Characterization of cyclic nucleotide phosphodiesterases with multiple separation techniques

Inwegen, R. G. Van; Pledger, W. J.; Strada, Samuel J.; Thompson, W. J.

doi:10.1016/0003-9861(76)90562-2

Cited by 31 publications

(12 citation statements)

References 39 publications

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“…Because we wished to compare total enzyme activity under different physiological conditions the subcellular fractions obtained by differential centrifugation were not further purified since this procedure usually leads to large and variable losses in activity (Singer, Dunn & Appleman, 1978). Furthermore, isolation procedures may change enzyme specificity (Goldberg & Haddox, 1977) and cause markedly different fractionation profiles (Van Inwegen, Pledger, Strada & Thompson, 1976). The kinetic values for the high-and low-ÄTm PDE in these semi-purified preparations were undoubtedly influenced by the presence of the other enzyme.…”

Section: Discussionmentioning

confidence: 99%

Regulation of cyclic nucleotide phosphodiesterase activity in myometrium from pregnant and spayed rhesus monkeys

Beatty¹,

Bocek²,

Herrington³

1979

Reproduction

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The activities of myometrial cyclic nucleotide phosphodiesterases (PDEs) and the sensitivity of these enzymes to the effector molecules, cGMP and cAMP, were determined in the 100,000 g supernatant of homogenates from pregnant and spayed rhesus monkeys. The specific activities (per mg nitrogen) of the myometrial cyclic nucleotide PDEs in the supernatant from spayed monkeys were higher than those from pregnant monkeys at all substrate levels studied. However, when calculated on the basis of the DNA content of the myometrium, which was 8 times higher in the spayed than in the pregnant animals, the specific activities were lower in the tissue from spayed animals. At substrate levels of 2 . 5 micron-cAMP, low levels of cGMP (0 . 1-1 . 0 micron) caused the same percentage increase in cGMP-PDE activity in both tissues. At high substrate levels of 100 micron-cAMP, 1 micron-cGMP inhibited only the cAMP-PDE from spayed monkeys, and the enzyme from spayed monkeys was more effectively inhibited by 10 and 40 micron-cGMP than was the enzyme from pregnant animals. The cGMP-PDE activity was inhibited by cAMP (1 . 0-50 . 0 micron), and the percentage inhibition with increasing levels of cAMP appeared to be similar in the two series. The levels of cGMP and cAMP that modify the rate of hydrolysis of the other nucleotide in rhesus myometrium seem to be within the physiological range for these compounds in situ. It therefore appears possible that cAMP and cGMP are each involved in regulating the degradation of the other nucleotide in rhesus myometrium.

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Section: Discussionmentioning

confidence: 99%

Regulation of cyclic nucleotide phosphodiesterase activity in myometrium from pregnant and spayed rhesus monkeys

Beatty¹,

Bocek²,

Herrington³

1979

Reproduction

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“…The number and activity of the different forms of PDE observed depend on the method of isolation and on the conditions of storage and assay. Slight alterations in procedure can lead to changes in activity and, therefore, to apparent variations in distribution (Van Inwegen et al, 1976). The concentration of substrate used in the assay of fractions from sucrose density gradients can alter the number of forms detected (Pichard and Cheung, 1976).…”

Section: Discussionmentioning

confidence: 99%

Phosphodiesterase Activities for Cyclic Nucleotides in Nerve Endings from the Bovine Posterior Pituitary Gland

