DETERMINATION OF PHOSPHOLIPID CHANGES AT FERTILIZATION OF XENOPUS LAEVIS
B. J. STITH LAB, UNIVERSITY OF COLORADO-DENVER
Phospholipids were extracted by the Bligh-Dyer (cholorform:methanol) method, and lipids separated on a normal phase column. The Rainin HPLC used has dual pumps and is driven by a Macintosh computer (see illustration below). There is a UV flow cell and a second method of detection (since some phospholipids are transparent to UV and the solvent absorbs): an evaporative light scattering detector. In this detector, the organic solvent is evaporated, leaving the phospholipid and intensity of a defected beam of light is quantified. We have obtained good standard lines for various phospholipids.
Phosphatidylcholine (a possible source of DAG) decreased by ~200 pmoles/cell and sphingomyelin decreased by ~230 pmoles/cell. Within 5 min of insemination, choline (a product of phospholipase D activity) increased by ~134 pmol/cell, whereas DAG increased 48 pmol/cell at 10-15 min and IP3 increased 170 fmol/cell at 5 min. Molecular species analysis suggests that DAG derived from non-PIP2 sources. Sphingomyelinase may be activated at fertilization as sphingomyelin decreased and ceramide increased. A phospholipase D inhibitor (1-butanol) inhibited gravitational rotation and first cleavage (control groups were in 2-butanol). Normal phase separation (HPLC) and an evaporative light scattering detector were used to separate and detect the mass of phospholipids whereas IP3, DAG, ceramide and choline were measured by enzymatic assays.
Dual pump HPLC on far left; on right under monitor is ELSD
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