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), Vol. 3
• Country of Publication: United States
• Language: English

Related Subjects

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