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Solubilization and cleavage of human neutrophil (N) affinity-labeled receptors for leukotriene B/sub 4/ (LTB/sub 4/)

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:6966049
; ; ; ;
LTB/sub 4/ chemotactic receptors in purified N plasma membranes (PMs) have been affinity-labeled with (/sup 3/H)-C-1 aminopropylamide-LTB/sub 4/ ((/sup 3/H)APA-LTB/sub 4/) by disuccinimidyl suberate (DSS) cross-linking. Intact Ns were pretreated with diisopropylfluorophosphate, suspended at 10/sup 7//ml in Hanks' solution-10 mM HEPES (pH 7.4), incubated for 30 min at 4/sup 0/C with 30 nM (/sup 3/H)APA-LTB/sub 4/ and 25 min with 1 mM bis(2-(succinimidooxycarbonyloxy)-ethyl) sulfone, an impermeant analog of DSS, and sonified for 30 sec at 4/sup 0/C. The 10,000 g supernatant of the sonicate was centrifuged at 40,000 g for 30 min at 4/sup 0/C on a discontinuous gradient of 10-50 g % sucrose, from which a mean of 78% of the radiolabel was recovered with PM markers. The extent and specificity of labeling of intact N receptors were similar to those of receptors in PMs. The radioactively-labeled receptors appeared as a single band of 35-40 kd in sodium dodecyl sulfate (SDS) 10 g % polyacrylamide gel electrophoresis. Cleavage of radiolabeled receptors with 1 mg/ml of cyanogen bromide in 70% formic acid for 18 hr at room temperature or with 30 mM HCl under N/sub 2/ for 4 hr at 105/sup 0/C converted a mean of 18-32% of the radioactivity to a band of 14 kd in SDS-15 g % PAGE. N receptors for LTB/sub 4/, thus, are localized in the PM and can be isolated for structural studies.
Research Organization:
Howard Hughes Medical Institute, San Francisco, CA
OSTI ID:
6966049
Report Number(s):
CONF-8604222-
Conference Information:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:4
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ARACHIDONIC ACID
BIOLOGICAL LOCALIZATION
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CLEAVAGE
CROSS-LINKING
CRYSTAL STRUCTURE
ELECTROPHORESIS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LABELLING
LEUKOCYTES
MATERIALS
MEMBRANE PROTEINS
MEMBRANES
MICROSTRUCTURE
MONOCARBOXYLIC ACIDS
NEUTROPHILS
ORGANIC ACIDS
ORGANIC COMPOUNDS
POLYMERIZATION
PROTEINS
RECEPTORS
TRACER TECHNIQUES
TRITIUM COMPOUNDS