Purification and immunological analysis of phospholipase D from castor bean endosperm.
Phospholipase D (EC 3.1.4.4) has been implicated in diverse cellular processes, but its physiological role is not well established in plants. In order to develop immunological and molecular biology approaches to address the problem, we report here the immunological analysis and N-terminal amino acid sequence of a cytosolic phospholipase D from castor bean (Ricinus communis L.). The enzyme was purified to apparent homogeneity from germinating castor bean endosperm. The specific activity of the purified enzyme was enhanced by approximately 670-fold with an overall yield of 4%. Its molecular mass was estimated at 92 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this enzyme was KLVENIEETVGFGKG. Polyclonal antibodies were raised against the purified enzyme. The antibodies inhibited the activity of transphosphatidylation more than that of hydrolysis of phospholipase D. The differential effect on the two activities of this enzyme implies that different active sites on this enzyme may be involved in the two reactions. Immunoblot analyses showed that the amounts of phospholipase D protein relative to the total endosperm proteins increased during the first 5 days of germination. The antibodies cross-reacted to proteins from several tested plant species, and those proteins had molecular masses similar to that of castor bean phospholipase D. These results indicate that the expression of phospholipase D in castor bean changes according to growth stages and that phospholipase D enzymes of different plant species are structurally related.
Wang
X
X
Department of Biochemistry, Kansas State University, Manhattan 66506.
Dyer
J H
JH
Zheng
L
L
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007123:Immunoelectrophoresis, Two-Dimensional; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010739:Phospholipase D / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D010947:Plants, Toxic; D011817:Rabbits / Q000276:immunology; D012278:Ricinus / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology
D000906:Antibodies; D010739:Phospholipase D