Purification of human plasma platelet-activating factor acetylhydrolase

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5333695

Stafforini, D M; Prescott, S M; McIntyre, T M

Platelet-activating factor (PAF;1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine is synthesized by a variety of cells. It induces hypotension, and activates platelets, neutrophils, and macrophages at nanomolar concentrations. Removal of the acetate abolishes biological activity, and is catalyzed by a specific PAF acetylhydrolase present in plasma and tissues. The authors developed a rapid assay, based on separation of (/sup 3/H)acetate from (/sup 3/H-acetyl)PAF by reversed-phase chromatography. In human plasma the enzyme exhibits an apparent Km of 5.7..mu..M, with a Vmax of 0.027..mu..mol/h/mg. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins (HDL) and 70% with low density lipoproteins (LDL). The enzyme was purified from LDL by precipitation with Na phosphotungstate and MgCl/sub 2/, solubilization with Tween 20, column chromatography and electrophoresis. This procedure resulted in a preparation that was 21,000-fold purified from plasma (spec. act. 575..mu..mol/h/mg) with a recovery of 10%. The purified enzyme has a molecular weight of about 43,000, a broad pH optimum (peak 7.5-8.0), and a pl of 4.6. It has greater activity when PAF is in a micellar, as compared to monomeric, and exhibits surface dilution kinetics, which may be important in vivo. The purification and characterization of this enzyme will allow detailed studies of its role in PAF metabolism.

• Research Organization: Univ. of Utah, Salt Lake City
• OSTI ID: 5333695
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETATES
ANIMAL CELLS
ANIMALS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BLOOD COAGULATION FACTORS
BLOOD PLASMA
BLOOD PLATELETS
BODY FLUIDS
CARBOXYLIC ACID SALTS
CENTRIFUGATION
CHROMATOGRAPHY
COAGULANTS
CONNECTIVE TISSUE CELLS
DRUGS
ELECTROPHORESIS
ENZYMES
HEMATOLOGIC AGENTS
HYDROLASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LEUKOCYTES
LIPIDS
LIPOPROTEINS
MACROPHAGES
MAMMALS
MAN
MATERIALS
MOLECULAR WEIGHT
NEUTROPHILS
ORGANIC COMPOUNDS
PHAGOCYTES
PRIMATES
PROTEINS
PURIFICATION
SEPARATION PROCESSES
SOMATIC CELLS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ULTRACENTRIFUGATION
VERTEBRATES