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Purification of NEM‐sensitive Mg 2+ ‐dependent phosphatidate phosphatase from yeast
Author(s) -
Han SeungHee,
Carman George M.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a238-d
Subject(s) - phosphatidate , yeast , phosphatase , chemistry , biochemistry , enzyme , protein kinase c , diacylglycerol kinase
The Mg 2+ ‐dependent phosphatidate (PA) phosphatase catalyzes the dephosphorylation of PA to synthesize diacylglycerol and Pi. The diacylglycerol generated in this reaction is used for the synthesis of triacylglycerol. There are at least two genes that encode Mg 2+ ‐dependent PA phosphatases in yeast. The PAH1 gene encodes a NEM‐insensitive enzyme whereas the gene that encodes the NEM‐sensitive enzyme has yet to be identified. To purify the NEM‐sensitive enzyme, we used a pah1 Δ dpp1 Δ lpp1 Δ triple mutant that lacks the PAH1 ‐encoded NEM‐insensitive enzyme as well as the DPP1 ‐ and LPP1 ‐encoded Mg 2+ ‐independent PA phosphatase activities. Mg 2+ ‐dependent PA phosphatase was extracted from the membrane fraction with buffer containing 1 M NaCl. The salt was then removed from the enzyme preparation by dialysis, which caused enzyme aggregation. The aggregated enzyme was suspended in buffer with sodium cholate, and then purified on phosphocellulose, MonoQ, and Superose 6 columns. The enzyme was enriched several thousand fold, and a single peptide was isolated. The procedure is being scaled up to obtain enough protein for amino acid sequence determination. Supported by NIH grant GM‐28140.