Purification and characterization of a cytosolic Ca(2+)-independent phospholipase A(2) from bovine brain

Abstract

The Ca(2+)-independent phospholipase A(2) (iPLA(2)) subfamily of enzymes is associated with arachidonic acid (AA) release and the subsequent increase in fatty acid turnover. This phenomenon occurs not only during apoptosis but also during inflammation and lymphocyte proliferation. In this study, we purified and characterized a novel type of iPLA(2) from bovine brain. iPLA(2) was purified 4,174-fold from the bovine brain by a sequential process involving DEAE-cellulose anion exchange, phenyl-5PW hydrophobic interaction, heparin-Sepharose affinity, Sephacryl S-300 gel filtration, Mono S cation exchange, Mono Q anion exchange, and Superose 12 gel filtration. A single peak of iPLA(2) activity was eluted at an apparent molecular mass of 155 kDa during the final Superose 12 gel-filtration step. The purified enzyme had an isoelectric point of 5.3 on two-dimensional gel electrophoresis (2-DE) and was inhibited by arachidonyl trifluoromethyl ketone (AACOCF(3)), Triton X-100, iron, and Ca(2+). However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA(2), and adenosine triphosphate (ATP). The spot with the iPLA(2) activity did not match with any known protein sequence, as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Altogether, these data suggest that the purified enzyme is a novel form of cytosolic iPLA(2).

Fig. 1.. Column chromatography profiles of iPLA₂ from bovine brain. iPLA₂ was prepared from bovine brain homogenates by precipitation at pH 5.0 with acetic acid followed by a series of chromatography steps. DEAE-cellulose anion-exchange chromatography (A), Phenyl-5PW hydrophobic chromatography (B), heparin-Sepharose CL-6B affinity chromatography (C), Sephacryl S-300 gel-filtration chromatography (D), Mono S cation-exchange FPLC (E), Mono Q anion-exchange FPLC (F), and Superose 12 FPLC (G). The inset in (G) shows the calibration curve for estimation the apparent molecular mass of iPLA₂. Molecular mass standard proteins were β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). Data is representative of at least ten independent experiments.

Fig. 2.. Superose 12 FPLC of iPLA₂ from the active eluate obtained from Mono Q FPLC. Equal volumes of the fractions from the Superose 12 FPLC were assayed for iPLA₂ activity, and separated by SDS-PAGE, and visualized by silver staining, as described in the “Materials and Methods”. These data are representative of at least five independent experiments.

Fig. 3.. 2-DE analysis of Superose 12 FPLC fractions. The fractions [20 (A), 21 (B), 22 (C), and 25 (D)] obtained from Superose 12 FPLC were analyzed by 2-DE, as described in the “Materials and Methods”. The positions of external marker proteins are shown on the left side of the gels. The encircled areas indicate spot A.

Fig. 4.. Characterization of the purified iPLA₂. Purified iPLA₂ was pre-incubated with the indicated concentrations of Ca²⁺ (A) or metal ions (B) for 10 min at 37℃ and then incubated with 2-[1-¹⁴C]AAGPC for 30 min at 37℃. Each data point represents the mean ± SD of at least five independent experiments. (C) The purified iPLA₂ activity was assayed in buffers at pH 4-11 by incubation with 2-[1-¹⁴C]AA-GPC for 30 min a t 37℃. Each data point represents the mean ± SD of at least three independent experiments. (D) Purified iPLA₂ was pre-incubated with the indicated concentration of BEL or AACOCF₃ for 10 min at 37℃, followed by incubation with 2-[1- ¹⁴C]AA-GPC for 30 min. (E) Purified iPLA₂ was pre-incubated with the indicated concentrations of ATP and Triton X-100 for 10 min at 37℃, and then incubated with 2- [1-¹⁴C]AA-GPC for 30 min at 37℃. Statistical significance was assessed by one-way ANOVA in (A, C, and E) and Student’s t-test in (D). *** P < 0.001; ** P < 0.01.