The regional and cellular distribution of gua- Considerable evidence now suggests that guanosine 3',5'-cyclic monophosphate (cGMP) may play a role in neuronal function (1-3) and that many effects of this nucleotide are mediated by activation of cGMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) (4). However, the distribution of this enzyme in neuronal tissue has not been closely examined. Cerebellum is the only neuronal tissue that has been reported to contain a high concentration of cGMP-dependent protein kinase (5-7) and of a substrate for this enzyme (8). In addition, the activity of cGMP-dependent protein kinase has been reported to be reduced in the cerebellum of several strains of mutant mice deficient in Purkinje cells (9). In the present study, we have determined the concentration of this enzyme in various regions of brain. We have also studied the cellular localization of cGMP-dependent protein kinase and a substrate (8) animals of the appropriate strain. PCD animals were killed at 3 months of age, nervous and weaver at 2-2.5 months, and staggerer at 21-28 days. Frozen cerebella of X-irradiated rats were a generous gift of Hermes Yeh.Preparation of Tissue Extracts for Photoaffinity Labeling. Adult cats, weighing 4-4.5 kg, were anesthesized with pentobarbital at 30 mg/kg intravenously and ventilated through a tracheotomy tube after administration of Flaxedil at 3 mg/kg. Various locations in the brain were biopsied and each specimen was immediately immersed in 10 ml of ice-cold "homogenization buffer" containing 10 mM Hepes (pH 7.0), 5 mM 2-mercaptoethanol, 1 mM EDTA, 30,M phenylmethylsulfonyl fluoride, 2% (vol/vol) ethanol, and 0.25-M sucrose. Mice were killed by cervical dislocation and individual cerebella were placed on ice in homogenization buffer. Brain microvessels were prepared from whole rabbit brain by the technique of Nathanson and Glaser (18) and suspended in homogenization buffer. All subsequent steps were carried out at 4°C. The tissue was homogenized by hand in 4-6 vol of homogenization buffer, and the homogenate was centrifuged for 45 min at 150,000 X g. The resulting cytosol was used immediately for photoaffinity labeling or pretreated by a 10-min incubation at 30°C with 50 ,ug of beef heart phosphodiesterase (Boehringer Mannheim) Abbreviations: cGMP, guanosine 3',5'-cyclic monophosphate; cAMP, adenosine 3',5'-cyclic monophosphate; 8-N3-cIMP, 8-azidoinosine 3',5'-cyclic monophosphate; PCD, Purkinje cell degeneration mouse strain; R-I and R-II, regulatory subunits of the type I and type II cyclic AMP-dependent protein kinases, respectively; Dal, dalton; 23-kDal G-substrate, 23,000-dalton substrate for cyclic GMP-dependent protein kinase. 5537The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" intaccordance with 18 U. S. C. §1734 solely to indicate this fact.
Nuclear extracts prepared from the livers of rats treated with or without 8-bromo-cAMP were tested for their ability to bind to various fragments from the flanking region of the gene encoding phosphoenolpyruvate carboxykinase (GTP) [GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] known to contain the element that confers transcriptional regulation by cAMP. Using the nitrocellulose-filtration method, concentration-dependent, apparently saturable binding was seen that is both specific and cAMP dependent. Analysis of various fragments pinpointed the active binding region to positions within the -67 to -111 region, which coincides with the functional regulatory element as shown by recent transfection studies. Formation of an apparently single complex between a synthetic oligomer containing the region from -67 to -111 of the phosphoenolpyruvate carboxykinase gene and a factor in nuclear extracts from cAMP-treated rat liver was visualized by the gel-retardation method. Complex formation is both concentration and cAMP dependent and can be prevented by excess specific but not nonspecific competitor DNA. The congruity of the results with the two different methods suggests that the factor we have detected has properties consistent with a possible role as mediator of the transcriptional control exerted by cAMP in eukaryotic cells.Many recent reports have demonstrated that cAMP controls the expression of a large number of genes at the level of transcription (1-6). In contrast to the case with steroid hormones, however, little is known about the link between formation of the cyclic nucleotide and its ultimate impact on the transcriptional process. Work from this laboratory has shown (7) that direct introduction of the catalytic subunit of the cAMP-dependent protein kinase into rat hepatoma cells triggers increased expression of the gene for tyrosine aminotransferase. In addition, administration of the kinase inhibitor protein blocked the effects of added 8-bromo-cAMP (8-Br-cAMP) but not those of adrenal steroids on the expression of this gene. Reisine et al. (8) These results strongly implicate the catalytic subunit of the cAMP-dependent protein kinase as the mediator of cAMP action on gene expression, but they do not provide any indication as to the next step in the process. The type II regulatory subunit of the cAMP-dependent kinase (RI,) has been reported to exhibit topoisomerase activity (10). Whether or not the R subunit is involved (10, 11), it seems reasonable that some protein factor exists, that can link kinase activation with transcription of cAMP-responsive genes. By analogy with other eukaryotic transcriptional regulatory systems (12), such a trans-acting factor should show high-affinity binding to a domain(s) most likely localized in the immediate 5'-flanking region of these genes. Indeed, at least four groups have reported that such domains exist in the 5'-flanking region of five different genes, each of which can confer responsiveness to cAMP on foreign genes in eukaryotic cells t...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.