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PMID: 8948459 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

The Biochemical journal ·Vol. 308 ( Pt 3) ·1995-06-15 ·Pages 983-9

Fleming IN, Yeaman SJ

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.

MeSH Terms
Amines/pharmacology Animals Chromatography, Affinity Chromatography, Gel Diglycerides/pharmacology Electrophoresis, Polyacrylamide Gel Ethylmaleimide/pharmacology Fatty Acids/chemistry Glycerides/pharmacology Liver/enzymology Lysophospholipids/metabolism,pharmacology Metals/pharmacology Molecular Weight Phosphatidate Phosphatase/chemistry,isolation & purification,metabolism Rats Substrate Specificity Triglycerides/pharmacology
Chemicals
Amines Diglycerides Fatty Acids Glycerides Lysophospholipids Metals Triglycerides Phosphatidate Phosphatase Ethylmaleimide
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Fleming I N
Department of Biochemistry and Genetics, Medical School, University of Newcastle-upon-Tyne, UK.
Yeaman S J
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj3080983
Published
1995-06-15
Pages
983-9
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1136819
Subset
IM
Grants
Wellcome Trust · United Kingdom
External Links
PubMed source DOI: 10.1042/bj3080983 PMC Full Text View journal details
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