Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.1.1.1 (alcohol dehydrogenase)
 9,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The substrate specificity of the cAMP-dependent protein kinase (cAPK) from Saccharomyces cerevisiae has been investigated using synthetic peptides corresponding to the local phosphorylation site sequence around Ser-230 in the yeast transcriptional activator ADR1. ADR1 is required for the expression of the glucose-repressible alcohol dehydrogenase. Yeast cAPK (encoded by the TPK1 gene) phosphorylated Ser-230 in the synthetic peptide ADR1-217-234, VRKRYLKKLTRRASFSAQ-NH2, with a Km of 5.3 microM compared with 46 microM for LRRASLG (Kemptide). Porcine heart cAPK phosphorylated the ADR1 peptide and Kemptide with the considerable lower Km values of 0.23 and 1.6 microM, respectively. These results indicate that the ADR1 peptide is an excellent substrate for cAPK. Both the yeast and mammalian protein kinases qualitatively shared a number of substrate specificity determinants in common involving residues on the proximal NH2-terminal side and up to the +4 position of the COOH-terminal side of the phosphoacceptor. The mammalian enzyme, however, had a much higher affinity for its substrates than did the yeast enzyme. In addition, the yeast and mammalian enzymes displayed several quantitative differences in their preferences for particular peptide substrates. In particular, the mammalian enzyme strongly preferred substrates with NH2-terminal extensions beyond the -4 position relative to the phosphoacceptor. These results suggest that all eukaryotic cAPKs recognize similar but not identical substrate specificity determinants. They also suggest that the different affinities for substrates that inhere to the individual enzymes could influence their physiological roles.
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PMID:Substrate specificities for yeast and mammalian cAMP-dependent protein kinases are similar but not identical. 191 32

Arginine vasopressin (antidiuretic hormone, ADH) stimulation of sodium transport in high electrical resistance epithelia is accompanied by adenylate cyclase stimulation and cAMP accumulation. The hypothesis of direct phosphorylation of the purified amiloride-blockable epithelial Na+ channel protein by cAMP-dependent protein kinase A after ADH treatment of cultured cells was investigated in this study. Phosphate-depleted A6 cells (a cell line derived from toad kidney) were exposed to 32PO4(3-) in the absence or presence of basolateral ADH (100 milliunits/ml). After 20 min (the time needed for ADH to increase maximally Na+ transport), the Na+ channels were extracted from the cells and purified. At every stage of purification, only one subunit of the Na+ channel, namely, the 315-kDa subunit, was specifically phosphorylated as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography or scintillation counting. In addition, a polyclonal antibody raised against purified epithelial Na+ channel protein was able to immunoprecipitate the phosphorylated channel protein from a detergent-solubilized fraction of vasopressin-treated A6 cells. This same subunit was also specifically phosphorylated in vitro when the purified Na+ channel protein was incubated with gamma-[32P]ATP and the purified catalytic subunit of the cAMP-dependent protein kinase. Thus, only a single component, the 315-kDa subunit, of the Na+ channel protein complex (which is composed of six subunits) can be phosphorylated both in vivo and in vitro. This subunit is selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 2-3 mol of 32P/mol of protein.
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PMID:Phosphorylation of a single subunit of the epithelial Na+ channel protein following vasopressin treatment of A6 cells. 245 53

ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide- and chloride-sensitive 22Na+ uptake and barium-sensitive, voltage-dependent 86Rb+ influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1 mM ATP, and 50 mM KCl, the barium-sensitive 86Rb+ influx increased from 361 +/- 138 to 528 +/- 120 pM/mg prot.30 sec (P less than 0.01). This increase was inhibited completely when heat-stable protein kinase inhibitor (1 microgram/ml) was also present in the vesicle solutions. The stimulation of 86Rb+ uptake by protein kinase required ATP rather than ADP. It also required opening of the vesicles by hypotonic shock, presumably to allow the kinase free access to the cytoplasmic face of the membranes. We conclude that cAMP-dependent protein kinase-mediated phosphorylation of apical membranes from the renal medulla increases the potassium conductance of these membranes. This mechanism may account for the ADH-mediated increase in potassium conductance in the mouse mTALH.
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PMID:Activation of K+ channels in renal medullary vesicles by cAMP-dependent protein kinase. 276 36

Wistar rats were injected just once, intraperitoneally with cortisol (1 microgram/g) or saline at the age of 5 days. The cortisol-treated rats did not differ significantly in the (U/P)osm ratio from the saline-treated controls before 15 days of life. Their response to ADH was distinct but weaker than in the saline controls aged 30 days. This reduced response persisted to 60 days of life. In the collecting tubule fragments, (3H)AVP specific binding was lower in the cortisol-treated rats than in the controls at the age of 20 and 60 days. There was no (3H)AVP specific binding in the proximal convoluted tubules in the cortisol- and saline-injected rats of both ages. The ontogenetic patterns of cAMP specific binding in the papillary cytosolic fraction were different: the early increase in cAMP binding was protracted in the cortisol-treated rats, and no peak appeared at the age of 25 days. Cytosolic protein kinase activity was lower, no peak appeared at 30 days, no activation of protein kinase occurred to the end of weaning in the cortisol-treated rats. The difference between the cortisol and saline groups was abolished by day 30. The interference of cortisol with the ontogenetic changes in AVP binding capacity and cAMP-dependent protein kinase appears to be a plausible cause of the altered development of the response to ADH.
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PMID:Decrease in the response to ADH of the rat kidney as a result of early postnatal treatment with cortisol. 303 81

