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PLANT PHYSIOLOGY , Vol 105, Issue 2 715-724, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Multiple Forms of Phospholipase D following Germination and during Leaf Development of Castor Bean
J. H. Dyer, S. B. Ryu and X. Wang
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506
Multiple molecular forms of phospholipase D (PLD; EC 3.1.4.4) were
identified and partially characterized in endosperm of germinated seeds and
leaves of castor bean (Ricinus communis L. var Hale). The different PLD
forms were resolved by nondenaturing polyacrylamide gel electrophoresis,
isoelectric focusing, and size-exclusion chromatography. PLD was detected
with both a PLD activity assay and immunoblots with PLD-specific
antibodies. There were three major forms of PLD, designated types 1, 2, and
3, based on their mobility during nondenaturing polyacrylamide gel
electrophoresis. Molecular masses of the PLD variants were estimated at
330, 230, and 270 kD for the types 1, 2, and 3, respectively. Isoelectric
points of the native type 1, 2, and 3 PLDs were approximately 6.2, 4.9, and
4.8. Under the in vitro assay conditions used, the three forms of PLD
exhibited the same substrate specificity, hydrolyzing phosphatidylcholine
(PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but not
phosphatidylserine (PS) and phosphatidylinositol (PI). The three forms of
PLD differed in their substrate preferences, and the order of activities
was: PLD 1, PE > PG = PC; PLD 2, PE > PG > PC; PLD 3, PE = PG =
PC. The Km values of PLDs 1, 2, and 3 for PC were 1.92, 2.62, and 5.18 mM,
respectively. These PLDs were expressed differentially following seed
germination and during leaf development. Type 1 was found in the early
stages of seedling growth and in young leaves, type 2 was present in all
the tissues and growth stages examined, and type 3 was expressed in
senescent tissues. The PLDs shifted from largely cytosolic to predominantly
membrane-associated forms during leaf development. The present studies
demonstrate the structural heterogeneity of plant PLD and growth
stage-specific expression of different molecular forms. The possible role
for the occurrence of multiple molecular forms of PLD in cellular
metabolism is discussed.
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