Characterization of (/sup 3/H)leukotriene D4 binding sites in guinea-pig ventricular myocardium

Journal Article · Sat Jun 01 00:00:00 EDT 1985 · J. Pharmacol. Exp. Ther.; (United States)

OSTI ID:5253918

Hogaboom, G K; Mong, S; Stadel, J M; Crooke, S T

(/sup 3/H)Leukotriene (LT) D4 was used to identify specific LTD4 binding sites in guinea-pig ventricular myocardial membranes. High-performance liquid chromatography analyses indicated that, in the presence of the gamma-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 3% of membrane-bound (/sup 3/H)LTD4 was converted to (/sup 3/H)LTC4 or (/sup 3/H)LTE4 at 30 degrees C. The specific (/sup 3/H) LTD4 binding, assayed in the presence of 80 mM L-serine-borate, reached a stable steady state within 45 min at 30 degrees C (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded an apparent dissociation constant (Kd) of 3.4 +/- 2.1 nM and a maximum number of binding sites of 850 +/- 91 fmol/mg of protein. Competition binding studies with (/sup 3/H)LTD4, synthetic 5S, 6R-LTD4 (LTD4) and its diastereoisomer 5R,6S-LTD4, LTE4, LTC4 and the putative LT antagonists FPL 55712, 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid (2-nor-LTD1) and SKF 88046 demonstrated a potency order of LTD4 greater than LTE4 greater than LTC4 greater than 5R,6S-LTD4 much greater than 2-nor-LTD1. FPL 55712 and SKF 88046 were ineffective in displacing the specific (/sup 3/H)LTD4 binding. Pretreatment of the heart membranes with the sulfhydryl reducing reagent dithiothreitol decreased the specific (/sup 3/H)LTD4 binding in a concentration-dependent manner. Scatchard analyses of saturation isotherms indicated that 0.3 mM dithiothreitol pretreatment of heart membranes decreased the maximum number of binding sites of the (/sup 3/H)LTD4 binding to 368 +/- 61 fmol/mg of protein with minimal effects on the apparent Kd.

• Research Organization: Smith Kline and French Labs., Philadelphia, PA
• OSTI ID: 5253918
• Journal Information: J. Pharmacol. Exp. Ther.; (United States), Journal Name: J. Pharmacol. Exp. Ther.; (United States) Vol. 3; ISSN JPETA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
ARACHIDONIC ACID
BODY
CARBOXYLIC ACIDS
CARDIOVASCULAR SYSTEM
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL BONDS
CHEMICAL COMPOSITION
CHROMATOGRAPHY
ENZYME INHIBITORS
ENZYMES
GUINEA PIGS
HEART
HYDROLASES
IN VITRO
ISOTHERMS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
MEMBRANES
METABOLITES
MONOCARBOXYLIC ACIDS
MUSCLES
MYOCARDIUM
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PEPTIDE HYDROLASES
RODENTS
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES