Purification and characterization of a low-molecular-weight phospholipase A2 from developing seeds of elm.
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.
Ståhl
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U
Department of Plant Biology, P.O. Box 7080, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden. ulf.stahl@vbiol.slu.se
Ek
B
B
Stymne
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S
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry* / Q000302:isolation & purification*; D054467:Phospholipases A2; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012639:Seeds / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014197:Trees / Q000201:enzymology*
D010940:Plant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2