Purification and Immunological Analysis of Phospholipase D from Castor Bean Endosperm

Xuemin Wang, Jim Dyer, Ling Zheng

Research output: Contribution to journalArticleResearchpeer-review

88 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)486-494
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume306
Issue number2
DOIs
StatePublished - 1 Jan 1993

Fingerprint

Castor Bean
Phospholipase D
Endosperm
Purification
Enzymes
Molecular mass
4 alpha-glucanotransferase
Antibodies
Amino Acid Sequence
Proteins
Physiological Phenomena
Ricinus
Amino Acids
Molecular biology
Germination
Electrophoresis
Sodium Dodecyl Sulfate
Molecular Biology
Polyacrylamide Gel Electrophoresis
Hydrolysis

Cite this

@article{ade77f41e7f14b1185999db164e14b31,
title = "Purification and Immunological Analysis of Phospholipase D from Castor Bean Endosperm",
abstract = "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.",
author = "Xuemin Wang and Jim Dyer and Ling Zheng",
year = "1993",
month = "1",
day = "1",
doi = "10.1006/abbi.1993.1541",
language = "English",
volume = "306",
pages = "486--494",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "2",

}

Purification and Immunological Analysis of Phospholipase D from Castor Bean Endosperm. / Wang, Xuemin; Dyer, Jim; Zheng, Ling.

In: Archives of Biochemistry and Biophysics, Vol. 306, No. 2, 01.01.1993, p. 486-494.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Purification and Immunological Analysis of Phospholipase D from Castor Bean Endosperm

AU - Wang, Xuemin

AU - Dyer, Jim

AU - Zheng, Ling

PY - 1993/1/1

Y1 - 1993/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0027496504&partnerID=8YFLogxK

U2 - 10.1006/abbi.1993.1541

DO - 10.1006/abbi.1993.1541

M3 - Article

VL - 306

SP - 486

EP - 494

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 2

ER -