Characterization of arachidonate 5-lipoxygenase and leukotriene A4 synthetase from RBL-1 cells

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

OSTI ID:7189947

Cook, M; Hogaboom, G K; Sarau, H M; Foley, J J; Crooke, S T

5-lipoxygenase (LO) and leukotriene (LT) A4 synthetase from RBL-1 high speed (105,000 x g for 60 min) supernatants were partially purified by protein-high performance liquid chromatography (HPLC) and characterized in detail. The partially purified preparation contained only 5-LO and LTA4 synthetase and was isolated from 12-LO, peroxidase and LTA4 hydrolase activities. Reaction products were separated by reversed phase HPLC and quantitated by absorption spectrophotometry and radiochemical detection. The enzyme preparation rapidly converted (/sup 14/C)arachidonate to (/sup 14/C)5-hydroperoxyeicosatetraenoic acid (HPETE) and (/sup 14/C)5,12-dihydroperoxyeicosatetraenoic acids (diHETEs). The 5,12-diHETEs were primarily non-enzymatic breakdown products of LTA4 (e.g., 6-trans-LTB4 and 6-trans-12-epi-LTB4). Both the 5-LO and LTA4 synthetase activities were Ca/sup 2 + -/ and ATP-dependent. For both enzyme activities, the CA/sup 2 +/ stimulation required the presence of ATP. The fatty acid hydroperoxides, 5-,12-, and 15-HPETE, both stimulated ((< 1 ..mu..M)) and inhibited ((> 3 ..mu..M)) 5-LO and LTA4 synthetase activities. The rapid isolation and subsequent characterization of 5-LO and LTA4 synthetase provide the bases for the further understanding of the role of the LO pathway in biological processes.

• Research Organization: Smith Kline and French Labs., Philadelphia, PA
• OSTI ID: 7189947
• Report Number(s): CONF-8606151-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ABSORPTION SPECTROSCOPY
ALKALINE EARTH METAL COMPOUNDS
ARACHIDONIC ACID
ATP
BIOLOGICAL PATHWAYS
CALCIUM COMPOUNDS
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CATIONS
CHARGED PARTICLES
CHROMATOGRAPHY
ENZYME ACTIVITY
ENZYMES
IONS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIGASES
LIQUID COLUMN CHROMATOGRAPHY
MONOCARBOXYLIC ACIDS
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
OXYGENASES
PURIFICATION
SEPARATION PROCESSES
SPECTROSCOPY
TRACER TECHNIQUES