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Biomedical subjects

M M Appleman

Publications and source records attributed to M M Appleman.

At least 19 recordsLinked to original sources
PubMed1986

Insulin stimulation of cyclic AMP phosphodiesterase is independent from the G-protein pathways involved in adenylate cyclase regulation.

The intact rat adipocyte was used to investigate the possibility of common intermediates in the insulin stimulation of cyclic AMP phosphodiesterase and the beta-adrenergic/adenosine regulation of adenylate cyclase. A five minute incubation of the isolated adipocytes with insulin produced a 50-100% increase in the phosphodiesterase activity found in the particulate fraction of homogenates. The insulin stimulation was not impaired by the presence of either agonist or antagonists of the inhibitory adenosine receptor which acts on adenylate cyclase. Phosphodiesterase activation by insulin was also observable above the level of stimulation produced by the beta-adrenergic agent isoproterenol and forskolin. The validity of the enzyme activity measurements was supported by measurements of the hormonal actions on cyclic AMP levels within the cells. Possible crossover between the adenylate cyclase and phosphodiesterase regulation systems at a post-receptor site was investigated using adipocytes exposed to bacterial toxins specific for the modification of guanine nucleotide binding proteins. Both cholera toxin, which irreversibly activates Gs and pertussis toxin which inactivates Gi caused some stimulation of the phosphodiesterase activity and suppressed activation by isoproterenol, but neither toxin prevented the insulin stimulation of cyclic AMP phosphodiesterase. These results suggest, while common components may participate in the beta-adrenergic stimulation of both adenylate cyclase and phosphodiesterase, the mechanism of insulin activation of the phosphodiesterase does not involve the components of adenylate cyclase regulation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Extensive but reversible depletion of ATP via adenylate cyclase in rat adipocytes.

In adipocytes, adenylate cyclase is positively regulated by beta-adrenergic agents and negatively regulated by adenosine. Incubation of adipocytes with adenosine deaminase relieves the inhibition of adenylate cyclase by destroying the adenosine that the cells release into the medium. When adipocytes are incubated with adenosine deaminase and the beta-adrenergic agent isoproterenol, most of their ATP is converted to AMP in 5 min. Either isoproterenol or adenosine deaminase alone has little or no effect. In the additional presence of the phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724) cAMP accumulates instead of AMP. Under these conditions, cAMP represents 40-50% of the total intracellular adenine nucleotides, and ATP only 5%. N6-(L-2-phenylisopropyl)adenosine, a deaminase-resistant adenosine agonist, prevents beta-adrenergic stimulation. 8-(p-Sulfophenyl)theophylline and 3-isobutyl-1-methylxanthine are both adenosine antagonists that can replace the deaminase in permitting beta-adrenergic stimulation of adenylate cyclase, but only the latter also inhibits the phosphodiesterase and causes accumulation of cAMP. When the ATP-depleted adipocytes are washed with fresh medium, the nucleoside triphosphate level can be restored within 5 min. The ATP-restored adipocytes can respond rapidly to a second dose of isoproterenol and adenosine antagonist. These findings point out the important role of adenosine in controlling adenylate cyclase activity and the possible involvement of adenylate cyclase in the control of energy flow in rat adipocytes.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1982

Insulin-dependent and insulin-independent low Km cyclic AMP phosphodiesterase from rat adipose tissue.

Chromatographic analysis of a soluble extract of rat adipose tissue on DEAE-Sephacel resolves four distinct peaks of 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) activity. Kinetic investigation indicates that two of these fractions have a high affinity for cyclic AMP and show negative cooperative kinetic behavior at high substrate concentration. They differ in the degree of inhibition by cyclic GMP and in their response to insulin. If rat epididymal fat pads are incubated with insulin prior to homogenization, only one of the low Km cyclic AMP phosphodiesterase forms is stimulated.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Purification, characterization and production of rabbit antibodies to rat liver particulate, high-affinity, cyclic AMP phosphodiesterase.

