Determination of flavin contents in neutrophils by a sensitive chemiluminescence assay: evidence for no translocation of flavoproteins from the cytosol to the membrane upon cell stimulation.
A sensitive and specific chemiluminescence (CL) method with bacterial luciferase was adapted for accurate measurement of the flavins FAD and FMN in the membrane and cytosolic fractions of neutrophils prepared from pig and human blood. The FAD and FMN contents (FAD/FMN = 100:2) in the membranes were essentially the same in resting (R) and myristate-stimulated (S) cells, although O2(-)-generation was markedly enhanced exclusively in S membranes. The O2(-)-forming activity of S samples remained unchanged or even increased after washing the membranes with buffer, although one-third of the FAD was lost during washing (a decrease from 140 to 95 pmol/10(8) cell-equivalent (CE) during washing). The cytosol is known to contain at least three components that are essential for O2- production (p47-phox, p67-phox, and a G-protein), and that are translocated to membranes upon activation, but its flavin content was one tenth of that of the membranes. The cytosol was treated with fatty acids in the absence of membranes to induce substantial precipitation of p47-phox, p67-phox and a protein of 32 kDa. No difference relative to a solvent-control was noted in the low flavin content of the precipitate indicating that these cytosolic components are not flavoproteins. These results do not support the possibility of translocation of a cytosolic flavoprotein to the membrane upon activation of the respiratory burst.
Yoshida
L S
LS
Department of Inflammation Research, Tokyo Metropolitan Institute of Medical Science, Japan.
Chiba
T
T
Kakinuma
K
K
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005227:Fatty Acids / Q000494:pharmacology; D005486:Flavin Mononucleotide / Q000097:blood*; D005182:Flavin-Adenine Dinucleotide / Q000097:blood*; D005420:Flavoproteins / Q000097:blood*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D019814:Myristic Acid; D009227:Myristic Acids / Q000494:pharmacology; D009504:Neutrophils / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D013481:Superoxides / Q000097:blood; D013552:Swine
D005227:Fatty Acids; D005420:Flavoproteins; D009227:Myristic Acids; D019814:Myristic Acid; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D008156:Luciferases