Examination of parathyroid hormone antisera for the presence of receptor antibodies.
Proteins from bovine kidney membranes were separated by denaturating polyacrylamide gel electrophoresis and blotted onto nitrocellulose paper. The blots were immunostained with parathyroid hormone (PTH) antisera, and the effect of the presence of PTH on immunostaining was determined. Immunostaining of membrane proteins by two specific antisera was altered by PTH. With one antiserum, the immunostaining of two specific proteins (apparent mass 90 and 105 kDa) was prevented by PTH. With the second antiserum the immunostaining of a 150 kDa protein was prevented by the hormone. These effects were strongest with the 90 and 150 kDa proteins and these were investigated further. Antibody binding was prevented either by co-incubation or by preincubation of the blots with PTH, followed by washing and subsequent exposure to the antisera. Concentrations of PTH as low as 1 nM prevented antibody binding to the 90 kDa species, but somewhat higher PTH concentrations were required with the 150 kDa protein. Oxidation of the PTH methionine residues in the amino terminal segment of PTH, and deletion of the first nine residues in the hormone greatly reduced the competition with the 90 kDa protein, but had no effect on immunostaining of the 150 kDa species. The 35-84 fragment of PTH was not a competitor for the 90 kDa species, while the 1-34 fragment was ineffective with the 150 kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Zull
J E
JE
Department of Biology, Case Western Reserve University, Cleveland, OH 44106.
Chuang
J
J
Smith
S K
SK
D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010281:Parathyroid Hormone / Q000276:immunology* / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone
D007106:Immune Sera; D008565:Membrane Proteins; D010281:Parathyroid Hormone; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone