Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

I N Fleming, S J Yeaman

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.
Original languageEnglish
Pages (from-to)983-9
Number of pages7
JournalBiochemical Journal
Volume308 ( Pt 3)
Publication statusPublished - 1995

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Phosphatidate Phosphatase
Ethylmaleimide
Liver
Purification
Rats
Enzymes
Cibacron Blue F 3GA
Gels
Gel Chromatography
Chromatography
Polyacrylamide Gel Electrophoresis
Monoglycerides
Peptides
Phosphatidic Acids
Column chromatography
Phosphorylation
Diglycerides
Octoxynol
Enzyme activity
Durapatite

Keywords

  • Amines
  • Animals
  • Chromatography, Affinity
  • Chromatography, Gel
  • Diglycerides
  • Electrophoresis, Polyacrylamide Gel
  • Ethylmaleimide
  • Fatty Acids
  • Glycerides
  • Liver
  • Lysophospholipids
  • Metals
  • Molecular Weight
  • Phosphatidate Phosphatase
  • Rats
  • Substrate Specificity
  • Triglycerides

Cite this

Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver. / Fleming, I N; Yeaman, S J.

In: Biochemical Journal, Vol. 308 ( Pt 3), 1995, p. 983-9.

Research output: Contribution to journalArticle

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abstract = "N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.",
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T1 - Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

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AU - Yeaman, S J

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N2 - N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

AB - N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

KW - Amines

KW - Animals

KW - Chromatography, Affinity

KW - Chromatography, Gel

KW - Diglycerides

KW - Electrophoresis, Polyacrylamide Gel

KW - Ethylmaleimide

KW - Fatty Acids

KW - Glycerides

KW - Liver

KW - Lysophospholipids

KW - Metals

KW - Molecular Weight

KW - Phosphatidate Phosphatase

KW - Rats

KW - Substrate Specificity

KW - Triglycerides

M3 - Article

C2 - 8948459

VL - 308 ( Pt 3)

SP - 983

EP - 989

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

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