Characterization of soluble and particulate parathyroid hormone receptors using a biotinylated bioactive hormone analog.
A bioactive biotin-containing derivative of the synthetic bovine parathyroid hormone analog [Nle8,Nle18,Tyr34]bovine parathyroid hormone-(1-34) (bPTH-(1-34] amide was prepared by reacting the peptide with N-biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide ester. The derivative was incubated with particulate renal plasma membranes or with detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) extracts of renal cortical membranes, and two membrane components were identified. Labeling of these components was competitively inhibited by underivatized bPTH-(1-34) or bPTH-(3-34) but not by insulin, adrenocorticotropin, or oxidized rat PTH-(1-34). PTH-binding components that were immobilized on nitrocellulose could be detected by incubating the membrane with biotinyl-bPTH-(1-34). Binding components of apparent molecular mass 68, 70, and 150 kDa were specifically labeled in plasma membranes derived from canine, human, and porcine renal cortex, rat liver, and human fibroblasts. The 68-kDa binding protein was found to be consistently more acidic than the 70-kDa binding protein in human, porcine, and canine renal membranes analyzed by two-dimensional electrophoresis. The 68-70-kDa receptor doublet could be specifically isolated by streptavidin-agarose chromatography of solubilized membrane extracts that had first been incubated with biotinyl-BPTH-(1-34). Biotinyl-bPTH-(1-34) should be useful as a tool for further characterization and purification of the PTH receptor.
Brennan
D P
DP
Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
Levine
M A
MA
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D002793:Cholic Acids / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007668:Kidney / Q000032:analysis; D010281:Parathyroid Hormone / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000032:analysis / Q000302:isolation & purification*; D018016:Receptors, Parathyroid Hormone; D012995:Solubility; D013552:Swine
D002793:Cholic Acids; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; C047977:biotinyl-parathyroid hormone; D001710:Biotin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate