Abstract
Cibacron Blue F3GA(CB-F3GA), which interacts with nucleotide-requiring enzymes, was used to determine the cellular location of the NADPH-binding site of the O 2 − generating enzyme in polymorphonuclear leukocytes(PMN). A fraction of plasma membranes, in which the O 2 − generating enzyme is located, was prepared from myristate(MA)-activated PMN by Percoll density gradient centrifugation. CB-F3GA was found to be a competitive inhibitor of the membrane-bound NADPH dependent O 2 − generating enzyme; the Ki value of CB-F3GA was about 0.8 μM. The dye did not inhibit O 2 − generation and release from PMN activated with MA or phorbol myristate acetate(PMA). These results suggest that the NADPH binding site of the O 2 − generating enzyme is located on the inner surface of the plasma membrane. The low Ki value of this enzyme for the dye suggests that the NADPH binding site has a dinucleotide fold.
Published Version
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