scholarly journals Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium

1987 ◽  
Vol 243 (2) ◽  
pp. 525-531 ◽  
Author(s):  
P M Epstein ◽  
D M Andrenyak ◽  
C J Smith ◽  
A J Pappano
Keyword(s):  
Cyclic Amp ◽  
Embryonic Development ◽  
Adenylate Cyclase ◽  
Cyclic Nucleotide ◽  
Sodium Acetate ◽  
Specific Activity ◽  
Ventricular Myocardium ◽  
Phosphodiesterase Activity ◽  
Ontogenetic Changes ◽  
Cyclic Amp Phosphodiesterase

The activities of cyclic AMP phosphodiesterase (3′,5′-cyclic nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.

scholarly journals Calcium-dependent protein modulator of cyclic nucleotide phosphodiesterases from mouse epidermis

1978 ◽  
Vol 176 (3) ◽  
pp. 727-732 ◽  
Author(s):  
A W Murray ◽  
A Rogers
Keyword(s):  
Cyclic Amp ◽  
Cyclic Nucleotide ◽  
Sodium Acetate ◽  
Deae Cellulose ◽  
Mouse Skin ◽  
Bovine Brain ◽  
Phosphodiesterase Activity ◽  
Mouse Epidermis ◽  
Calcium Dependent ◽  
Cyclic Amp Phosphodiesterase

1. A heat-stable modulator protein was partially purified from mouse epidermis. The protein stimulated modulator-depleted cyclic AMP phosphodiesterase from bovine brain in the presence of Ca2+. 2. DEAE-cellulose chromatography of epidermal extracts demonstrated the presence of two main phosphodiesterase activities that hydrolysed both cyclic AMP and cyclic GMP. A minor peak was eluted between 0.1 and 0.3 M-sodium acetate and a major peak was eluted between 0.3 and 0.45 M-sodium acetate. 3. Cyclic AMP phosphodiesterase activity eluted at low salt concentrations was markedly activated by the epidermal modulator protein in the presence of Ca2+. Storage of the enzyme led to a decrease in its sensitivity to the protein modulator. 4. Treatment of mouse skin with the tumour promoter 12-O-tetradecanoylphorbol 13-acetate, which leads to an increase in epidermal cyclic nucleotide phosphodiesterase activity, did not alter the amount of modulator present in soluble epidermal extracts. The tumour promoter decreased the amount of modulator extractable from particulate epidermal preparations with Triton X-100.

scholarly journals Identification and characterization of a Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in a human lymphoblastoid cell line

1987 ◽  
Vol 243 (2) ◽  
pp. 533-539 ◽  
Author(s):  
P M Epstein ◽  
S Moraski ◽  
R Hachisu
Keyword(s):  
Cyclic Amp ◽  
Cell Line ◽  
Cyclic Nucleotide ◽  
Sodium Acetate ◽  
Lymphoblastoid Cell Line ◽  
Human Peripheral Blood ◽  
Affinity Column ◽  
Phosphodiesterase Activity ◽  
Linear Kinetics ◽  
Non Linear

This study examines the pattern and regulatory properties of cyclic nucleotide phosphodiesterases in a human lymphoblastoid B-cell line (RPMI 8392) established from a patient with acute lymphocytic leukaemia. In this cell line, phosphodiesterase activity measured at 0.25 microM-cyclic AMP is approx. 7-fold greater than that in isolated human peripheral-blood lymphocytes, and 16% of the phosphodiesterase activity in RPMI 8392 cells is associated with particulate fractions. Phosphodiesterase activity in crude fractions of this cell line is reproducibly stimulated by about 60-80% by Ca2+-calmodulin. In the presence of 20 nM-calmodulin, half-maximal stimulation occurs at 0.7 microM-Ca2+. The cytosolic phosphodiesterase activity of RPMI 8392 cells is separated into two forms by DEAE-Sephacel chromatography. The first form is eluted at approx. 0.2 M-sodium acetate, catalyses the hydrolysis of both cyclic AMP and cyclic GMP, and is stimulated 3-fold by Ca2+-calmodulin. This form exhibits non-linear kinetics for cyclic AMP in the absence of calmodulin, with extrapolated Km values of 0.8 and 4 microM, and non-linear kinetics in the presence of calmodulin, with extrapolated Km values of 0.5 and 1 microM. The Vmax. values are increased approx. 3-fold by calmodulin. The second form is eluted at approx. 0.6 M-sodium acetate, is specific for cyclic AMP, and insensitive to stimulation by Ca2+-calmodulin. The Ca2+-calmodulin-sensitive phosphodiesterase from the DEAE-Sephacel column can be adsorbed to a calmodulin-Sepharose affinity column and eluted with EGTA. This enzymic activity can also be immunoprecipitated by a monoclonal antibody directed against a calmodulin-bovine heart phosphodiesterase complex. This study documents the existence of Ca2+-calmodulin-sensitive phosphodiesterase in a cultured lymphoblastoid cell line derived from a leukaemic patient.

