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Partial purification of a cytosolic Mg 2+ ‐dependent PA phosphatase from yeast
Author(s) -
Han SeungHee,
Carman George M.
Publication year - 2006
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.20.5.a948-b
Subject(s) - phosphatase , chemistry , hydroxylapatite , enzyme , chromatography , alkaline phosphatase , yeast , biochemistry , affinity chromatography , specific activity , potassium phosphate , dephosphorylation , sepharose , elution , phosphate , enzyme assay , cytosol
PA phosphatase catalyzes the dephosphorylation of PA to form the DAG used to synthesize phospholipids and triacylglycerols. Yeast contains both Mg 2+ ‐dependent and Mg 2+ ‐independent PA phosphatase enzymes. Soluble and membrane‐associated forms of the Mg 2+ ‐dependent PA phosphatase have been identified. In this work, we report progress in purifying the soluble form of the enzyme. A dpp1 Δ lpp1 Δ double mutant was used as the starting point because it lacks the major Mg 2+ ‐independent PA phosphatase enzymes that interfere with purification. A variety of chromatography resins were evaluated for their ability to bind the Mg 2+ ‐dependent PA phosphatase activity from the cytosolic fraction of the cell. Among the resins tested, Q‐Sepharose and hydroxylapatite afforded maximum enzyme binding. The PA phosphatase enzyme could be eluted from the Q‐Sepharose resin in a chromatography buffer containing with 0.3 M NaCl whereas it could be eluted from hydroxylapatite with 1 M potassium phosphate buffer. The purifications and yields of activity from these resins were typically 3‐ to 6‐fold and 60–80%, respectively. Further purification by affinity chromatography is being evaluated. Supported by NIH grant GM 28140.