]APP to the solubilized chicken cerebellar and porcine hippocampal membranes was found. The binding of [125I]APP to the solubilized receptors was dependent on incubation time, temperature, and protein concentrations and appeared to have a slightly acidic optimal pH. APP binding to chicken and porcine brain extracts showed a high specificity for APP, although the chicken receptors do not discriminate well between APP and its related peptides, neuropeptide Y and peptide YY. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the brain extracts as in membrane-bound receptors; however, the high affinity component of the chicken receptor showed a decreased affinity after extraction. APP receptors in chicken and porcine brain extracts retained their insensitivity to the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate). Cross-linking studies were performed with the homobifunctional cross-linker disuccinimidyl suberate and brain membrane receptors solubilized with n-octyl-beta-D-glucoside. An APP receptor species with a M(r) of 67,000, the same size as that of the labeled protein in native membrane homogenates of chicken and pig brains, was identified. However, in the canine brain we observed a M(r) 85,000 receptor protein, suggesting that species differences exist among the structures of brain APP receptors. The solubilized cross-linked APP receptors in these species were adsorbed by wheat germ agglutinin-agarose and by concanavalin A, indicating that they are glycoprotein in nature. The availability of the solubilized receptors from vertebrate brains with n-octyl-beta-D-glucoside represents an important step toward the purification and molecular characterization of the APP receptors.
Inui
A
A
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Miura
M
M
Teranishi
A
A
Hirosue
Y
Y
Nakajima
M
M
Okita
M
M
Nishimura
R
R
Baba
S
S
Kasuga
M
M
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D003902:Detergents; D004285:Dogs; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006624:Hippocampus / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D037102:Lectins; D008274:Magnesium / Q000494:pharmacology; D010191:Pancreatic Polypeptide / Q000378:metabolism*; D011964:Receptors, Gastrointestinal Hormone / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013045:Species Specificity; D013552:Swine
D003902:Detergents; D007457:Iodine Radioisotopes; D037102:Lectins; D011964:Receptors, Gastrointestinal Hormone; C029438:receptors, pancreatic polypeptide-specific; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C009725:pancreatic polypeptide, avian; D010191:Pancreatic Polypeptide; D008274:Magnesium