Purification of phospholipase D from Dacus carota by three-phase partitioning and its characterization.
Phospholipase D from Dacus carota (carrot) was purified by subjecting it to three-phase partitioning. The single step of three phase partitioning led to 13-fold purification with an activity recovery of 72%. SDS-PAGE analysis showed a single band with minimum molecular weight corresponding to nearly 60 kDa. The purified enzyme had a pH optimum in the range of 6.0--6.5 and was unstable above 30 degrees C. Kinetic studies showed a K(m) value of 9.5 mM and a V(max) of 0.35 mL min(-1). The enzyme purified by three-phase partitioning was found to resolve into two isoenzymes on a DEAE-cellulose column.
Chemistry Department, Indian Institute of Technology, Hauz Khas, New Delhi 110 016, India.
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D000645:Ammonium Sulfate / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D002848:Chromatography, DEAE-Cellulose; D018552:Daucus carota / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000494:pharmacology; D010739:Phospholipase D / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature; D020002:tert-Butyl Alcohol / Q000378:metabolism
D007527:Isoenzymes; D010713:Phosphatidylcholines; D013501:Surface-Active Agents; D010739:Phospholipase D; D020002:tert-Butyl Alcohol; D000645:Ammonium Sulfate; D002118:Calcium