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Several lines of experimental evidence indicate the involvement of a guanine nucleotide-dependent protein (G-protein) in the hormone-stimulated hydrolysis of phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2). However, the shortcomings of available procedures for cell-free assay of hormone-stimulated phosphoinositidase C (PIC) have limited our current understanding of the molecular and mechanistic details of PIC regulation. We recently have proposed that turkey erythrocyte membranes may provide a valuable model system for studies of G-protein-dependent PtdIns(4,5)P2 hydrolysis. The membranes can be simply prepared from [3H]inositol-labelled erythrocytes and they contain a PIC activity that hydrolyses endogenous phosphoinositides and is exquisitively sensitive to guanine nucleotides. PtdIns(4,5)P2 is the principal substrate for this enzyme, there being relatively little direct hydrolysis of phosphatidylinositol 4-phosphate and no detectable hydrolysis of PtdIns. The membranes also contain a purinoceptor of the P2y subclass that is efficiently coupled to PtdIns(4,5)P2 hydrolysis both in intact cells and in the isolated membranes. 2-Methylthioadenosine trisphosphate (2-methyl-S-ATP), a specific P2y receptor agonist, has no effect upon PtdIns(4,5)P2 hydrolysis in the absence of guanine nucleotides, but greatly enhances both the potency and efficacy of PIC activation by guanine nucleotides such as GTP gamma S. GTP gamma S alone stimulates PIC activity only after a prolonged time-lag; the effect of increasing doses of 2-methyl-S-ATP is progressively to shorten this lag phase. These results suggest that the mechanism of G-protein activation involves acceleration of a nucleotide exchange reaction as has been demonstrated for the activation of adenylate cyclase in the same membrane preparation. As well as contributing valuable information on the substrate specificity of PIC and its mode of regulation by hormones, turkey erythrocytes provide a plentiful source of plasma membranes and may be useful for purification of the appropriate G-protein and PIC activities.
C P Downes, and
C P Berrie, and
P T Hawkins, and
L Stephens, and
J L Boyer, and
T K Harden
April 1989,
The Journal of biological chemistry,
C P Downes, and
C P Berrie, and
P T Hawkins, and
L Stephens, and
J L Boyer, and
T K Harden
January 1991,
Pharmacology & therapeutics,
C P Downes, and
C P Berrie, and
P T Hawkins, and
L Stephens, and
J L Boyer, and
T K Harden
July 1993,
The Journal of biological chemistry,
C P Downes, and
C P Berrie, and
P T Hawkins, and
L Stephens, and
J L Boyer, and
T K Harden
November 1987,
FEBS letters,
C P Downes, and
C P Berrie, and
P T Hawkins, and
L Stephens, and
J L Boyer, and
T K Harden
August 1990,
The Journal of biological chemistry,
C P Downes, and
C P Berrie, and
P T Hawkins, and
L Stephens, and
J L Boyer, and
T K Harden
December 1989,
Trends in pharmacological sciences,
C P Downes, and
C P Berrie, and
P T Hawkins, and
L Stephens, and
J L Boyer, and
T K Harden
October 1996,
European journal of pharmacology,
C P Downes, and
C P Berrie, and
P T Hawkins, and
L Stephens, and
J L Boyer, and
T K Harden
July 1991,
Biochemical and biophysical research communications,
C P Downes, and
C P Berrie, and
P T Hawkins, and
L Stephens, and
J L Boyer, and
T K Harden
May 1997,
FEBS letters,
C P Downes, and
C P Berrie, and
P T Hawkins, and
L Stephens, and
J L Boyer, and
T K Harden
December 1994,
The Biochemical journal,
