Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been proposed elsewhere [Meeker, R.B. & Harden, T. K. (1982) Mol. Pharmacol. 22, 310-319] that muscarinic cholinergic receptor-mediated attenuation of cAMP accumulation occurs through activation of phosphodiesterase in 1321N1 human astrocytoma cells. Pertussis toxin, which ADP-ribosylates the guanine nucleotide regulatory protein involved in receptor-mediated inhibition of adenylate cyclase (Ni), has been utilized to further differentiate between the mechanism of cholinergic regulation of cAMP metabolism in 1321N1 cells and the mechanism involving inhibition of adenylate cyclase in other tissues. Muscarinic receptor-mediated regulation of cAMP accumulation in NG108-15 neuroblastoma-glioma cells occurs through inhibition of adenylate cyclase. Pretreatment of these cells with pertussis toxin completely blocked the capacity of carbachol to attenuate cAMP accumulation. In contrast, concentrations of pertussis toxin two to three orders of magnitude higher than those effective in NG108-15 cells had no effect on muscarinic receptor-mediated attentuation of cAMP accumulation in 1321N1 cells. In addition, no effect of pertussis toxin was observed either on the control rate or the carbachol-stimulated rate of cAMP degradation measured directly in intact 1321N1 cells. A 41,000 Mr protein previously proposed to be the alpha subunit of Ni was labeled during incubation of a plasma membrane fraction from 1321N1 cells with [32P]NAD and pertussis toxin. Pertussis toxin is apparently active in 1321N1 cells, since this protein substrate was not labeled in plasma membrane preparations from cells previously incubated with toxin. Functional activity of Ni was demonstrated by the observation that guanosine 5'-[gamma-thio]triphosphate- and GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity occurred in cell-free preparations from 1321N1 cells. The inhibitory activity of these guanine nucleotides was lost in membrane preparations from pertussis toxin-treated cells. The data suggest that adenylate cyclase is not involved in cholinergic action in 1321N1 cells and, furthermore, Ni is not involved in muscarinic receptor-mediated activation of phosphodiesterase in these cells. Thus, pertussis toxin can be used to differentiate between two mechanisms of cholinergic regulation of cAMP metabolism.
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PMID:Pertussis toxin differentiates between two mechanisms of attenuation of cyclic AMP accumulation by muscarinic cholinergic receptors. 609 Nov 3

Transducin, a guanine nucleotide regulatory protein found in the bovine retinal rod outer segment, mediates the signal coupling between rhodopsin and a cyclic GMP phosphodiesterase. Previous studies have demonstrated that photolyzed rhodopsin catalyzed the exchange of GTP for GDP bound to transducin. The transducin-GTP complex, in turn, activates the phosphodiesterase. Purified transducin (T) has been resolved by omega-amino octylagarose chromatography into two functional subunits: T alpha (Mr approximately 39,000) and T beta gamma (Mr approximately 36,000 and approximately 10,000). The guanine nucleotide binding site is on the T alpha subunit. Neither the T alpha nor the T beta gamma subunit showed significant GTPase activity, Gpp(NH)p-GDP exchange, and ability to bind to rhodopsin when assayed in the presence of reconstituted membranes containing photolyzed rhodopsin. However, all the above activities were restored if the two subunits were recombined. Analysis of the reconstituted GTPase activity as a function of the T alpha subunit concentration revealed a linear relationship. On the other hand, GTPase activity rapidly saturated at T beta gamma concentration much lower than the T alpha concentration, indicating that the two subunits were dissociated during GTP hydrolysis. These findings strongly suggest that the T beta gamma subunit is an activator of the GTPase activity. Its mode of action is to enable the T alpha subunit to interact with rhodopsin effectively.
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PMID:Characterization of transducin from bovine retinal rod outer segments. I. Separation and reconstitution of the subunits. 613 9

The first stage of amplification in the cyclic GMP cascade in bovine retinal rod is carried out by transducin, a guanine nucleotide regulatory protein consisting of two functional subunits, T alpha (Mr approximately 39,000) and T beta gamma (Mr approximately 36,000 and approximately 10,000). Limited trypsin digestion of the T beta gamma subunit converted the beta polypeptide to two stable fragments (Mr approximately 26,000 and approximately 14,000). The GTPase and Gpp(NH)p binding activities were not significantly affected by the cleavage. Trypsin digestion of the T alpha subunit initially removed a small segment from the polypeptide terminus and resulted in the formation of a single 38,000-Da fragment. When this fragment was recombined with the intact T beta gamma subunit in the presence of membranes containing photolyzed rhodopsin, the reconstituted transducin exhibited greatly reduced GTPase and Gpp(NH)p binding activities. The loss in activities was due to the inability of the cleaved T alpha to bind to the photolyzed rhodopsin. Prolonged digestion converted the 38,000-Da fragment to a transient 32,000-Da fragment and then to two stable 23,000-Da and 12,000-Da fragments. The cleavage of the 32,000-Da fragment, however, can be blocked by bound Gpp(NH)p. The 32,000-Da fragment contains the Gpp(NH)p binding site and retains the ability to activate phosphodiesterase. These results indicate that the guanine nucleotide binding and rhodopsin binding sites are located in topologically distinct regions of the T alpha subunit and proved evidence that a large conformational transition of the molecule occurs upon the conversion of the bound GDP to GTP.
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PMID:Characterization of transducin from bovine retinal rod outer segments. II. Evidence for distinct binding sites and conformational changes revealed by limited proteolysis with trypsin. 613 10

The mechanism of hCG-induced desensitization of the cAMP system was studied in Percoll-purified mouse Leydig cells. Pretreatment of Leydig cells with hCG resulted in a time- and dose-dependent decrease in the capacity of hCG-induced cAMP formation. Maximal desensitization (approximately 90%) was induced by only partial prior stimulation. Desensitization, however, was not observed without a prior increase in cAMP or testosterone production. Pretreatment of the cells with N6,O2'-dibutyryl cAMP (DBcAMP) also induced a dose- and time-dependent densensitization. cAMP was only effective in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX). Cholera toxin desensitized the hormone-induced cAMP response as drastically as hCG. Cholera toxin was unable to reverse the refractory state induced by one of the agonists. hCG-induced desensitization was not associated with a loss in [125I]hCG binding or an increase in maximal phosphodiesterase activity, and appeared not to be dependent on protein synthesis. Membranes from hCG, cholera toxin of DBcAMP-desensitized cells showed an impaired adenylate cyclase activity in response to hCG, hCG plus beta-gamma-imidoguanosine 5'-triphosphate (GPPNP) and NaF. In conclusion, hCG-induced desensitization of the adenylate cyclase system in mouse Leydig cells can be mimicked by cholera toxin, DBcAMP and cAMP, indicating a cAMP-mediated process. The site of the 'lesion' has to be localized to the guanine nucleotide regulatory protein-adenylate cyclase complex rather than to its uncoupling from the hormone receptor.
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PMID:Desensitization of the cAMP system in mouse Leydig cells by hCG, cholera toxin, dibutyryl cAMP and cAMP: localization of the 'lesion' to the guanine nucleotide regulatory protein-adenylate cyclase complex. 619 38

Adenylate cyclase was solubilized from washed particulate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9 and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio-Gel A. By both procedures the enzyme was resolved into two components, one insensitive to guanyl 5'-yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca2+ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca2+ and calmodulin. The data support the possibility that two independent forms of adenylate cyclase exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca2+-calmodulin. Fractions containing the guanylnucleotide-sensitive activity were found to contain a factor that inhibited basal and Ca2+-stimulated adenylate cyclase in the Ca2+-sensitive fraction. The inhibitor was inactivated by heating at 60 degrees C and by incubation with trypsin. Inhibition was not time-dependent, and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and therefore it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co-sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.
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PMID:Properties of detergent-dispersed adenylate cyclase from cerebral cortex. Presence of an inhibitor protein. 626 48

Challenge of intact hepatocytes with amylin only succeeded in elevating intracellular cyclic AMP levels and activating phosphorylase in the presence of the cAMP phosphodiesterase inhibitor IBMX. Both amylin and CGRP similarly activated adenylate cyclase, around 5-fold, although approximately 400-fold higher levels of amylin were required to elicit half maximal activation. Amylin activated adenylate cyclase though apparently simple Michaelien kinetics whereas CGRP elicited activation by kinetics indicative of apparent negative co-operativity. Use of the antagonist CGPP(8-37) showed that both CGRP and amylin activated hepatocyte adenylate cyclase through a common receptor by a mnemonical mechanism where it was proposed that the receptor co-existed in interconvertible high and low affinity states for CGRP. It is suggested that this model may serve as a paradigm for G-protein linked receptors in general. Amylin failed to both stimulate inositol phospholipid metabolism in hepatocytes and to elicit the desensitization of glucagon-stimulated adenylate cyclase. Amylin did, however, elicit the phosphorylation of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes and prevented the action of insulin in reducing the level of phosphorylation of this G-protein.
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PMID:Regulation of hepatocyte adenylate cyclase by amylin and CGRP: a single receptor displaying apparent negative cooperatively towards CGRP and simple saturation kinetics for amylin, a requirement for phosphodiesterase inhibition to observe elevated hepatocyte cyclic AMP levels and the phosphorylation of Gi-2. 792 19

PDE4C is one of four mammalian genes that encode multiple PDE4 cyclic AMP-specific phosphodiesterase isoforms that are inhibited by rolipram. Fluorescent in situ hybridisation localised PDE4C to the p13.1 region of human chromosome 19. Overlapping cosmid clones spanning the human PDE4C gene were identified and characterised. Analysis of this locus indicated that the PDE4C gene spans at least 38 kb, consists of at least 18 exons, and contains the marker D19S212 within an intron. Comparison of published human PDE4C cDNA sequences with those of the genomic DNA identified four alternatively spliced exons and the possibility that the PDE4C locus contains at least three alternative promoters. PDE4C-containing cosmids also contained the genes for the growth regulatory transcription factor, JUND, and the mini guanine nucleotide regulatory protein, RAB3A. The RAB3A gene was shown to consist of 5 exons spanning 7.9 kb, while the JUND gene was found to contain no introns. Analysis of cosmids containing PDE4C, JUND, and RAB3A showed that 27 kb separate JUND and PDE4C, while only 3.7 kb separate PDE4C and RAB3A. The three genes share the same orientation of transcription and are arranged in the order cen- 5'- JUND-PDE4C-RAB3A-3'-tel.
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PMID:Genomic organisation of the human cyclic AMP-specific phosphodiesterase PDE4C gene and its chromosomal localisation to 19p13.1, between RAB3A and JUND. 1057 28


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