2-Mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB) was originally introduced as a reversible inhibitor of RNA synthesis, but subsequent findings made this suggestion doubtful. We examined the effect of MPB on active sodium transport, measured as short-circuit current (scc), across the isolated urinary bladder of the toad (Bufo marinus). The drug caused a rapid, dose-dependent inhibition of baseline scc; 25 micrograms/ml MPB reduced it by 70%. Sensitivity to MPB was the same in the presence and absence of metabolizable substrate. The transport stimulation by aldosterone (7 X 10(-8)M) was abolished entirely when MPB was introduced 30 min before the hormone. In bladders incubated with MPB with or without aldosterone, removal of both agents resulted in a rise in scc, which was more rapid in the aldosterone-pretreated hemibladders; a significant difference was observed after 30 min. This suggests that MPB inhibited transport at a site distal to messenger RNA accumulation. The effect of 3 hr of pretreatment with MPB on the response of the bladders to antidiuretic hormone (ADH, 20 mU) and cyclic AMP (cAMP, 10 mM) was then examined. The absolute increment in scc due to these agents was the same as in the absence of MPB, though the baseline was much reduced by the drug. After challenging MPB-pretreated bladders with theophylline (22.5 mM), sodium transport rose continuously for 90 min, in contrast to the small, short-lived rise in the absence of MPB. It is proposed that, in the toad bladder, MPB may: (1) inhibit cAMP-dependent protein kinase, as found by us in other tissues; and (2) counteract the accumulation of a transport inhibitor, possibly calcium or cyclic GMP, in tissues treated with endogenous or exogenous cAMP.
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PMID:2-Mercapto-1-(beta-4-pyridethyl) benzimidazole inhibition of basal and aldosterone-stimulated sodium transport but prolongation of the transient theophylline-induced stimulation in the toad bladder. 619 73

Cell differentiation is essential for normal growth and homeostasis, and drug-induced differentiation of tumor cells into benign or normal cells is an important approach for anticancer chemotherapy. Studies of induction mechanisms for cell differentiation and discovery of differentiation-inducing factors are thus critical components of drug development. The Screening of differentiation-inducing factors, such as purified aldehyde reductase, a xenobiotic metabolite enzyme, that induces differentiation of human acute myeloid leukemia HL60 cells into monocyte/macrophage cells is described. Mechanisms of all-trans-retinoic acid (RA)-induced differentiation are also covered. RA is a potent inducer of HL60 cell differentiation and when used as a sole agent it can induce complete remission in patients with acute promyelocytic leukemia (APL). While one mechanism of the effect of RA involves RA nuclear receptors, retinoylation (a posttranslational modification of proteins by RA) may be a new nongenomic mechanism by which RA acts on cells. An early event in RA-induced differentiation may be retinoylation of RII alpha (regulatory subunits of cAMP-dependent protein kinase), in which RII alpha units are retinoylated and the retinoylated RII alpha is then translocated to the nucleus. Drugs can also be combined with RA in RA-differentiation therapy. Cytodifferation therapy by RA in APL patients exhibits limitations due to the resistance of relapsed patients to further RA treatment. This may occur through the induction of expression of various genes that reduce RA blood concentrations. Treatment with combinations of RA and other agents may be one way to reduce induction of those genes. Good candidates for such agents include cAMP-elevating agents, retinoids, steroids, and fatty acids that synergistically induce differentiation of HL60 cells. Two derivatives of falconensone A, falconensone A p-bromophenylhydrazone, which has a bromophenyl residue, and falconensone A dioxime, which possesses a hydroxy residue, were synthesized to incorporate features of RA and N-[4-hydroxyphenyl] retinamide. Both derivatives have exhibited more potent biological activity than the parent falconensone A in vitro and in vivo.
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PMID:[Induction of cell differentiation and development of new anticancer drugs]. 1218 70

In order to better understand ligand-induced closure in domain enzymes, open unliganded X-ray structures and closed liganded X-ray structures have been studied in five enzymes: adenylate kinase, aspartate aminotransferase, citrate synthase, liver alcohol dehydrogenase, and the catalytic subunit of cAMP-dependent protein kinase. A sequential model of ligand binding and domain closure was used to test the hypothesis that the ligand actively drives closure from an open conformation. The analysis supports the assumption that each enzyme has a dedicated binding domain to which the ligand binds first and a closing domain. In every case, a small number of residues are identified to interact with the ligand to initiate and drive domain closure. In all cases except adenylate kinase, the backbone of residues located in an interdomain-bending region (hinge site) is identified to interact with the ligand to aid in driving closure. In adenylate kinase, the side-chain of a residue located directly adjacent to a bending region drives closure. It is thought that by binding near a hinge site the ligand is able to get within interaction range of residues when the enzyme is in the open conformation. Interdomain bending regions not involved in inducing closure are involved in control, helping to determine the location of the hinge axis. Similarities have been discovered between aspartate aminotransferase and citrate synthase that only come to light in the context of their dynamical behaviour in response to binding their substrate. Similarity also exists between liver alcohol dehydrogenase and cAMP-dependent protein kinase whereby groups on NAD and ATP, respectively, mimic the backbone of a single amino acid residue in a process where a three residue segment located at the terminus of a beta-sheet, moves to form hydrogen bonds with the mimic that resemble those found in a parallel beta-sheet. This interaction helps to drive domain closure in a process that has analogy to protein folding.
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PMID:Identification of specific interactions that drive ligand-induced closure in five enzymes with classic domain movements. 1516 65