Partial purification from macrophages of a phospholipase A/sub 2/ that hydrolyzes arachidonic acid from phosphatidylcholine

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6073627

Leslie, C; Wall, M; Zelarney, P; Voelker, D

The first step in platelet-activating factor production and arachidonic acid release from stimulated inflammatory cells is thought to be the activation of a phospholipase A/sub 2/ (PLA/sub 2/). The mouse macrophage cell line, RAW 264, was found to contain PLA/sub 2/ activity that hydrolyzed /sup 3/H-arachidonic acid from sonicated dispersions of 1-0-alkyl-linked phosphatidylcholine (PC). The PLA/sub 2/ was cytosolic, required calcium, and exhibited a broad pH optimum between 6 and 10. When the hydrolysis of /sup 3/H-arachidonic acid from 1-palmitoyl- and 1-hexadecyl-linked PC (15 ..mu..M) was compared, slightly higher activity against 1-acyl-linked PC was found. However, the activity against 1-palmitoyl-linked PC substrates containing oleic or linoleic acids (sn-2) was considerably lower. To understand the properties of the PLA/sub 2/ in more detail, purification was undertaken. After ammonium sulfate precipitation the PLA/sub 2/ was fractionated over G150, on which it eluted slightly before an albumin marker. The enzyme bound to DEAE cellulose at pH 8.0 and was eluted in 0.3M NaCl. Further purification was achieved by hydrophobic chromatography over phenyl Sepharose followed by ion-exchange chromatography using Q-Sepharose resulting in 200-700-fold increases in specific activity. These results demonstrate the presence of a cytosolic PLA/sub 2/ in macrophages that shows specificity for arachidonic acid at the sn-2 position of PC, has an apparent molecular weight greater than or equal to albumin, is anionic at pH 8.0 and is relatively hydrophobic.

• Research Organization: National Jewish Center, Denver, CO
• OSTI ID: 6073627
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMALS
ARACHIDONIC ACID
BIOSYNTHESIS
BLOOD COAGULATION FACTORS
CARBOXYLESTERASES
CARBOXYLIC ACIDS
CHROMATOGRAPHY
COAGULANTS
CONNECTIVE TISSUE CELLS
DRUGS
ENZYME ACTIVITY
ENZYMES
ESTERASES
ESTERS
FRACTIONATION
HEMATOLOGIC AGENTS
HYDROLASES
ION EXCHANGE CHROMATOGRAPHY
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPASE
LIPIDS
MACROPHAGES
MAMMALS
METABOLISM
MICE
MONOCARBOXYLIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PH VALUE
PHAGOCYTES
PHOSPHOLIPIDS
RODENTS
SEPARATION PROCESSES
SOMATIC CELLS
SYNTHESIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES