scholarly journals The insulin-stimulated cyclic AMP phosphodiesterase binds to a single class of protein sites on the liver plasma membrane

1981 ◽  
Vol 198 (3) ◽  
pp. 703-706 ◽  
Author(s):  
M D Houslay ◽  
R J Marchmont
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Plasma Membranes ◽  
Receptor Site ◽  
Single Class ◽  
Liver Plasma Membrane ◽  
Cyclic Amp Phosphodiesterase ◽  
Receptor Sites ◽  
Rat Liver Plasma Membranes ◽  
Dependent Protein

The peripheral cycle AMP phosphodiesterase from rat liver plasma membranes binds with high affinity (2.4 nM) to a single class of receptor sites on the liver plasma membrane. These receptor sites appear to be proteins, as they are trypsin- and heat-labile. The sensitivity of these sites to denaturation by trypsin and heat is a first-order process. The presence of Ca2+ (5 mM) increases the affinity of these sites for the enzyme, but does not alter their total number. The receptor sites and the cyclic AMP phosphodiesterase occur in similar numbers, at around 2 pmol/mg of plasma-membrane protein. It is proposed that the peripheral, liver plasma-membrane cyclic AMP phosphodiesterase is attached to a specific site on the insulin receptor and that the binding of insulin to the receptor site triggers a conformational change in the enzyme such that the enzyme can be phosphorylated and activated by an endogenous cyclic AMP-dependent protein kinase.

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 The action of islet activating protein (pertussis toxin) on insulin's ability to inhibit adenylate cyclase and activate cyclic AMP phosphodiesterases in hepatocytes

1986 ◽  
Vol 235 (1) ◽  
pp. 145-149 ◽  
Author(s):  
C M Heyworth ◽  
A M Grey ◽  
S R Wilson ◽  
E Hanski ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pertussis Toxin ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Guanine Nucleotide ◽  
Cyclic Amp Phosphodiesterase ◽  
Peripheral Plasma ◽  
Guanine Nucleotide Regulatory Protein

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the ‘dense-vesicle’ cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and ‘dense-vesicle’ cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in 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.

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.

scholarly journals A peripheral and an intrinsic enzyme constitute the cyclic AMP phosphodiesterase activity of rat liver plasma membranes

1980 ◽  
Vol 187 (2) ◽  
pp. 381-392 ◽  
Author(s):  
R J Marchmont ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Plasma Membranes ◽  
High Salt ◽  
Phosphodiesterase Activity ◽  
Cyclic Amp Phosphodiesterase ◽  
Rat Liver Plasma Membranes ◽  
Solubilized Enzyme ◽  
Single Exponential ◽  
Burk Plot

1. Approx. 10% of the rat liver cellular cyclic AMP phosphodiesterase activity was associated with a plasma-membrane fraction. 2. Lineweaver-Burk plots of this activity were clearly non-linear, yielding extrapolated Km values of 0.7 and 60.6 microns. 3. Treatment of these membranes with high-ionic-strength NaCl solutions apparently released 80% of this activity assayed at 0.4 micron-cyclic AMP, and 15% of the activity assayed at 1 mM-cyclic AMP. 4. The high-salt-solubilized enzyme gave a non-linear Lineweaver-Burk plot. 5. The cyclic AMP phosphodiesterase activity of the washed high-salt-treated membranes exhibited a linear Lineweaver-Burk plot, yielding a Km of 60 microns. 6. The high-salt-solubilized enzyme exhibited a single peak of activity upon polyacrylamide-gel electrophoresis, a single peak upon sucrose-density-gradient centrifugation (3.9 S) and decayed as a single exponential upon heat-treatment (half-life 1 min at 55 degrees C). 7. The activity of washed high-salt-treated membranes decayed as a single exponential upon heat-treatment (half-life 42 min at 55 degrees C), and was solubilized in the detergent Triton X-100. 8. Cytosol-derived cyclic AMP phosphodiesterase activity could bind to washed high-salt-treated plasma membranes, but was totally eluted by washing with 1 mM-KHCO3, unlike the high-salt-solubilized enzyme, which required high salt concentrations to elute it. 9. We suggest that the cyclic AMP phosphodiesterase activity of rat liver plasma membranes can be resolved into two components: a single peripheral protein exhibiting apparent negative co-operativity, that is distinct from cytosol forms, and an intrinsic protein exhibiting normal Michaelis kinetics.

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).

Axonal Transport of the Ca2+-Dependent Protein Modulator of 3':5'-Cyclic-AMP Phosphodiesterase in the Rabbit Visual System

1980 ◽  
Vol 35 (1) ◽  
pp. 242-248 ◽  
Author(s):  
Paul F. Erickson ◽  
Kenneth B. Seamon ◽  
Blake W. Moore ◽  
Robert S. Lasher ◽  
Lee N. Minier
Keyword(s):  
Cyclic Amp ◽  
Visual System ◽  
Axonal Transport ◽  
Cyclic Amp Phosphodiesterase ◽  
Dependent Protein

scholarly journals Distribution of insulin receptor sites among liver plasma membrane subfractions

FEBS Letters ◽  
1973 ◽  
Vol 34 (2) ◽  
pp. 259-262 ◽  
Author(s):  
W.H. Evans ◽  
J.J.M. Bergeron ◽  
I.I. Geschwind
Keyword(s):  
Plasma Membrane ◽  
Insulin Receptor ◽  
Liver Plasma Membrane ◽  
Receptor Sites
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