Identification and characterization of phospholipase A/sub 2/ (PLA/sub 2/) in bovine pulmonary endothelial cells (BPEC)
Phosphatidylcholine labeled in the sn-2 position with /sup 3/H-oleic acid was used to measure PLA/sub 2/ in cell sonicates (CS) prepared from confluent cultures of BPEC. Substrate at 10-200 ..mu..M was incubated with 5-30 ..mu..g of CS protein in HEPES buffer at 37/sup 0/C. A plot of /sup 3/H-oleic acid release vs time was linear and proportional to the amount of CS protein. Lineweaver-Burk plots of the data were linear with V/sub max/ = 22.2 nmole/mg protein/hr and K/sub d/ = 121 ..mu..M. Under these conditions, phospholipase C activity was 20-fold lower, and phospholipase A/sub 1/ activity was not detectable. PLA/sub 2/ activity was pH-dependent with optima at 4.5 and 7.5. Ca/sup + +/ was not required for activity, and addition of up to 10 mM Ca/sup + +/ to CS in EDTA increased activity by only 10-20%. After centrifugation of CS at 100,000 g for 90 min, 62% of the PLA/sub 2/ activity was recovered in the particular fraction. Triton X-100 (0.006-0.4%) inhibited PLA/sub 2/ up to 90%, whereas 2 mM deoxycholate produced nearly 3-fold activation. Of several agents tested, bromophenacylbromide (BPB) was the most effective inhibitor. Treatment of CS with BPB at 37/sup 0/C for 30 min produced up to 9% inhibition (K/sub i/ = 5 ..mu..M). Phenylmethanesulfonyl fluoride at 200 ..mu..m produced 41% inhibition. Quinacrine at 1 mM inhibited PLA/sub 2/ by 18%. These data define characteristics of BPEC PLA/sub 2/ that should prove useful in studies of the role of this enzyme in specific cellular functions.
- Research Organization:
- St. Louis Univ. School of Medicine, MO
- OSTI ID:
- 5450860
- Report Number(s):
- CONF-8604222-; TRN: 86-028373
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:3; Conference: 70. annual meeting of the Federation of American Society for Experimental Biology, St. Louis, MO, USA, 13 Apr 1986
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
LIPASE
ENZYME ACTIVITY
PURIFICATION
CALCIUM
CATTLE
CELL CULTURES
CENTRIFUGATION
CHOLINE
ENDOTHELIUM
ENZYME INHIBITORS
LUNGS
OLEIC ACID
PH VALUE
PROTEINS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ALCOHOLS
ALKALINE EARTH METALS
AMINES
AMMONIUM COMPOUNDS
ANIMAL TISSUES
ANIMALS
BODY
CARBOXYLESTERASES
CARBOXYLIC ACIDS
DOMESTIC ANIMALS
DRUGS
ELEMENTS
ENZYMES
ESTERASES
HYDROLASES
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPOTROPIC FACTORS
MAMMALS
METALS
MONOCARBOXYLIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
QUATERNARY COMPOUNDS
RESPIRATORY SYSTEM
RUMINANTS
SEPARATION PROCESSES
TISSUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques