scholarly journals Characterization of a rat liver cyclic GMP-activated phosphodiesterase by chromatography on hexyl-agarose. Inhibition of phosphodiesterase activity by hexyl-agarose

1981 ◽  
Vol 199 (2) ◽  
pp. 441-446 ◽  
Author(s):  
D Couchie ◽  
C Erneux ◽  
J E Dumont
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Cyclic Gmp ◽  
Hydrophobic Interactions ◽  
Phosphodiesterase Activity ◽  
Hydrophobic Matrix ◽  
The Matrix ◽  
Two Peaks ◽  
Assay Conditions

Chromatography on hexyl-agarose resolved a partially purified cyclic GMP-activated phosphodiesterase from rat liver into two peaks of activity: the first was eluted with 0.5 M-KCl and was cyclic AMP-specific. The second was tightly bound to hexyl-agarose and was not eluted with KCl (0--2.0 M), which enhanced the hydrophobic interactions of this form with the matrix. It was eluted with 0.5 M-Tris, hydrolysed cyclic AMP and cyclic GMP and was specifically activated by cyclic GMP. The cyclic GMP-activated phosphodiesterase was immobilized on hexyl-agarose. Enzyme activity, quantitatively bound to hexyl-agarose, was not released from the hydrophobic matrix in the presence of cyclic AMP or cyclic GMP, under our assay conditions. The immobilized form of the enzyme retained catalytic activity, was inhibited by 0.1 mM-cyclic AMP and was activated by micromolar concentrations of cyclic GMP to a lesser extent (7-fold) than the control, i.e. the enzyme mixed with unsubstituted agarose (15-fold). When the enzyme was immobilized, inhibition of cyclic AMP phosphodiesterase activity was only observed in the presence of cyclic GMP (at 3 microM); in its absence, activity remained unchanged. The kinetic behaviour of the immobilized enzyme is consistent with the hypothesis of a binding site distinct from the hydrolytic and activating sites.

scholarly journals Function of calmodulin in postsynaptic densities. I. Presence of a calmodulin-activatable cyclic nucleotide phosphodiesterase activity.

1981 ◽  
Vol 89 (3) ◽  
pp. 433-439 ◽  
Author(s):  
D J Grab ◽  
R K Carlin ◽  
P Siekevitz
Keyword(s):  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
Postsynaptic Density ◽  
Maximal Activity ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Phosphodiesterase Activity ◽  
Native Structure ◽  
Fraction I ◽  
Two Peaks

The postsynaptic density (PSD) fraction from canine cerebra cortex was found to contain an endogenous cyclic nucleotide-phosphodiesterase activity that was independent on Mn2+ and/or Mg2+ but not on Ca2+. Maximal activity was obtained at 1 micrometer Mn2+. This cyclic nucleotide phosphodiesterase activity was not decreased upon removal of the calmodulin from the PSD fraction, nor was it increased by the addition of calmodulin to a postsynaptic density fraction deficient in calmodulin. The enzymatic activity could be extracted by sonication, with the soluble enzyme having properties similar to those found in the native structure. Two peaks of cyclic nucleotide phosphodiesterase activities could be obtained after S-300 Sephacryl column chromatography of this soluble fraction: fraction I (excluded peak) and fraction II (215,000 mol wt). The fraction I activity preferred cyclic AMP over cyclic GMP and was not activated by calmodulin. The fraction II activity has an approximately fourfold lower Km for cyclic GMP over cyclic AMP. This fraction II activity was activatable by calmodulin, which increased the Vmax and decreased the Km in the case of both cyclic nucleotides. We conclude that two activities are present in the PSD, one activatable, and one not activatable, by calmodulin.

Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae

1987 ◽  
Vol 7 (10) ◽  
pp. 3629-3636
Author(s):  
J Nikawa ◽  
P Sass ◽  
M Wigler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cyclic Amp ◽  
Copy Number ◽  
Growth Arrest ◽  
Yeast Cells ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
High Affinity ◽  
Cyclic Amp Phosphodiesterase

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.

scholarly journals Characterization of a calmodulin-dependent high-affinity cyclic AMP and cyclic GMP phosphodiesterase from male mouse germ cells

1984 ◽  
Vol 217 (3) ◽  
pp. 693-700 ◽  
Author(s):  
R Geremia ◽  
P Rossi ◽  
D Mocini ◽  
R Pezzotti ◽  
M Conti
Keyword(s):  
Cyclic Amp ◽  
Germ Cells ◽  
Cyclic Gmp ◽  
Salt Concentration ◽  
Male Mouse ◽  
Competitive Inhibition ◽  
Sedimentation Coefficient ◽  
Phosphodiesterase Activity ◽  
Thermal Stabilities ◽  
High Affinity

Two cyclic nucleotide phosphodiesterase activities were separated by ion-exchange chromatography of cytosol from male mouse germ cells. A form eluted at low salt concentration showed high affinity (Km congruent to 2 microM) and low affinity (Km congruent to 20 microM) for cyclic AMP, and high affinity (Km congruent to 3.5 microM) for cyclic GMP. A second form, eluted at high salt concentration, showed high affinity (Km congruent to 5 microM) for cyclic AMP and was similar to a phosphodiesterase activity described in rat germ cells. The present study was performed to characterize the first form, which represents most of the phosphodiesterase activity in mouse germ cells. The enzyme was sensitive to Ca2+ and calmodulin stimulation, which increased its activity 3-4-fold. Calmodulin stimulation depended on direct interaction of the activator with the enzyme, as indicated by the reversible changes in the chromatographic elution pattern in the presence of Ca2+, as well as by the increase in the sedimentation coefficient in the presence of calmodulin. Reciprocal inhibition kinetics between cyclic AMP and cyclic GMP for the calmodulin-dependent form demonstrated a non-competitive inhibition between the two substrates, suggesting the presence of separate catalytic sites. This is in agreement with kinetic parameters and different thermal stabilities of cyclic AMP- and cyclic GMP-hydrolysing activities. Furthermore, the relevant change in s value, depending on the absence or presence of Ca2+ and calmodulin, suggested that the enzyme is composed of subunits, which aggregate in the presence of the activator. A model for catalytic site composition and reciprocal interaction is also proposed.

scholarly journals Specific antibodies and the selective inhibitor ICI 118233 demonstrate that the hormonally stimulated ‘dense-vesicle’ and peripheral-plasma-membrane cyclic AMP phosphodiesterases display distinct tissue distributions in the rat

1987 ◽  
Vol 248 (3) ◽  
pp. 897-901 ◽  
Author(s):  
N J Pyne ◽  
N Anderson ◽  
B E Lavan ◽  
G Milligan ◽  
H G Nimmo ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Plasma Membrane ◽  
Cyclic Amp ◽  
Rat Liver ◽  
White Adipose Tissue ◽  
Tissue Homogenate ◽  
Phosphodiesterase Activity ◽  
Cyclic Amp Phosphodiesterase ◽  
Peripheral Plasma ◽  
Membrane Enzyme

Polyclonal-antibody preparations DV1 and PM1, raised against purified preparations of rat liver insulin-stimulated ‘dense-vesicle’ and peripheral-plasma-membrane cyclic AMP phosphodiesterases, were used to analyse rat liver homogenates by Western-blotting techniques. The antibody DV1 identified only the 63 kDa native subunit of the ‘dense-vesicle’ enzyme, and the antibody PM1 only the 52 kDa subunit of the plasma-membrane enzyme. These antibodies also detected the subunits of these two enzymes in homogenates of kidney, heart and white adipose tissue from rat. Quantitative immunoblotting demonstrated that the amount of these enzymes (by wt.) varied in these different tissues, as did the expression of these two enzymes, relative to each other, by a factor of as much as 7-fold. The ratio of the dense-vesicle enzyme to the peripheral-plasma-membrane enzyme was lowest in liver and kidney and highest in heart and white adipose tissue. ICI 118233 was shown to inhibit selectively the ‘dense-vesicle’ cyclic AMP phosphodiesterase in liver. It did this in a competitive fashion, with a Ki value of 3.5 microM. Inhibition of tissue-homogenate cyclic AMP phosphodiesterase activity by ICI 118233 was used as an index of the contribution to activity by the ‘dense-vesicle’ enzyme. By this method, a tissue distribution of the ‘dense-vesicle’ enzyme was obtained which was similar to that found by using the immunoblotting technique. The differential expression of isoenzymes of cyclic AMP phosphodiesterase activity in various tissues might reflect a functional adaptation, and may provide the basis for the different physiological actions of compounds which act as selective inhibitors.

