Identification and Purification of Calcium‐Independent Phospholipase A<sub>2</sub> from Bovine Brain Cytosol

Journal of Neurochemistry - Tập 59 Số 2 - Trang 708-714 - 1992
Yutaka Hirashima1, A.A. Farooqui, John Mills, Lloyd A. Horrocks
1Department of Medical Biochemistry, Ohio State University, Columbus 43210.

Tóm tắt

Abstract: Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KC1. However, when buffer with 1 M KC1 was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero‐3‐phosphoethanolamine, whereas the latter was active with both diacyl‐glycero‐3‐phosphoethanolamine and 1‐alk‐1′‐enyl‐2‐acylglycero‐3‐phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel nitration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high‐resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40‐kDa molecular mass and two minor bands with 14‐ and 25‐kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high‐resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The optimal pH values of phospholipase A2 obtained from peaks I and II were 7.4 and 8.0, respectively. Neither phospholipase required Ca2+ for activity. The optimal concentrations of Triton X‐100 for peak I and peak II enzymes were 0.05 and 0.1%, respectively.

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Thông tin xuất bản

Journal of Neurochemistry

Tập 59 Số 2

708-714

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