Covalent labeling of a high-affinity, guanyl nucleotide sensitive parathyroid hormone receptor in canine renal cortex

Journal Article · Tue Apr 07 04:00:00 EDT 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00381a013· OSTI ID:5962750

Nissenson, R A; Karpf, D; Bambino, T; Winer, J; Canga, M; Nyiredy, K; Arnaud, C D

Putative parathyroid hormone (PTH) receptors in canine renal membranes were affinity labeled with /sup 125/I-bPTH(1-34) using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 4-azido-benzoate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a major 85,000 molecular weight (M/sub r/) PTH binding component, the labeling of which was inhibited by nanomolar concentrations of unlabeled PTH and by micromolar concentrations of 5'-guanylyl imidodiphosphate (Gpp-(NH)p). Labeling was not influenced by the unrelated peptides insulin and arginine vasopressin. Minor PTH binding components of M/sub r/ 55,000 and 130,000 were also seen, and labeling of these was likewise sensitive to unlabeled PTH and to Gpp(NH)p. Omission of protease inhibitors during the isolation of plasma membranes resulted in the loss of the M/sub r/ 85,000 PTH binding species and the appearance of an M/sub r/ 70,000 form. Several minor PTH binding components also were observed. Equilibrium binding studies showed that such membranes had an affinity for PTH indistinguishable from that in membranes isolated with protease inhibitors and displaying a major M/sub r/ 85,000 PTH binding species. The authors conclude that the major form of the adenylate cyclase coupled PTH receptor in canine renal membranes is an M/sub r/ 85,000 protein. An endogenous enzyme, probably a lysosomal cathepsin, can cleave this form to produce an M/sub r/ 70,000 receptor that retains full functional activity with respect to high-affinity, guanyl nucleotide sensitive PTH binding. The ability to covalently label the PTH receptor in high yield represents a major step toward the structural characterization of this important detector molecule.

• Research Organization: Univ. of California, San Francisco
• OSTI ID: 5962750
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:7; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ANIMALS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BODY
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CROSS-LINKING
CYCLASES
DAYS LIVING RADIOISOTOPES
DOGS
DOSE-RESPONSE RELATIONSHIPS
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
HORMONES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KIDNEYS
LYASES
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
PARATHORMONE
PEPTIDE HORMONES
POLYMERIZATION
PROTEINS
RADIOISOTOPES
RECEPTORS
VERTEBRATES