scholarly journals The roles of phospholipase D and a GTP-binding protein in guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes

1990 ◽  
Vol 272 (3) ◽  
pp. 749-753 ◽  
Author(s):  
K M Hurst ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Low Concentrations ◽  
Rat Liver Plasma Membranes ◽  
Triton X ◽  
Hydrolysis Of

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5′-[beta gamma-imido]triphosphate and guanosine 5′-[alpha beta-methylene]triphosphate, but not adenosine 5′-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5′-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

scholarly journals Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements

1987 ◽  
Vol 248 (3) ◽  
pp. 791-799 ◽  
Author(s):  
S J Taylor ◽  
J H Exton
Keyword(s):  
Phospholipase C ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Maximal Effect ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes ◽  
Membrane Bound ◽  
Hydrolysis Of

The effect of the GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5′-[beta gamma-imido]triphosphate, but not with adenosine 5′-[gamma-thio]triphosphate, and was inhibited by guanosine 5′-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is proposed that GTP analogues and hormones, acting through a guanine nucleotide-binding protein, activate the enzyme mainly by lowering its Ca2+ requirement.

scholarly journals Effect of guanine nucleotides on polyphosphoinositide synthesis in rat liver plasma membranes

1990 ◽  
Vol 271 (3) ◽  
pp. 591-597 ◽  
Author(s):  
C Benistant ◽  
A P Thomas ◽  
R Rubin
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Guanine Nucleotides ◽  
The Other ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes ◽  
Stimulation Of ◽  
Rapid Incorporation

The effect of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) on PtdIns and PtdIns(4)P kinase activities was measured in rat liver plasma membranes. The addition of [32P]ATP resulted in the rapid incorporation of 32P into PtdIns(4)P and PtdIns(4,5)P2, with maximal levels reached within 30 s. GTP[S] (25-500 microM) increased the rate and magnitude of [32P]PtdIns(4)P and [32P]PtdIns(4,5)P2 formation by 50 and 120% respectively. Similar stimulatory effects were induced by guanosine 5′-[beta gamma-imido]triphosphate, GTP, GDP and guanosine 5′-[beta-thio]diphosphate. The stimulation of PtdIns phosphorylation by GTP[S] occurred in the presence of 2 mM-EGTA, a condition which fully inhibited phosphoinositide-specific phospholipase C. GTP[S] did not stimulate phosphomonoesterase activity, and its action was not due to the binding of magnesium. However, the overall ATP-hydrolysing activity of the membrane preparation was inhibited by GTP[S] and the other guanine nucleotides. There was a direct correlation between the extent of this inhibition and the stimulation of polyphosphoinositide formation. The results indicate that stimulation of polyphosphoinositide formation by guanine nucleotides in rat liver plasma membranes can be accounted for by an inhibition of ATP hydrolysis. These data are inconsistent with a specific GTP-binding protein (G-protein)-mediated stimulation of PtdIns or PtdIns(4)P kinase.

scholarly journals Lysophosphatidylcholines can modulate the activity of the glucagon-stimulated adenylate cyclase from rat liver plasma membranes

1979 ◽  
Vol 178 (1) ◽  
pp. 217-221 ◽  
Author(s):  
M D Houslay ◽  
R W Palmer
Keyword(s):  
Adenylate Cyclase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Hormone Receptors ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes ◽  
Increased Sensitivity

1. Synthetic lysophosphatidylcholines inhibit the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes at concentrations two to five times lower than those needed to inhibit the fluoride-stimulated activity. 2. Specific 125I-labelled glucagon binding to hormone receptors is inhibited at concentrations similar to those inhibiting the fluoride-stimulated activity. 3. At concentrations of lysophosphatidylcholines immediately below those causing inhibition, an activation of adenylate cyclase activity or hormone binding was observed. 4 These effects are essentially reversible. 5. We conclude that the increased sensitivity of glucagon-stimulated adenylate cyclase to inhibition may be due to the lysophosphatidylcholines interfering with the physical coupling between the hormone receptor and catalytic unit of adenylate cyclase. 6. We suggest that, in vivo, it is possible that lysophosphatidylcholines may modulate the activity of adenylate cyclase only when it is in the hormone-stimulated state.

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