scholarly journals Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities of rat mammary tissue

1984 ◽  
Vol 219 (3) ◽  
pp. 801-809 ◽  
Author(s):  
I Mullaney ◽  
R A Clegg
Keyword(s):  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
Deae Cellulose ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Mammary Tissue ◽  
Phosphodiesterase Activity ◽  
High Affinity ◽  
Low Concentrations ◽  
Maximal Activation

Cyclic nucleotide phosphodiesterase activity in mammary tissue from rats in midlactation was resolved by DEAE-cellulose chromatography into three functionally distinct fractions: a Ca2+/calmodulin-stimulated cyclic GMP phosphodiesterase, a cyclic GMP-stimulated low-affinity cyclic nucleotide phosphodiesterase, and a high-affinity cyclic AMP-specific phosphodiesterase. The absolute activities and relative proportions of high- and low-affinity enzymes resemble those found, for example, in liver, as distinct from those in excitable tissues. Three functional characteristics are described which are peculiar to mammary-tissue phosphodiesterases. Firstly, the concentration of free Ca2+ required to achieve half-maximal activation of the Ca2+/calmodulin-stimulated phosphodiesterase is somewhat higher than for the analogous enzyme in other tissues; secondly, the activity of this enzyme towards cyclic AMP relative to that towards cyclic GMP is unusually low, and thirdly, the low-affinity cyclic nucleotide phosphodiesterase is inhibited by low concentrations of free Ca2+.

scholarly journals Cyclic nucleotide phosphodiesterase activities in Neurospora crassa

1982 ◽  
Vol 203 (3) ◽  
pp. 611-616 ◽  
Author(s):  
M T Téllez-I ñón ◽  
G C Glikin ◽  
H N Torres
Keyword(s):  
Cyclic Amp ◽  
Neurospora Crassa ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Sucrose Density Gradient Centrifugation ◽  
Molecular Weights

Cyclic nucleotide phosphodiesterase activities in soluble Neurospora crassa mycelial extracts were resolved into two peaks, phosphodiesterase I and II, by chromatography on DEAE-cellulose columns. Phosphodiesterase I hydrolysed cyclic AMP and cyclic GMP equally well. Phosphodiesterase II was active on cyclic GMP but scarcely active on cyclic AMP. Phosphodiesterase I was resolved by gel filtration and sucrose-density-gradient centrifugation into three peaks having molecular weights of about 57 000, 125 000 and 225 000. This suggests that this enzyme activity has at least three aggregation forms, tentatively defined as monomeric, dimeric and tetrameric. Similarly, phosphodiesterase II was resolved into two forms, having molecular weights of about 170 000 and 320 000. Evidence on the interconversion between phosphodiesterase I forms was obtained.

scholarly journals Cyclic nucleotide phosphodiesterase of rat pancreatic islets. Effects of Ca2+, calmodulin and trifluoperazine

1981 ◽  
Vol 197 (2) ◽  
pp. 459-464 ◽  
Author(s):  
M C Sugden ◽  
S J Ashcroft
Keyword(s):  
Cyclic Amp ◽  
Pancreatic Islets ◽  
Islets Of Langerhans ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
Specific Inhibitor ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Phosphodiesterase Activity ◽  
Rat Pancreatic Islets ◽  
Rat Islets Of Langerhans

Cyclic nucleotide phosphodiesterase activity towards cyclic AMP and cyclic GMP was studied in extracts of rat islets of Langerhans. Biphasic Eadie plots [Eadie (1942) J. Biol. Chem. 146, 85-93] were obtained with either substrate suggesting the presence of both ‘high’- and ‘low’-Km components. The apparent Km values were 6.2 +/- 0.5 (n = 8) microM and 103.4 +/- 13.5 (6) microM for cyclic AMP and 3.6 +/- 0.3 (12) microM and 61.4 +/- 7.5 (13) microM for cyclic GMP. With cyclic AMP as substrate, phosphodeisterase activity was increased by calmodulin and Ca2+ and decreased by trifluoperazine, a specific inhibitor of calmodulin. With cyclic GMP as substrate, phosphodiesterase activity was decreased by omission of Ca2+ or addition of trifluoperazine. Addition of exogenous calmodulin had no effect on activity. The data suggest that Ca2+ may influence the islet content of cyclic AMP and cyclic GMP via effects on calmodulin-dependent cyclic nucleotide phosphodiesterase(s).

