SINGLE-STEP ISOLATION OF BOVINE SEMINAL PLATELET-ACTIVATING-FACTOR ACETYLHYDROLASE

Bovine seminal platelet-activating factor acetylhydrolase was isolated to >90% purity in a single step using a butyl sepharose column. The p rocedure involves the elution of the activity by use of an ethanol gra dient. Protein binds readily to the resin in the absence of high ionic strength and elutes as a peak centered at 30% ethanol. Approximately 2 mg (by Bradford; 5 mg by weight) of the enzyme can thus be easily ob tained from 9 mi of seminal plasma. The specific activity of the purif ied protein was 22 mu mol/minute/mg. About 10% of the loaded activity systematically passed unadsorbed through the column, even after repass ing. Most of this activity, however, was attributed to the same or a v ery similar enzyme that cross-reacts with polyclonal antibodies direct ed against the highly purified platelet-activating factor acetylhydrol ase. The enzyme was acid-labile but very resistant to freezing and lyo philization. This purification procedure should constitute a valuable asset to investigators interested in platelet-activating factor and pl atelet-activating factor acetylhydrolase roles in reproductive biology .