Is cytochrome b558 translocated into the plasma membrane from granules during the activation of neutrophils?

Abstract

Two laboratories (Borregaard et al. (1984) J. Biol. Chem. 259, 47; Ohno et al. (1985) J. Biol. Chem. 260, 2409) have reported that a b-type cytochrome (b558) was translocated into plasma membranes from specific granules in activated neutrophils. In an attempt to examine the cytochrome b translocation, porcine neutrophils were activated by treatment with surface-active agents such as myristate (MA) and phorbol myristate acetate (PMA), and then the postnuclear supernatants of both activated and unactivated cells were fractionated by Percoll density gradient centrifugation with a Zonal rotor. In activated neutrophils, high O2- generating activity was found in the plasma membrane fraction, which showed a peak of Na, K-ATPase activity as a marker enzyme. Cytochrome b558 was recovered 74 to 78% in the plasma membrane fraction and 14 to 16% in granules in either activated or unactivated cells. No change in specific content of cytochrome b558 was observed in plasma membranes before and after activation of cells. Furthermore, in both activated and unactivated cells, vitamin B12-binding protein, a specific granule marker, was mainly found in the bottom fractions and scarcely at all in plasma membranes. These results suggest that no translocation of cytochrome b558 occurs during activation of neutrophils.