scholarly journals Isolation of a 5.3-S calmodulin-deficient 3′:5′-cyclic nucleotide phosphodiesterase from cardiac muscle

1982 ◽  
Vol 205 (2) ◽  
pp. 427-435 ◽  
Author(s):  
A Mohindru ◽  
A R Rhoads
Keyword(s):  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Guanosine Monophosphate ◽  
Cyclic Nucleotide Phosphodiesterase

1. In the presence of Ca2+, a 5.3-S 3′:5′-cyclic nucleotide phosphodiesterase (EC 3.1.4.17) from bovine ventricle was isolated and purified by (NH4)2SO4 precipitation and DEAE-cellulose and Affi-Gel Blue chromatography. The enzyme activity was enriched 800-fold by these procedures. 2. Sucrose-density gradient centrifugation, gel filtration and non-denaturing polyacrylamide-gel electrophoresis resolved a single enzyme species with an Mr of 89 000. 3. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the purified enzyme demonstrated a prominent protein band at Mr 59000 and a minor band of Mr 28000. Calmodulin was not detected. 4. The hydrolysis of micromolar concentrations of 3′:5′-cyclic guanosine monophosphate (cyclic GMP) but not 3′:5′-cyclic adenosine monophosphate (cyclic AMP) was stimulated by calmodulin. 5. Anomalous biphasic kinetics plots were observed for both the catalysis of cyclic AMP and cyclic GMP hydrolysis. Kinetic plots became linear in the presence of calmodulin. 6. After several months of storage at −20 degrees C, the 5.3-S enzyme was transformed into a 6.2-S cyclic GMP-specific enzyme and a 4.4-S non-specific form.

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.

scholarly journals Isoelectric-focusing patterns of cyclic nucleotide phosphodiesterase from rat heart

1981 ◽  
Vol 199 (1) ◽  
pp. 113-119 ◽  
Author(s):  
G Némoz ◽  
A F Prigent ◽  
J F Pageaux ◽  
H Pacheco
Keyword(s):  
Cyclic Amp ◽  
Isoelectric Focusing ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
Rat Heart ◽  
Specific Form ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Phosphodiesterase Activity ◽  
New Information ◽  
Overlapping Peaks

1. Isoelectric focusing on a flat gel bed of the rat heart cytosolic fraction resolved cyclic nucleotide phosphodiesterase activity into several forms, characterized by their substrate specificity, kinetic constants and dependence towards Ca2+ and calmodulin. A peak of pI 4.9 displayed 20 times more affinity for cyclic GMP than for cyclic AMP and was markedly inhibited by EGTA. A less substrate-specific form, only slightly sensitive to EGTA inhibition, focused at pH 5.45. Several overlapping peaks detected between pH 5.55 and pH6 specifically hydrolysed cyclic AMP, with non-Michaelian kinetics; these peaks were insensitive to Ca2+ chelation. 2. Isoelectric focusing did not dissociate enzyme-calmodulin complexes, as none of the resulting peaks was activatable by calmodulin plus Ca2+. 3. Some new information on rat cardiac phosphodiesterase is obtained with this technique, which is convenient for routine analytical studies of phosphodiesterase, as well as for preparative purposes.

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 The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

1980 ◽  
Vol 186 (2) ◽  
pp. 491-498 ◽  
Author(s):  
Patricia Methven ◽  
Marius Lemon ◽  
Kanti Bhoola
Keyword(s):  
Cyclic Amp ◽  
Guinea Pig ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
Metal Ion ◽  
Gel Filtration ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Physiological Control ◽  
Cyclic Amp Phosphodiesterase ◽  
Stimulus Secretion Coupling

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (Km 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (Km 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg2+. Mn2+ (3mm) was as effective as Mg2+ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca2+ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg2+ was 3mm. At concentrations of Mg2+ between 0.1 and 1mm, 1mm-Ca2+ was activatory, and at concentrations of Mg2+ below 0.1mm, 1mm-Ca2+ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.

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