Guanine Nucleotide Regulatory Proteins in Receptor-Mediated Polyphosphoinositide Hydrolysis in Human Leukocytes

Charles D. Smith, Ralph Snyderman

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

This chapter discusses the methods used for analyzing the involvement of N proteins in the chemoattractant-induced breakdown of phosphatidylinositol 4, 5-bisphosphate (PIP2) in human polymorphonuclear leukocytes (PMNs). The study utilizes three systems: intact cells, detergent-permeabilized cells, and isolated plasma membranes. These approaches provide increasing ability to define the biochemical mechanisms of the coupling of occupied receptors to phospholipase C activation and prove to be generally applicable to other cells and receptor systems. The chapter discusses the quantitation of polyphosphoinositide hydrolysis in intact PMNs. Radiolabeled PMNs are exposed to hormones or to buffer. Hormone-stimulated polyphosphoinositide hydrolysis is measured as the decrease in the amount of radioactivity in phosphatidylinositol 4-phosphate (PIP) and PIP2 or the increase in radioactivity in inositol phosphates. Radioactivity in PIP and PIP2 is measured by liquid-scintillation counting after the isolation of these lipids by affinity chromatography using immobilized neomycin. Inositol phosphates are analyzed by anion-exchange chromatography followed by liquid-scintillation spectrometry.

Original languageEnglish (US)
Pages (from-to)261-271
Number of pages11
JournalMethods in enzymology
Volume141
Issue numberC
DOIs
StatePublished - Jan 1987

Fingerprint

Phosphatidylinositol Phosphates
Radioactivity
GTP-Binding Proteins
Hydrolysis
Leukocytes
Inositol Phosphates
Scintillation
Hormones
Affinity chromatography
Scintillation Counting
Neomycin
Chemotactic Factors
Liquids
Type C Phospholipases
Cell membranes
Phosphatidylinositols
Chromatography
Affinity Chromatography
Liquid Chromatography
Detergents

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

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Guanine Nucleotide Regulatory Proteins in Receptor-Mediated Polyphosphoinositide Hydrolysis in Human Leukocytes. / Smith, Charles D.; Snyderman, Ralph.

In: Methods in enzymology, Vol. 141, No. C, 01.1987, p. 261-271.

Research output: Contribution to journalArticle

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