The cyclic nucleotide phosphodiesterase (EC 3.4.16) activities of a rat liver particulate fraction were analyzed after solubilization by detergent or by freeze-thawing. Analysis of the two extracts by DEAE-cellulose chromatography revealed that they contain different complements of phosphodiesterase activities. The detergent-solubilized extract contained a cyclic GMP phosphodiesterase, a low affinity cyclic nucleotide phosphodiesterase whose hydrolysis of cyclic AMP was activated by cyclic GMP and a high affinity cyclic AMP phosphodiesterase. The freeze-thaw extract contained a cyclic GMP phosphodiesterase and two high affinity cyclic AMP phosphodiesterase, but no low affinity cyclic nucleotide phosphodiesterase. The cyclic AMP phosphodiesterase activities from the freeze-thaw extract and from the detergent extract all had negatively cooperative kinetics. One of the cyclic AMP phosphodiesterases from the freeze-thaw extract (form A) was insensitive to inhibition by cyclic GMP; the other freeze-thaw solubilized cyclic AMP phosphodiesterase (form B) and the detergent-solubilized cyclic AMP phosphodiesterase were strongly inhibited by cyclic GMP. The B enzyme appeared to be converted into the A enzyme when the particulate fraction was stored for prolonged periods at -20 degrees C. The B form was purified extensively, using DEAE-cellulose, a guanine-Sepharose column and gel filtration. The enzyme retained its negatively cooperative kinetics and high affinity for both cyclic AMP and cyclic GMP throughout the purification, although catalytic activity was always much greater for cyclic AMP. Rabbit antiserum was raised against the purified B enzyme and tested via a precipitin reaction against other forms of phosphodiesterase. The antiserum cross-reacted with the A enzyme and the detergent-solubilized cyclic AMP phosphodiesterase from rat liver. It did not react with the calmodulin-activated cyclic GMP phosphodiesterase of rat brain, the soluble low affinity cyclic nucleotide phosphodiesterase of rat liver or a commercial phosphodiesterase preparation from bovine heart. These results suggest a possible interrelationship between the high affinity cyclic nucleotide phosphodiesterase of rat liver.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Biochemical characterization of postsynaptically localized cyclic nucleotide phosphodiesterase.

This study demonstrates the postsynaptic localization of one of the isozymes of cyclic nucleotide phosphodiesterase (PDE) activity at asymmetrical, axospinous terminals in the rat corpus striatum and neocortex. Characterization of this enzymatic activity demonstrates that the PDE form surviving aldehyde fixation for electron cytochemistry can be considered to preferentially hydrolyze cyclic 3'5'-guanosine monophosphate, and it requires calcium and a heat-stable calcium-dependent regulator protein (CDR) for full hydrolytic activity. Ion exchange chromatographic analysis of extracts of corresponding unfixed brain regions demonstrates that only one enzyme activity peak exhibits similar aldehyde resistance and calcium and regulator protein activatibility.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1978

Cyclic AMP phosphodiesterase in human lymphocytes and lymphoblasts.

Cyclic nucleotide phosphodiesterase activities were examined in lymphocytes from 12 transformed human B cell lines, two T cell lines, six patients with lymphocytic leukemia, and 10 normal donors. A consistent difference bwtween cells from the normal and leukemic state was observed. The cyclic AMP phosphodiesterase activity from normal lymphocytes is inhibited greater than 80% by muM cyclic GMP while this concentration of nucleotide has little or no effect on the enzyme from transformed lymphocytic cell lines or from lymphocytic cells of leukemia patients. The reported lack of cyclic GMP phosphodiesterase in human lymphocytes from several sources is confirmed. The apparent absence of a cyclic GMP degradation mechanism and of cyclic GMP control of cyclic AMP hydrolysis may be related to defective lymphocyte growth control.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1976

Cyclic nucleotide phosphodiesterases in neoplastic and nonneoplastic human mammary tissues.

Cyclic nucleotide phosphodiesterase activity was studied in 33 malignant neoplastic, 2 benign neoplastic, and 18 nonneoplastic human mammary tissues. Enzyme activity, using both cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monosphosphate as substrates, was measured in whole homogenates over a concentration range of 1 to 100 muM. Specific activity was calculated at substrate concentrations of 1 muM (low KM enzyme activity) and 100 muM (high KM activity). Diethylaminoethyl cellulose chromatography was used to separate the different enzyme species. The malignant neoplastic tissues had higher levels of both low-KM cyclic adenosine 3':5'-monophosphate and low-KM cyclic guanosine 3':5'-monophosphate phosphodiesterases. Further, the mean value of the ratio of low-km cyclic adenosine 3':5'-monophosphate to low-KM cyclic guanosine 3':5'-monophosphate activity was higher for the cancer tissues than for the nonneoplastic tissues. Diethylaminoethyl cellulose chromatography indicated the presence of three enzymes in both neoplastic and nonneoplastic mammary tissue. The kinetic as well as regulatory properties of the separated enzymes indicated that they are distinct enzyme activities. The phosphodiesterase properties were similar for neoplastic and nonneoplastic tissues and resembled those described previously in many other mammalian tissues. While both neoplastic and nonneoplastic tissues had detectable levels of the protein activator for phosphodiesterase, the cancer tissues appeared to have a higher level.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

The role of cyclic GMP in the regulation of cyclic AMP hydrolysis.

A rat-heart cyclic nucleotide phosphodiesterase has been chromatographically separated from related enzymes and its kinetic properties have been studied. The enzyme can hydrolyze both cyclic AMP and cyclic GMP and has about the same maximum velocity and apparent KM (greater than 10-5 M) for the two nucleotides. Kinetic plots indicate positive cooperative behavior for both substrates. Cyclic GMP at low concentrations is a potent activator of cyclic AMP hydrolysis and this activation, as well as the cooperativity, can be abolished by treatment with solvents or sulfydryl reagents under conditions which do not destroy the catalytic function. A kinetic model for this enzyme is porposed and the physiologic role is discussed.

Allosteric Regulation↗