Cyclic AMP phosphodiesterase activity in mouse pancreatic islets Effects of calmodulin and phospholipids

1986 ◽  
Vol 111 (4) ◽  
pp. 533-538 ◽  
Author(s):  
Kirsten Capito ◽  
Carl Jørgen Hedeskov ◽  
Peter Thams
Keyword(s):  
Enzyme Activity ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pancreatic Islets ◽  
Total Activity ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Phosphodiesterase Activity ◽  
Cyclic Amp Phosphodiesterase ◽  
Mouse Pancreatic Islets

Abstract. The activity of cyclic AMP phosphodiesterase in mouse pancreatic islets was investigated. 85% of the total activity was found in a 27 000 g supernatant fraction. The phosphodiesterase activity in the supernatant fraction, but not in the particulate fraction, was stimulated approximately 20% by Ca2+ (10−5m) and calmodulin (1 μm). The Km (cyclic AMP) of the unstimulated enzyme in the supernatant fraction was 20 μm, and the Vmax was 2 nmol/min × mg protein−1. The possible influence of a range of phospholipids was investigated. PI* and PS (150 μg/ml) inhibited the enzyme 20–30% both in the absence and presence of Ca2+/calmodulin, whereas PE, PC and PA did not affect the enzyme activity. ATP (1 mm) did not affect the particulate or supernatant fraction phosphodiesterase either in the absence or presence of Ca2+/calmodulin or Ca2+/phospholipid. It is concluded that, contrary to islet adenylate cyclase, islet cyclic AMP phosphodiesterase may be regulated by Ca2+/calmodulin.

scholarly journals Regulation by forskolin of cyclic AMP phosphodiesterase and protein kinase C activity in LLC-PK1 cells

1991 ◽  
Vol 279 (1) ◽  
pp. 23-27 ◽  
Author(s):  
R J Anderson ◽  
R Breckon ◽  
D Colston
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Phosphodiesterase Activity ◽  
Cyclic Amp Phosphodiesterase ◽  
Kinase C ◽  
Naturally Occurring ◽  
Protein Kinase C Activity ◽  
Structural Identity

Forskolin, a naturally occurring activator of adenylate cyclase, inhibits total and high-affinity cyclic AMP phosphodiesterase activity in soluble and particulate fractions of cultured LLC-PK1 renal epithelial cells. The naturally occurring forskolin analogue 1,9-dideoxyforskolin, which does not stimulate adenylate cyclase activity, is a more potent inhibitor of cyclic AMP phosphodiesterase activity than forskolin. To clarify the structural feature of the forskolin molecule responsible for inhibition of cyclic AMP phosphodiesterase activity, the effects of two agents which share structural identity with portions of the forskolin ring were tested. The steroid 5-pregnenolone, but not the hexose alpha-D-galactose, inhibited cyclic AMP phosphodiesterase activity in LLC-PK1 cells. Forskolin and 1,9-dideoxyforskolin both stimulate protein kinase C activity in LLC-PK1 cells. The effect of 1,9-dideoxyforskolin in stimulating LLC-PK1 protein kinase C activity can be attenuated by staurosporine. Both 5-pregnenolone and alpha-D-galactose also stimulate protein kinase C activity in LLC-PK1 cells. 5-Pregnenolone and the phorbol ester phorbol 12-myristate 13-acetate cause translocation of protein kinase C from a soluble to a particulate fraction, while both 1,9-dideoxyforskolin and alpha-D-galactose increase protein kinase C activity in both soluble and particulate fractions. Our results demonstrate that forskolin exerts diverse enzymic effects in cultured LLC-PK1 cells.

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.

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