Biochem. J. (1996) 314
(469–475) (Printed in Great Britain)
Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils
R. Alexander BLACKWOOD*‡, James E. SMOLEN†, Ronald J. HESSLER†, Donna M. HARSH* and Amy TRANSUE*
Department of Pediatrics, Divisions of *Infectious Diseases and †Hematology, University of Michigan Medical Center, 1500 E. Medical Center Drive, Ann Arbor, MI 48109-0244, U.S.A.
Several models have been developed to study neutrophil degranulation. At the
most basic level, phospholipid vesicles have been
used to investigate the lipid interactions occurring during membrane fusion.
The two major forms of assays used to measure
phospholipid vesicle fusion are based either on the dilution of
tagged phospholipids within the membrane of the two fusing
partners or the mixing of the aqueous contents of the vesicles.
Although problems exist with both methods, the latter is considered to be more
accurate and representative of true fusion.
Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a
fluorescent marker, we have taken advantage of the quenching
properties of p-xylenebispyridinium bromide ('DPX') to develop
a simple aqueous-space mixing assay that can be used with any
sealed vesicle. We compared our new assay with more conventional assays using
liposomes composed of phosphatidic acid
(PA) and phosphatidylethanolamine (PE), obtaining comparable
results with respect to Ca2+-dependent fusion. We extended our
studies to measure the fusion of neutrophil plasma-membrane
vesicles as well as azurophil and specific granules with PA/PE
(1:3) liposomes. Both specific granules and plasma-membrane
vesicles fused with PA/PE liposomes at [Ca2+] as low
as 500 mM,
while azurophil granules showed no fusion at [Ca2+] as high as
12 mM. These differences in the ability of Ca2+ to induce fusion
may be related to differences observed in whole cells with respect
to secretion.
Abbreviations used: ANTS, 8-aminonaphthalene-1,3,6-trisulphonic acid; DPX,
p-xylenebispyridinium bromide; PA, phosphatidic acid; PE,
phosphatidylethanolamine; LUV, large unilamellar vesicles; NBD-PE,
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PE; Rh-PE, N-(lissamine rhodamine B
sulphonyl)-PE; DFP, di-isopropyl fluorophosphate.
‡ To whom correspondence should be sent.
Received 26 April 1995/24 October 1995; accepted 9 November 1995
The Biochemical Society, London © 1996
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