Characterization of recombinant rat brain phospholipase

D. S. Min, S. K. Park, J. H. Exton

Research output: Contribution to journalArticle

Abstract

We have recently cloned a cDNA encoding a phosphatidylcholine-specific PLD from rat brain and named it rPLD (submitted to J. Biol. Chem.) It shows 90 percent amino acid identiy with the human PLD isoform hPLD1B. We have expressed rPLD as a histidine-taGged fusion protein in insect cells using the expression vector bluebasrlfis. The recombinant protein was purified to homogeniy by Ni 2 agarose affinity chromatography. The activity and expression of rPLD in Sf9 cells wre detected in both cytos9ol and membrane fractions. Pl(4.5)P 2 and PI(3.4.5)P 3activated rPLD equipotentiy, but other acide phospholipids were ineffective, The activity of rPLD was dependent on both M 2+ and Ca 2+and showed a broad dependence on PH wish optinum activety at pH 6.5-7.5 The enzyme was activated by the small G protens but kinase C and 3 but not activated PLD in a manner that was stimulated by phorbol ester but did not require ATP. The purified rPLD did not show wynergistic interactins betweem ARF and R ho and between these proteins and PKC or Purified PKC and phosptorylated parified PL in vitro and the phosphorylated PLD exhibited a mobility shift on SDS PAGE. Phosphorylation of PLD by PKC Proxo another regutatoryu mechanism for the enzyme.

Original languageEnglish
Pages (from-to)A1350
JournalFASEB Journal
Volume11
Issue number9
Publication statusPublished - 1997 Dec 1

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Phospholipases
Rats
Brain
Insect Proteins
Affinity chromatography
Sf9 Cells
Agarose Chromatography
Phosphorylation
Phorbol Esters
Enzymes
Phosphatidylcholines
Affinity Chromatography
Recombinant Proteins
Histidine
Sepharose
Polyacrylamide Gel Electrophoresis
Phospholipids
Protein Isoforms
Proteins
Phosphotransferases

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Min, D. S., Park, S. K., & Exton, J. H. (1997). Characterization of recombinant rat brain phospholipase. FASEB Journal, 11(9), A1350.
Min, D. S. ; Park, S. K. ; Exton, J. H. / Characterization of recombinant rat brain phospholipase. In: FASEB Journal. 1997 ; Vol. 11, No. 9. pp. A1350.
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Min, DS, Park, SK & Exton, JH 1997, 'Characterization of recombinant rat brain phospholipase', FASEB Journal, vol. 11, no. 9, pp. A1350.

Characterization of recombinant rat brain phospholipase. / Min, D. S.; Park, S. K.; Exton, J. H.

In: FASEB Journal, Vol. 11, No. 9, 01.12.1997, p. A1350.

Research output: Contribution to journalArticle

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