Studies on the pH gradient and histamine uptake of isolated mast cell granules

Journal Article · Thu May 01 00:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:7025088

De Young, M B; Nemeth, E F; Scarpa, A

A purified preparation of mast cell granules with intact perigranular membranes was obtained using a method involving probe sonication of rat serosal mast cells followed by differential centrifugation and Percoll gradient separation of the granules. Purification was assessed with histamine and mast cell granule protease assays. Granule integrity was demonstrated by light and electron microscopy and quantitated with a ruthenium red binding assay. The low yield of granules (20 ..mu..g protein/4 rats) necessitated the development of two microanalytical techniques to demonstrate the existence of a pH gradient across the membrane: 9-aminoacridine fluorescence studies in a cuvet with 50 ..mu..l capacity and /sup 14/C-methylamine distribution studies on microgram quantities of granule protein. Quantitation of results from isotope studies were confounded by the presence of oil used for separating granules from the aqueous phase. Nonetheless, an extrapolation procedure calibrated by external pH yielded an internal pH value of 5.46 +/- .03 (n = 4), consistent with values observed in granules obtained from other secretory cells. Collapse of the pH gradient by NH/sub 4//sup +/ or nigericin/KCl was demonstrated using either technique. Addition of histamine depressed intragranular pH, suggesting that histamine transport may utilize the ..delta..pH as a driving force.

• Research Organization: Univ. of Pennsylvania, Philadelphia
• OSTI ID: 7025088
• Report Number(s): CONF-8606151-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
HISTAMINE
UPTAKE
MAST CELLS
PH VALUE
PURIFICATION
CARBON 14 COMPOUNDS
CELL MEMBRANES
FLUORESCENCE
TRACER TECHNIQUES
AMINES
ANIMAL CELLS
AZOLES
CELL CONSTITUENTS
CONNECTIVE TISSUE CELLS
HETEROCYCLIC COMPOUNDS
IMIDAZOLES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LUMINESCENCE
MEMBRANES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
SOMATIC CELLS
550201* - Biochemistry- Tracer Techniques