The reaction rate constant
k of phospholipase D on L-α-dimyristoyl phosphatidylcholine (DMPC) liposome with cholesterol and L-α-phosphatidylinositol (PI) was measured by 8-anilino-1-naphtalenesulfonate fluorometry at pH 5.6 in Hepes buffer.
The concentration of cholesterol which represents the maximum velocity of reaction of the phospholipase D was markedly affected by the addition of the L-α-PI. The optimum concentration of Ca
2+ which is essential to the enzyme reaction of the phospholipase D decreased by 1/6 from 60 mM to 10mM after the addition of the L-α-PI. The decrease in the optimum Ca
2+ concentration may be caused by phosphatidic acid which a decomposed product of the L-α-PI. The concentration of cholesterol showing the maximum rate constant
k of phospholipase D decreased by the addition of L-α-PI from 26 mol% to 18 mol% in the presence of 60mm Ca
2+ at pH 5.6.
The rate constants of phospholipase D were largely dependant on the reaction temperature when a small amount of cholesterol was added, while those were almost independent of the reaction temperature when the concentration of Ca
2+ was low.
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