Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells |
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Authors: | Jean F. Emly Wendy A. Ratcliffe Elaine Green Sarah J. Bowden David A. Heath Ann Blight Susan Hughes John G. Ratcliffe |
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Affiliation: | 1. Wolfson Research Laboratories, Department of Clinical Chemistry, Queen Elizabeth Medical Centre, Birmingham, UK;2. Department of Medicine, Queen Elizabeth Medical Centre, Birmingham, UK;3. Skin Culture Laboratory, Birmingham Accident Hospital, Birmingham, UK |
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Abstract: | The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments. |
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Keywords: | Parathyroid hormone-related protein Immunoassay Bioactivity PTHRP parathyroid hormone-related protein PTH parathyroid hormone RIA radioimmunoassay IBMXa isobuthyl-methylxanthine IRMA immunoradiometric assay PBS phosphate buffered saline PMSF phenylmethylsulphonyl fluoride FCS foetal calf serum PNA peanut agglutinin |
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