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.

scholarly journals Characterization of the phosphorylated form of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes

1981 ◽  
Vol 195 (3) ◽  
pp. 653-660 ◽  
Author(s):  
R J Marchmont ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Enzyme Treatment ◽  
Hill Coefficient ◽  
Activation Process ◽  
Native State ◽  
Cyclic Amp Phosphodiesterase ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes

Incubation of intact purified rat liver plasma membranes with insulin, cyclic AMP and ATP led to the activation of the peripheral “low-Km” cyclic AMP phosphodiesterase. When (gamma-32P]ATP was included in the incubation mixture, after purification of this enzyme to homogeneity it was found to contain 1 mol of alkali-labile 32P/mol of enzyme. Treatment of the homogeneous phosphorylated enzyme with alkaline phosphatase released all of the 32P from the protein while restoring its activity to the native state. The reversibility of the activation that is achieved by the phosphorylation of this enzyme could also be demonstrated with a high-speed supernatant from rat liver. This restored the activity of the activated membrane-bound enzyme to its native state. The Ka for the cyclic AMP-dependence of this process (1.6 micrometer) was unaffected by a range of ATP concentrations (1-10 mM) and by a range of membrane protein concentrations (0.2-2 mg/ml). Adenylyl imidodiphosphate could not substitute for ATP, and concanavalin A could not substitute for insulin, as essential ligands in the activation process. The purified activated enzyme exhibited Km 0.6 microM, Vmax 10.9 units/mg of protein and Hill coefficient (h) 0.47. The Vmax. for this activated enzyme was much higher than that of the native enzyme, yet h was much lower.

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 Evidence that the peripheral cyclic AMP phosphodiesterase of rat liver plasma membranes is a metalloenzyme

1985 ◽  
Vol 225 (1) ◽  
pp. 143-147 ◽  
Author(s):  
J Londesborough
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Cyclic Nucleotide ◽  
Plasma Membranes ◽  
Metal Chelators ◽  
Phosphodiesterase Activity ◽  
Cyclic Amp Phosphodiesterase ◽  
Liver Plasma Membranes ◽  
Isomer Activity ◽  
Rat Liver Plasma Membranes

Cyclic nucleotide phosphodiesterase activity in salt extracts of rat liver plasma membranes was progressively inactivated by treatment with the metal chelators 8-hydroxyquinoline and o-phenanthroline, but not the non-chelating m-phenanthroline isomer. Activity at 20 microM-cyclic AMP was lost more slowly than activity at 0.4 microM-cyclic AMP. The activity of treated preparations was partially restored by incubation with Zn2+ or Mn2+ ions (in the presence of 1 mM-MgCl2) but not with Ca2+, Cd2+, Co2+, Cu2+ or Fe2+ ions, nor by MgCl2 alone. The results suggest the presence in the membrane extracts of a cyclic AMP phosphodiesterase containing tightly bound metal, possibly Zn or Mn, that affects the enzyme's affinity for cyclic AMP.

Distribution of membrane-bound cyclic AMP-dependent protein kinase in plasma membranes of cells of the kidney cortex

1975 ◽  
Vol 24 (1) ◽  
pp. 145-159 ◽  
Author(s):  
Rolf Kinne ◽  
Linda J. Shlatz ◽  
Evamaria Kinne-Saffran ◽  
Irving L. Schwartz
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Plasma Membranes ◽  
Kidney Cortex ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Membrane Bound

scholarly journals Purification and properties of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes

1981 ◽  
Vol 195 (3) ◽  
pp. 645-652 ◽  
Author(s):  
R J Marchmont ◽  
S R Ayad ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Hill Coefficient ◽  
Affinity Column ◽  
Cyclic Amp Phosphodiesterase ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes

The peripheral high-affinity cyclic AMP phosphodiesterase from rat liver plasma membranes was purified to apparent homogeneity. The procedure used involved the initial purification of liver plasma membranes and the solubilization of the enzyme by using a high-ionic-strength medium. This was followed by chromatography of the enzyme on DEAE-cellulose, Affi-Gel Blue, a novel affinity column and Sephadex G-100. A 9500-fold purification of the enzyme with a 24% yield was achieved by this procedure. The purified enzyme was apparently monomeric (Mr 52000) as it exhibited identical molecular weights on analysis by gel filtration, sedimentation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It is suggested that the non-Michaelis kinetics exhibited by the enzyme are due to it obeying a mnemonical mechanism, where it displays Km 0.7 micrometer, Vmax. 9.1 units/mg of protein and Hill coefficient (h) 0.62. Cyclic GMP acts as a poor substrate for the enzyme, with Km 120 micrometer and Vmax. 0.4 unit/mg of protein, and also as an inhibitor of the enzyme, with I50 (concentration giving 50% inhibition) 150 micrometer when assayed at 0.4 micrometer-cyclic AMP. Inhibition by 5′-AMP is unlikely to be of physiological importance, as it is only a weak inhibitor of the enzyme (I50 47 mM assayed at 0.4 micrometer-cyclic AMP).

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