Purification and characterization of a low-molecular-weight phospholipase A2 from developing seeds of elm

Abstract

Phospholipase A2 (PLA2) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca2+ for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA2, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA2s and, to our knowledge, is the first plant enzyme of this type to be described.

Figure 1

Elution profiles of protein (——) and PLA₂ activity (- - - - -) from the different chromatographic steps in the purification of the elm PLA₂: Q-Sepharose Fast Flow (A), Superose 12 (B), C₄ reverse-phase HPLC (C), and C₂/C₁₈ reverse-phase SMART (D).

Figure 2

SDS-PAGE analysis of fractions from the three last purification steps of the elm PLA₂ purification, along with a commercial PLA₂ from cobra venom. Lane 1, Twenty-five micrograms of Superose 12 eluate; lane 2, 2 μg of C₄-reverse phase eluate; lane 3, 0.1 μg of C₂/C₁₈ reverse-phase eluate; lane 4, molecular size markers; and lane 5, 0.1 μg of cobra venom PLA₂. Samples were reduced with DTT and separated on an 8 to 18% gradient gel.

Figure 3

Recovery of activity from elm and cobra PLA₂ from a SDS-PAGE gel. Duplicate samples of 50 ng of PLA₂ from cobra venom and of purified elm-seed PLA₂, unreduced, were separated on an 8 to 18% gradient gel. One lane with cobra venom and one lane with elm PLA₂ were immediately sliced into 2- to 3-mm-wide pieces, from which proteins were eluted, precipitated, and assayed for PLA₂ activity, as described in Methods. Remaining lanes were stained overnight with colloidal Coomassie blue. Lane 1, Cobra venom PLA₂; lane 2, elm seed PLA₂.

Figure 4

Molecular mass determination of purified PLA₂ fraction by MALDI-TOF MS. Purified elm PLA₂ (1–2 pmol/μL) was analyzed in a MALDI-TOF MS instrument, as described in Methods.

Figure 5

A, The effect of pH. Incubations were carried out with 1 ng of purified PLA₂, 0.2 mm 1-palmitoyl-2-[¹⁴C]palmitoyl-PC, 1 mm lubrol PX, and 10 mm CaCl₂ in 75 mm buffer at 30°C for 15 min in a final volume of 50 μL. Buffers used were: acetic acid, pH 4.5 to 5.5; Mes, pH 5.5 to 6.5; Bis-Tris propane, pH 6.5 to 9.5, and Caps, pH 9.5 to 11. Bis-Tris propane, 1,3-bis(Tris[hydroxymethyl]methylamino)propane; Caps, 3-(cyclohexylamino)-1-propanesulfonic acid. B, The effect of Ca²⁺ concentration on PLA₂ activity. Incubations were carried out with 1 ng of purified PLA₂ in 50 mm Tris-HCl buffer, pH 8.0, in the presence of 0.2 mm 1-palmitoyl-2-[¹⁴C]palmitoyl-PC, 1 mm lubrol PX, and with Ca²⁺ concentrations as indicated in the figure in a final volume of 50 μL at 30°C for 15 min. C, Time-course incubations of purified PLA₂ with and without BSA. Purified PLA₂, 0.5 ng, was incubated in the presence of 0.2 mm 1-palmitoyl-2-[¹⁴C]palmitoyl-PC, 1 mm lubrol PX, 10 mm CaCl₂, 50 mm Tris-HCl, pH 8.0, in the absence of BSA (•), with 50 μg of BSA (○), or with 250 μg of BSA (×), in a final volume of 50 μL for the indicated times at 30°C. D, Effect of substrate concentration at various lubrol PX/phosphatidylcholine molar ratios. Purified PLA₂, 1 ng, was incubated in the presence of 10 mm CaCl₂, 50 mm Tris-HCl, pH 8.0, 0.02 to 1 mm 1-palmitoyl-2-[¹⁴C]palmitoyl-PC without detergent (•) or with a lubrol PX/phosphatidylcholine molar ratio of 2 (○) or 6 (×) in a final volume of 50 μL for 10 min at 30°C. conc., Concentration.

Figure 6

Alignment of the N-terminal amino acid sequence of the purified elm PLA₂ with the deduced amino acid sequences of the rice EST sequences D47653 and C27540 (accession nos.) and the 49 first N-terminal amino acids of animal secretory group I (cobra) and group II (human synovial fluid) PLA₂. The last amino acid in the predicted signal peptide (certainty 0.76 by PSORT World Wide Web server) of the D47653 sequence is underlined. The consensus line shows identical amino acids for the three plant sequences. Stars denote amino acid residues that are conserved among all animal secretory PLA₂s (Chen et al., 1994b).