Measurement of Subcellular Sites of Polyphosphoinositide Metabolism in Isolated Rat Hepatocytes

William W. Wells, Mark A. Seyfred, Charles D. Smith, Masahiro Sakai

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Abstract

The products of phosphatidylinositol bisphosphate (PIP2) degradation––diacylglycerol and D-myo-inositol 1, 4, 5-trisphosphate (IP3)––have both been identified as intracellular second messengers. Numerous cell types respond to a variety of stimuli with the enhanced hydrolysis of PIP2 and to a lesser extent of phosphatidylinositol 4-phosphate (PIP). The plasma membrane PIP2 phosphodiesterase does not appear to hydrolyze phosphatidylinositol (PI) in liver. One approach to the investigation of the subcellular location of polyphosphoinositide metabolic reactions is to fractionate cell homogenates by the best procedures available and to assay the respective enzymes with added substrates under Vmax conditions. A second approach for determining the subcellular sites of the metabolism of PIP and PIP2 is to analyze the distribution of PIP and PIP2, the products of PI and PIP kinase, in a cell; however, their low levels make analysis by existing chemical means virtually impossible. The chapter explains the preparation of phosphatidylinositol [4-32P] phosphate and phosphatidylinositol [4,5-32p]bisphosphate.

Original languageEnglish (US)
Pages (from-to)92-99
Number of pages8
JournalMethods in enzymology
Volume141
Issue numberC
DOIs
Publication statusPublished - Jan 1987

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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