Characteristics of calmodulin binding to purified human lymphocyte plasma membranes.
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.
D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D002746:Chlorpromazine / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D008565:Membrane Proteins; D013449:Sulfonamides; D014268:Trifluoperazine; C017967:W 7; D002118:Calcium; D002746:Chlorpromazine