Characteristics of calmodulin binding to purified human lymphocyte plasma membranes
| Autor: | T P Lee, J Venuti, I Macara, R Kawauchi, P J Davis, B K Mookerjee |
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| Rok vydání: | 1987 |
| Předmět: | |
| Zdroj: | The Journal of Immunology. 139:42-48 |
| ISSN: | 1550-6606 0022-1767 |
| DOI: | 10.4049/jimmunol.139.1.42 |
| Popis: | We have explored the role of calmodulin in plasma membrane-related phenomena in lymphocyte activation by measurement of [125I]calmodulin binding to highly purified plasma membrane of human peripheral blood lymphocytes. Calcium-dependent calmodulin binding to lymphocyte membrane was found to reach equilibrium within 5 min of incubation at 37 degrees C and to be saturable and specific. A single class of high affinity-binding sites was identified, with a dissociation constant (Kd) of 1 to 3 X 10(-8) M and a total binding capacity (Bt) of 1 to 2 pmol/mg membrane protein. The free calcium concentration necessary for half-maximal binding was 100 to 300 nM. This was strikingly similar to the cytoplasmic-free calcium activity [Ca2+]i measured by the Quin-2 fluorescence technique, particularly after stimulation with phytomitogens. Calmodulin binding was inhibitable by trifluoperazine (TFP), W-7, and chloropramazine, all of which are calmodulin antagonists. The concentration of TFP that caused 50% inhibition of lymphocyte proliferative responses to phytomitogens was found to be identical to the concentration of TFP which causes 50% inhibition of calmodulin binding to lymphocyte plasma membrane. SDS-polyacrylamide gel electrophoresis followed by gel overlay and autoradiography with iodinated calmodulin revealed five calcium-dependent, TFP-inhibitable, calmodulin-binding polypeptides. |
| Databáze: | OpenAIRE |
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