Hepoxilin A3-specific binding in human neutrophils.
Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 degrees C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 degrees C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3 +/- 9.1 nM and 8.86 +/- 1.4 pmol/ml per 2 x 10(6) cells (+/- S.E.M.) respectively reflecting approx. 2.67 x 10(6) sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.
Reynaud
D
D
Research Institute, Hospital for Sick Children, Toronto, Canada.
Demin
P
P
Pace-Asciak
C R
CR
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D001665:Binding Sites; D019286:Endopeptidase K; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009504:Neutrophils / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D014316:Tritium
D014316:Tritium; C038243:8-hydroxy-11,12-epoxyeicosa-5,9,14-trienoic acid; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D015126:8,11,14-Eicosatrienoic Acid