Isolation of a gastrin releasing peptide receptor from normal rat pancreas.
The presence of a putative GRP receptor on rat pancreatic particulate membranes was demonstrated by covalent cross-linking to 125I-gastrin releasing peptide (GRP), which revealed a radioactive band with Mr = 80-90 kDa on reduced SDS-PAGE. Fresh rat pancreatic membranes contained a GRP receptor which was solubilized with Triton X-100 as assessed by its failure to sediment at 100,000 x g for one hour and its ability to pass through a 0.22 mu filter. When 125I-GRP binding was studied using Sephadex G50 gel filtration chromatography to separate bound from unbound ligand, substantial amounts of 125I-GRP binding were observed in rat crude solubilized pancreatic membranes, but essentially no specific binding was observed until the crude solubilized membranes were fractionated by ammonium sulfate precipitation. Specific 125I-GRP binding was 500, 700 and 1400 fmol/mg protein, respectively, in the 0-25%, 25-50% and 50-80% saturated ammonium sulfate fractions (125I-GRP concentration = 1 nM). Specific binding was temperature dependent, saturable and of high affinity, (KD = 2.3 nM). A unique 70 kDa band was visualized by silver staining of the SDS-PAGE of eluates of GRP(14-27) affinity gel compared with eluates of control affinity gels incubated with the 25-50% (NH4)2SO4 fraction. The lower Mr than that observed with covalent cross-linking may represent the binding subunit of a larger receptor protein. This ligand-affinity isolated protein is thus a good candidate for the GRP receptor, or the binding subunit of it, from normal rat pancreas.
Kane
M A
MA
Medical Oncology Section, Denver Veterans Affairs Medical Center, CO 80220.
Kelley
K
K
Ross
S E
SE
Portanova
L B
LB
D000645:Ammonium Sulfate; D000818:Animals; D001839:Bombesin / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D051381:Rats; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility
D003432:Cross-Linking Reagents; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D001839:Bombesin; D000645:Ammonium Sulfate