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.

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.

Influence of dopamine on cyclic nucleotide enzymes in bovine retinal membrane fractions

1993 ◽  
Vol 10 (6) ◽  
pp. 991-996 ◽  
Author(s):  
Ari Sitaramayya ◽  
Lorraine Lombardi ◽  
Alexander Margulis
Keyword(s):  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
D2 Receptors ◽  
D1 Receptors ◽  
Metabolizing Enzymes ◽  
D2 Agonist ◽  
Hydrolysis Of ◽  
Cyclic Amp Synthesis

AbstractDopamine is a major neurotransmitter and neuromodulator in vertebrate retina. Although its pharmacological and physiological actions are well understood, the biochemical mechanisms of its signal transduction are less clear. Acting via D1 receptors, dopamine was shown to increase cyclic AMP levels in intact retina and to activate adenylate cyclase in retinal homogenates. The action via activation of D2 receptors is controversial: it was reported to decrease cyclic AMP levels in intact retina but inhibition of cyclase could not be demonstrated in retinal homogenates; also it was reported to activate rod outer segment cyclic GMP phosphodiesterase in vitro but did not decrease cyclic GMP levels in aspartate-treated retinas. We made an attempt to fractionate bovine retinal membranes and to investigate the effects of dopamine, via Dl and D2 receptors, on the synthesis and hydrolysis of cyclic AMP and cyclic GMP. Activation of cyclic AMP synthesis was noted in all fractions, but no effects were evident on cyclic nucleotide hydrolysis or cyclic GMP synthesis in any fraction. Also, D2 agonist did not inhibit cyclic AMP synthesis. These observations suggest that D2 receptors may not be directly coupled to cyclic nucleotide metabolizing enzymes in bovine retina.

Rod outer segment phosphodiesterase: Factors affecting the hydrolysis of cyclic-AMP and cyclic-GMP

1974 ◽  
Vol 18 (6) ◽  
pp. 509-515 ◽  
Author(s):  
G. Chader ◽  
R. Fletcher ◽  
M. Johnson ◽  
R. Bensinger
Keyword(s):  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Outer Segment ◽  
Factors Affecting ◽  
Rod Outer Segment ◽  
Hydrolysis Of

scholarly journals A study on sensing and adaptation in Dictyostelium discoideum: guanosine 3',5'-phosphate accumulation and light-scattering responses.

1983 ◽  
Vol 96 (6) ◽  
pp. 1566-1570 ◽  
Author(s):  
B Wurster ◽  
U Butz
Keyword(s):  
Light Scattering ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Rapid Decrease ◽  
Initial Increase ◽  
Steady State Concentration ◽  
Fast Reactions ◽  
State Concentration ◽  
Hydrolysis Of

Cells of Dictyostelium discoideum respond to extracellular cyclic AMP with marked changes in intracellular cyclic GMP levels and light scattering. In this work, defined temporal increases in cyclic AMP were produced by the continuous addition of cyclic AMP to agitated suspensions of cells; concomitant hydrolysis of cyclic AMP by the cells subsequently established a constant, steady state concentration. The cells responded to the initial increase in extracellular cyclic AMP with a rapid increase in the intracellular cyclic GMP concentration and a rapid decrease in light scattering. At cyclic AMP input rates of 0.5-5 nM X s-1, the fast reactions of cyclic GMP and light scattering had already relaxed while the cyclic AMP concentration in the cell suspension was still increasing. The cells responded to constant concentrations of cyclic AMP with constant elevated cyclic GMP concentrations and constant decreased levels of light scattering. Our results are consistent with the existence of two types of perception systems, one of which adapts to constant stimuli and one of which does not adapt.

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 Identification and properties of renal mineralocorticoid receptors in relation to glucocorticoid binders in rat liver and kidney

1976 ◽  
Vol 154 (3) ◽  
pp. 567-575 ◽  
Author(s):  
M K Agarwal
Keyword(s):  
Molecular Weight ◽  
Blood Serum ◽  
High Molecular Weight ◽  
Rat Liver ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Competitive Binding ◽  
Mineralocorticoid Receptors ◽  
Liver And Kidney

The binding of the natural mineralocorticoid aldosterone and the glucocorticoid corticosterone to macromolecules in rat liver and kidney cytoplasmic fractions was compared by various chromatographic procedures. Equilibration of kidney cytosol with 10nM-aldosterone, either alone or in the presence of a competing steroid, was ideal for ionexchange chromatography of DEAE-cellulose DE-52, and revealed the presence of four sorts of binding components. One of these, eluted in the 0.001M-phosphate pre-wash, and another, less abundant, forming a peak at 0.006M-phosphate, did not bind corticosterone at equimolar concentrations, and appear to constitute the mineralocorticoid-specific ‘MR‘ receptor in rat kidney. They could not be detected in the liver. Radioactivity eluted in the 0.02 and 0.06M-phosphate regions on DEAE-cellulose DE-52 appears to be due to [3H]aldosterone binding to glucocorticoid-specific ‘GR’ receptors and to transcortin respectively, since labelling was greater with corticosterone even at 10 nM than with the mineralocorticoid at 100nM and since [14C]corticosterone bound to blood serum transcortin was always co-chromatographed in the 0.06M-phosphate region. These two components appear to be identical with those in the liver and could be labelled maximally only by 100nM-corticosterone. The separation between specific mineralo- and glucocorticoid-binding species was less clear when chromatography was attempted on DEAE-Sephadex A-50 columns, possibly because of disaggregation into subunits in the presence of the high KC1 concentrations required for elution. Competitive binding followed by filtration through Sephadex G-200 gel indicated that cellular MR binders, unlike GR receptors, exist mostly as high-molecular-weight aggregates, although both appear to exhibit a comparable monomeric molecular weight of approx. 67000.

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+.

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