A SIMPLE PROCEDURE FOR THE STUDY OF INOSITOL PHOSPHATIDES IN CAT BRAIN SLICES

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Abstract
A procedure is described for the separation of monophosphoinositide (MPI), diphosphoinositide (DPI), and triphosphoinositide (TPI) from small samples of tissue. The method is readily applicable to experiments with tissue slices. It is based upon the finding that MPI is readily extracted into chloroform–methanol (2:1), and DPI and TPI are extracted when 1 part of concentrated hydrochloric acid is added to 300 parts of the solvent. The phosphoinositides are then separated chromatographically on formaldehyde-treated paper.In experiments in which slices of cat brain were incubated in the presence of inorganic32P, the phosphorus compounds of high specific radioactivity remaining in the tissue residue after extraction of lipids and nucleic acids, and referred to previously as "inositide P" or "residue organic P", were found to be chiefly protein-bound DPI and TPI, with small amounts of protein-bound phosphatidyl serine and phosphatidyl ethanolamine.Increasing the concentration of K+ion in the medium from 5.9 mM to 105 mM caused a decrease in the incorporation of inorganic32P into the "lipid extract", MPI, DPI, and TPI of cat brain slices. Addition of chlorpromazine (0.1 mM), or acetylcholine (10 mM) with eserine sulfate (0.5 mM), caused an increase in the incorporation of inorganic32P into the "lipid extract" and MPI, but with no corresponding increase in the incorporation into DPI and TPI.
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