Vale

Baker

Hope

1984

Journal of Neurochemistry

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The cyclic nucleotide phosphodiesterase (PDE) activities were studied in a nerve ending fraction from bovine neural lobes. Most of the activity was particulate and unaffected by calcium. Lineweaver-Burk plots for this fraction showed negative cooperativity with apparent Km values for cyclic AMP of 11 microM and for cyclic GMP of 4 microM. The soluble activities for both cyclic nucleotides were activated by calcium and inhibited by calmodulin-binding drugs (trifluoperazine and calmidazolium). The apparent Km values were 50 microM for cyclic AMP and 20 microM for cyclic GMP for the soluble activities. Sucrose density gradients resolved the soluble activities into two peaks. The activity with the higher sedimentation rate (MW 122,000 daltons) hydrolysed both cyclic nucleotides and was calcium-calmodulin-dependent. The other peak (MW 47,000 daltons) had a higher affinity for cyclic AMP than for cyclic GMP and was calcium-independent. Solubilized particulate activities gave two main peaks on the density gradient, both calcium-independent. One was mainly for cyclic AMP (MW 47,000 daltons) and the other mainly for cyclic GMP (MW 133,000 daltons). The function of PDEs in relation to secretion was discussed.

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Section: Methodsmentioning

confidence: 99%

“…This regulation is complex and involves a number of different control mechanisms. Hormones (reviewed Wells & Hardman, 1977; Thompson , growth conditions of cultured cells (Anderson et al, 1973; Russell & Pastan, 1974;Strada & Pledger, 1975;Pledger et al, 1975a,b), endogenous protein activators (Cheung, 1971; Kakiuchi et al, 1973;Watterson et al, 1976;Stevens et al, 1976), and cyclic nucleotides themselves (Beavo et al, 1971;Russell et al, 1972; Mangianello & Vaughn, 1972; Thompson et al, 1973;Russell et al, 1973;Van Inwegen et al, 1977)' have all been shown to affect enzyme activity. Furthermore, most mammalian tissues contain multiple forms of cyclic nucleotide phosphodiesterase with different kinetic and regulatory properties (Thompson & Appleman, 1971a,b;Appleman et al, 1973;Appleman & Terasaki, 1975; Wells & Hardman, 1977; Strada & Thompson, 1978).…”

mentioning

confidence: 99%

Characterization of soluble uterine cyclic nucleotide phosphodiesterase

Gardner¹,

Thompson²,

Strada³

et al. 1978

Biochemistry

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Soluble cyclic nucleotide phosphodiesterase of rat uterus displays distinct structural and regulatory properties. Like phosphodiesterases from many mammalian sources the soluble uterine enzyme system exhibits nonlinear Lineweaver--Burk kinetics with cyclic adenosine 3':5'-monophosphate (cAMP) as substrate (apparent Kms congruent to 3 and 20 micron) and linear kinetics with cyclic guanosine 3':5'-monophosphate (cGMP) as substrate (apparent Km congruent to 3 micron). Unlike most other mammalian phosphodiesterases, however, numerous separation procedures reveal only a single form of uterine phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP. A single form of the enzyme is observed upon sucrose gradient centrifugation (7.9 S), agarose gel filtration, and DEAE-cellulose chromatography at either pH 8.0 OR 6.0. Heat denaturation (50 degrees C) of soluble uterine phosphodiesterase causes the loss of both cAMP and cGMP hydrolytic activities at the same rate. Isoelectric focusing reveals major (pI = 5.2) and minor forms (pI = 5.8) of phosphodiesterase which both catalyze the hydrolysis of the two cyclic nucleotide substrates. In vivo administration of estradiol produces identical decreases in the activities of cAMP and cGMP phosphodiesterase. These results raise the possibility that the uterus contains a single form of soluble phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP.

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Characterization of cyclic nucleotide phosphodiesterases with multiple separation techniques

Cited by 31 publications

References 39 publications

Regulation of cyclic nucleotide phosphodiesterase activity in myometrium from pregnant and spayed rhesus monkeys

Regulation of cyclic nucleotide phosphodiesterase activity in myometrium from pregnant and spayed rhesus monkeys

Phosphodiesterase Activities for Cyclic Nucleotides in Nerve Endings from the Bovine Posterior Pituitary Gland

Characterization of soluble uterine cyclic nucleotide phosphodiesterase

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