Specific identification of tissue kallikrein in exocrine tissues and in cell-free translation products with monoclonal antibodies
Autor: | Lee Chao, J Chao, C M Woodley, H S Margolius |
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Rok vydání: | 1985 |
Předmět: |
Male
medicine.drug_class Pancreatic Extracts Submandibular Gland Tissue kallikrein Biology Monoclonal antibody Biochemistry Endopeptidases medicine Animals Pancreas Molecular Biology Binding Sites Cell-Free System urogenital system Serine Endopeptidases Antibodies Monoclonal Rats Inbred Strains Radioimmunoassay Cell Biology Kallikrein Molecular biology Rats Protein Biosynthesis Tissue Kallikreins biology.protein Electrophoresis Polyacrylamide Gel Kallikreins Antibody Clone (B-cell biology) Research Article circulatory and respiratory physiology |
Zdroj: | Biochemical Journal. 231:721-728 |
ISSN: | 1470-8728 0264-6021 |
DOI: | 10.1042/bj2310721 |
Popis: | A panel of six mouse monoclonal antibodies (IgG1) has been prepared against purified rat urinary kallikrein (EC 3.4.21.35) and characterized. In radioimmunoassay, the antibody titres of ascitic fluid giving 50% binding to 125I-kallikrein range from 1:2 × 10(3) to 1:1 × 10(6). Antibodies from four of the clones show no cross-reactivity with human urinary kallikrein, rat urinary esterase A or tonin. However, antibodies from a fifth clone cross-react with tonin and, from a sixth, with both urinary esterase A and tonin. Three of the kallikrein affinity-purified monoclonal antibodies inhibited, whereas one of the antibodies stimulated, kallikrein activity. Tissue kallikrein from rat submandibular-gland and pancreatic extracts and urine were labelled with [14C]di-isopropyl phosphofluoridate, immunoprecipitated with each of the six monoclonal antibodies and identified to be 38 kDa proteins, similar in size to purified rat urinary kallikrein. Western-blot analysis shows that 125I-labelled kallikrein monoclonal antibodies (V4D11) bind directly to a 38 kDa protein in submandibular-gland and pancreatic extracts and urine. Cell-free translation products of submandibular-gland polyadenylylated[poly(A)+]mRNA were immunoprecipitated with affinity-purified sheep anti-kallikrein antibodies and three monoclonal antibodies (V4D11, V4G6 and V1C3). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these immunoprecipitates revealed that two kallikrein precursors with Mr values of 37 000 and 35 000 are encoded by submandibular-gland mRNA. The third monoclonal antibody, V1C3, which binds to active kallikrein, did not recognize either precursor form. Collectively, the data show that these monoclonal antibodies comprise a set of powerful and specific reagents for studies of tissue kallikreins. |
Databáze: | OpenAIRE |
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