Identification and Partial Characterization of EctoATPase Expressed by Immortalized B Lymphocytes
| Autor: | Terence L. Kirley, James R. Maleckar, Judith A. Kapp, James W. Thomas, Kenneth A. Brewer, Kenneth E. Dombrowski |
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| Rok vydání: | 1997 |
| Předmět: |
Biophysics
Biology Biochemistry Epitope Substrate Specificity Mice Western blot medicine Extracellular Animals Humans Magnesium Enzyme Inhibitors Molecular Biology B cell Adenosine Triphosphatases chemistry.chemical_classification B-Lymphocytes medicine.diagnostic_test Precipitin Tests Molecular biology Cell Compartmentation Kinetics medicine.anatomical_structure Enzyme Membrane protein chemistry biology.protein Calcium Antibody Nucleoside |
| Zdroj: | Archives of Biochemistry and Biophysics. 340:10-18 |
| ISSN: | 0003-9861 |
| Popis: | EctoATPases are extracellular membrane-bound enzymes that catalyze the hydrolysis of the gamma phosphate from ATP. EctoATPase is expressed by activated and immortalized Epstein-Barr virus-transformed human peripheral blood B lymphocytes and murine B cell hybridomas. By contrast, ectoATPase activity is not expressed on nontransformed human peripheral blood B lymphocytes, murine spleen cells, or murine myeloma cells. The K(m) for ATP for the B cell ectoATPases ranged from 5 to 77 microM; the Vmax ranged from 48 to 129 pmol/ min/10(4) cells. The enzyme required Mg2+ for maximal activity with little dependence on Ca2+. ADP and purine and pyrimidine nucleoside triphosphates were competitive inhibitors of the catalytic reaction. A putative ectoATPase protein has been identified by Western blot analysis of membrane proteins from the immortalized B cells. Under reducing conditions, antiectoATPase antibodies cross-reacted with a 66-kDa protein from murine B cell hybridoma membranes. By contrast a 200-kDa protein from the B cell hybridoma membranes cross-reacted with the antibodies under nonreducing conditions, suggesting a disulfide-linked trimer. The antibodies also cross-reacted with a 66-kDa protein from human B cell membranes under reducing conditions, but did not cross-react with membrane proteins under nonreducing conditions. This suggests that the antibody epitope(s) recognized on the reduced human protein is masked under nonreducing conditions. Thus, this work demonstrates: (1) that ectoATPase may serve as a marker for B cell activation; and (2) mammalian and avian ectoATPases have conserved interspecies immunological epitopes and kinetic properties. |
| Databáze: | OpenAIRE |
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