scholarly journals Evidence for a messenger function of cyclic GMP during phosphodiesterase induction in Dictyostelium discoideum

1982 ◽  
Vol 152 (1) ◽  
pp. 232-238
Author(s):  
P J van Haastert ◽  
F J Pasveer ◽  
R C van der Meer ◽  
P R van der Heijden ◽  
H van Walsum ◽  
...  
Keyword(s):  
Computer Simulation ◽  
Folic Acid ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Close Correlation ◽  
Phosphodiesterase Activity ◽  
Transient Elevation ◽  
Lines Of Evidence ◽  
Stimulation Of

Chemotactic stimulation of vegetative or aggregative Dictyostelium discoideum cells induced a transient elevation of cyclic GMP levels. The addition of chemoattractants to postvegetative cells by pulsing induced phosphodiesterase activity. The following lines of evidence suggest a messenger function for cyclic GMP in the induction of phosphodiesterase: (i) Folic acid and cyclic AMP increased cyclic GMP levels and induced phosphodiesterase activity. (ii) Cyclic AMP induced both cyclic GMP accumulation and phosphodiesterase activity by binding to a rate receptor. (iii) The effects of chemical modification of cyclic AMP or folic acid on cyclic GMP accumulation and phosphodiesterase induction were closely correlated. (iv) A close correlation existed between the increase of cyclic GMP levels and the amount of phosphodiesterase induced, independent of the type of chemoattractant by which this cyclic GMP accumulation was produced. (v) Computer simulation of cyclic GMP binding to intracellular cyclic GMP-binding proteins indicates that half-maximal occupation by cyclic GMP required the same chemoattractant concentration as did half-maximal phosphodiesterase induction.

scholarly journals Characterization of the glucose 6-phosphate dehydrogenase activity in rat liver mitochondria

1976 ◽  
Vol 159 (3) ◽  
pp. 683-687 ◽  
Author(s):  
M Grunwald ◽  
H Z Hill
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
The Other ◽  
Rat Liver Mitochondria ◽  
Kinetic Behaviour ◽  
Two Peaks ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose 6-phosphate dehydrogenase activity in rat liver mitochondria can be released by detergent. The released activity is separated by chromatography into two peaks. One peak has the kinetic behaviour and mobility similar to the soluble sex-linked enzyme, whereas the other peak is similar to the microsomal hexose 6-phosphate dehydrogenase. There is no evidence for the existence of a new glucose 6-phosphate dehydrogenase activity in rat liver mitochondria.

scholarly journals Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver

1986 ◽  
Vol 234 (2) ◽  
pp. 325-334 ◽  
Author(s):  
N J Pyne ◽  
M E Cooper ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Cyclic Gmp ◽  
Integral Membrane Protein ◽  
Soluble Enzyme ◽  
Cyclic Amp Phosphodiesterase ◽  
Apparent Homogeneity ◽  
Low Concentrations ◽  
Considerable Homology ◽  
Hydrolysis Of

Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a ‘particulate enzyme’ found as an integral membrane protein associated with the plasma membrane, and a ‘soluble’ enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms.

scholarly journals Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells

1983 ◽  
Vol 213 (2) ◽  
pp. 379-386 ◽  
Author(s):  
G J Strewler ◽  
M A Danello ◽  
V C Manganiello ◽  
M Vaughan
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Cyclic Gmp ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Hepatoma Cells ◽  
Partial Hydrolysis ◽  
Kinetic Behaviour ◽  
Lysosomal Proteinase ◽  
Hydrolysis Of

We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.

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