Determination of PAF-2-acylhydrolase activity
Conference
·
· Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:6898869
A simple and rapid procedure has been developed to determine the PAF-2-acylhydrolase activity in various biological fluids. The assay was based upon the following biophysical properties of PAF (1-O-hexadecyl-2-/sup 3/H-acetyl-sn-glycero-3-phosphocholine, 10 Ci/mMole). In aqueous solution PAF is not soluble below its critical micellar concentration (1.1 ..mu..M) unless albumin (2.5 mg/ml) is present which binds and stabilizes PAF in solution. However, upon precipitation of the albumin with trichloroacetic acid (TCA), 100% of the PAF also co-precipitates. Thus, in a reaction mixture to assess PAF-2-acylhydrolase activity conducted in the presence of albumin, termination of the reaction by the addition of TCA (10% final concentration) precipitates all residual PAF and also inactivates all PAF-2-acylhydrolase activity. Under these conditions 100% of the released /sup 3/H-acetate is recovered in the supernatant after centrifugation, 10,000 x g for 60 sec. Therefore, determination of released /sup 3/H-acetate provides a highly accurate and reproducible measurement of both substrate (PAF) and product (/sup 3/H-acetate) concentrations. Kinetic studies assessing the PAF-2-acylhydrolase in human serum diluted in Hank's balance salt solution containing 2.5 mg/ml human albumin, pH 7.20 yielded an apparent Km = 5 nM.
- Research Organization:
- Univ. of Texas Health Science Center, San Antonio
- OSTI ID:
- 6898869
- Report Number(s):
- CONF-8604222-
- Conference Information:
- Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:4
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETATES
ALBUMINS
BIOASSAY
BIOCHEMICAL REACTION KINETICS
BLOOD COAGULATION FACTORS
CARBOXYLIC ACID SALTS
COAGULANTS
DRUGS
ENZYME ACTIVITY
ENZYMES
HEMATOLOGIC AGENTS
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
ORGANIC COMPOUNDS
PROTEINS
REACTION KINETICS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
59 BASIC BIOLOGICAL SCIENCES
ACETATES
ALBUMINS
BIOASSAY
BIOCHEMICAL REACTION KINETICS
BLOOD COAGULATION FACTORS
CARBOXYLIC ACID SALTS
COAGULANTS
DRUGS
ENZYME ACTIVITY
ENZYMES
HEMATOLOGIC AGENTS
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
ORGANIC COMPOUNDS
PROTEINS
REACTION KINETICS
TRACER TECHNIQUES
TRITIUM COMPOUNDS