PURIFICATION AND CHARACTERIZATION OF A LOW-MOLECULAR-WEIGHT PHOSPHOLIPASE A(2) FROM DEVELOPING SEEDS OF ELM

Phospholipase A(2) (PLA(2)) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus g labra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophor esis the purified fraction showed a single protein band with a mobilit y that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-f light 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 sequen ce of some rice (Oryza sativa) expressed sequence tag clones. The puri fied enzyme had an alkaline pH optimum and required Ca2+ for activity. It was unusually stable with regard to heat, acidity, and organic sol vents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA(2), neither hydrolyzing the sn-1 position of phosphatidy lcholine nor having any activity toward lysophosphatidylcholine or dia cylglycerol. The biochemical data and amino acid sequence alignments i ndicate that the enzyme is related to the well-characterized family of animal secretory PLA(2)s and, to our knowledge, is the first plant en zyme of this type to be described.