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 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.

Phospholipase C in rat liver plasma membranes

FEBS Letters ◽  
1986 ◽  
Vol 198 (1) ◽  
pp. 85-88 ◽  
Author(s):  
Per-Martin Melin ◽  
Roger Sundler ◽  
Bengt Jergil
Keyword(s):  
Phospholipase C ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes

scholarly journals Catalytic unit-independent phosphorylation and dephosphorylation of type II regulatory subunit of cyclic AMP-dependent protein kinase in rat liver plasma membranes

1986 ◽  
Vol 234 (1) ◽  
pp. 163-168 ◽  
Author(s):  
Z Kiss ◽  
Y Luo ◽  
G Vereb
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Liver Plasma Membranes ◽  
Catalytic Unit ◽  
Rat Liver Plasma Membranes ◽  
Dependent Protein ◽  
Membrane Bound

Rat liver plasma membranes contain a 55 kDa protein which proved to be identical with type II regulatory subunit (RII) of the cyclic AMP-dependent protein kinase (kinase A) by several criteria (gel electrophoretic behaviour, peptide map, position of the autophosphorylated site). Analysis of phosphopeptide maps revealed that the membrane-bound RII was phosphorylated by a kinase which is unrelated to the catalytic unit (C) of kinase A. Dephosphorylation of the membrane-bound RII by an endogenous phosphatase was stimulated by both cyclic AMP and fluoride. Addition of C did not stimulate dephosphorylation even in the presence of ADP; moreover, protein inhibitor of C did not modify the effects of cyclic AMP or fluoride. The effects of both cyclic AMP and fluoride were, however, inhibited by C. Results indicate that rat liver plasma membranes contain a phosphorylation-dephosphorylation system for which RII is a relatively specific substrate.

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