Interactions of phospholipase D with 1,2 diacyl-sn-glycerol-3-phosphorylcholine, dodecylsulfate, and Ca2+.
Some properties of the pure, soluble phospholipase D (phosphatidycholine phosphatido hydrolase, EC 3.1.4.4) interactions with phosphatidyl choline (1,2 diacyl-sn-glycerol-3-phosphoryl choline) in a system also containing dodecylsulfate and Ca2+ ions were studied. Concentrations of Ca2+ greater than 50 mM were necessary both for activity and adsorption of the enzyme to the "supersubstrate." Ethylenediamine tetraacetic acid caused inhibition of activity, greater than one would expect from its chelating capacity. A nonlinear increase in activity with the increase of enzyme protein was observed, suggesting a subunit aggregation into a higher mol wt protein, catalytically more active. Upon centrifugation of the supersubstrate-enzyme complex at 4.5 X 10(5) g-min at 30 C, most of the substrate molecules sedimented regardless of the pH. The reverse was true when centrifugation was done at 1 C. Phospholipase D hydrolyzed phosphatidylcholine molecules present in the supersubstrate at temperatures around 0 C at a rate 1/5 that of a maximal value measured at 30 C. The Arrhenius plot was linear in the range from 0 to 30 C, and at that temperature the curve broke with a smaller slope. Activation energy of 9.1 Kcal/mol, below 30 C, was calculated. Adsorption of the enzyme to the sedimentable supersubstrate occurred at pH 8.0, regardless of temperature. At pH 5.6, a considerable portion of phosphatidylcholine was degraded at 30 C, thus minimizing the capacity of the supersubstrate to adsorb the enzyme. Although Mg2+ could replace Ca2+ in the formation of sedimentable supersubstrate, it neither assists in adsorption of the enzyme nor in activation of the phosphatidylcholine hydrolysis.
D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010740:Phospholipases / Q000378:metabolism; D011485:Protein Binding; D012967:Sodium Dodecyl Sulfate
D012967:Sodium Dodecyl Sulfate; D008345:Manganese; D007811:Lanthanum; D010740:Phospholipases; D008274:Magnesium; D002118:Calcium