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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A mutation in the gene IRA1 (formerly called PPD1) was originally characterized as a deficiency of a phosphoprotein phosphatase. The IRA1 gene has been cloned and sequenced. A large open reading frame (8,817 base pairs) which can encode a protein of 2,938 amino acids was found. Northern (RNA) blot analysis detected a message of about 10 kilobases, and nuclease S1 protection demonstrated mRNA start points at 97 and 98 base pairs upstream from the putative initiator ATG codon. Disruption of the IRA1 gene resulted in sensitivity to nitrogen starvation and heat shock. Diploids homozygous for the disrupted IRA1 gene were deficient in sporulation. Disruption of the IRA1 gene suppressed the lethality of the cdc25 mutation but did not suppress the lethality of either the ras1 ras2 or the cyr1 mutations. Deficiency of the phosphoprotein phosphatase was not reproducible in the disruption mutant of the IRA1 gene. Moreover, the ira1 mutant showed an increased level of cyclic AMP. Our results suggest that the IRA1 protein inhibits the function of the RAS proteins in a fashion antagonistic to the function of the CDC25 protein in the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.
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PMID:IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. 254 Apr 26

Acetyl-CoA carboxylase from liver exhibits a linear inverse relationship between the ratio of enzymic activities at 0 and 2 mM citrate and the extent of phosphorylation by its kinase, and this citrate activity ratio method was used to examine the effect of nutritional conditions on the phosphorylation state of the enzyme. This method showed that the calculated phosphorylation state, being the extent of phosphorylation at sites accessible to carboxylase kinase, was highest in the livers of starved rats, lower in those fed normally, and lower still in starved rats which had been refed for 48 h on a fat-free diet. The actual values were 0.44, 0.26, and 0 mol of P/subunit, respectively, provided that liver samples were frozen rapidly to liquid nitrogen temperatures and extracted with stopping buffers at temperatures well below freezing. Normal homogenization with stopping buffers (containing inhibitors for protein kinases and phosphatases) resulted in much higher calculated phosphorylation states. The effect of nutritional conditions on the phosphorylation state as estimated reported above was confirmed by purifying the carboxylase from livers of rats, measuring the amount of phosphate which could be incorporated by carboxylase kinase, and comparing this with the phosphorylation state calculated from the citrate activity ratio method or the specific activity. Furthermore, treatment with protein phosphatase of carboxylase from starved rats resulted in the largest increase in specific activity, that from the starved/refed rats in the least. Finally, the effects of hyperglycemia on carboxylase and phosphorylase characteristics in the livers of intact rats were ascertained by taking liver samples and preparing crude extracts by the rapid freezing method described above. Hyperglycemia caused a rapid increase in the activity of the carboxylase and a rapid decrease in its putative phosphorylation state as measured by the citrate activity ratio method. Phosphorylase was also dephosphorylated, as indicated by a decrease in phosphorylase a activity. We conclude that the citrate activity ratio method is a valid test for the phosphorylation state of acetyl-CoA carboxylase in crude extracts of tissue.
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PMID:Phosphorylation state of acetyl-coenzyme A carboxylase. II. Variation with nutritional condition. 287 34

Calmodulin and its target enzymes are important regulators of numerous cellular processes, including reversible protein phosphorylation. The calmodulin-dependent protein phosphatase (calcineurin) has been suggested to play roles in activation of T cells and in the mating response of yeast. Recently, studies have shown it to be the target of immunosuppressant drugs such as cyclosporin and FK-506. In this study, we have cloned the gene for the catalytic subunit of calcineurin, CnA, from the yeast Schizosaccharomyces pombe. The gene (named ppb1+) has been mapped to chromosome II by analysis of the hybridization of a genomic DNA probe to an ordered library. The gene produces a single mRNA species of 2.5 kilobases, which varies during the cell cycle in exponentially growing cells. In addition, expression of ppb1+ mRA is induced by nitrogen starvation, a condition that favors mating in S. pombe. The ppb1+ gene promoter contains a cis-acting element for the ste11 transcription factor, and we have shown that induction of the ppb1+ mRNA during nitrogen starvation is dependent on the ste11 gene product. Together with earlier studies showing that disruption of the ppb1+ gene in S. pombe results in sterility (Yoshida, T., Toda, T., and Yanagida, M. (1994) J. Cell Sci., 107, 1725-1735), our studies suggest that the ppb1+ gene plays a role in the gene expression cascade that is essential for mating and sporulation in S. pombe.
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PMID:Regulation of calcineurin gene expression in Schizosaccharomyces pombe. Dependence on the ste11 transcription factor. 755 98

Organ transplantation offers new life to patients who suffer from incurable disease. The problem of rejection of the transplanted organ has been overcome with the use of potent immunosuppressive drugs. These drugs, although they allow graft tolerance and graft survival, also are associated with complications such as osteoporosis. Although factors such as nutrition, gonadal status, and ambulatory status are important, the use of immunosuppressive drugs appears to be the main factor in the development of osteoporosis. The drugs that are responsible for this bone loss are glucocorticoids and the calcineurin phosphatase inhibitors, cyclosporine and tacrolimus. The incidence of bone disease depends, in part, on which organ is transplanted. Kidney transplant recipients appear to be less susceptible to the development of overt osteoporosis than do heart or liver transplant recipients. The most critical period of bone loss in organ recipients appears to be within the first 6 months, with the most dramatic reduction occurring within the first 3 months following transplantation. Trabecular (cancellous) bone of the spine appears to be most at risk, with vertebral fractures occurring most commonly. Transplant recipients should be evaluated by bone mineral densitometry and measurement of vitamin D metabolites, blood urea nitrogen, creatinine, calcium, and phosphate. Markers of bone turnover may help in assessing the rate of remodeling. Gonadal function should be ascertained by measurement of serum testosterone (males) or estradiol (females) levels. Therapy should be directed toward prevention of bone loss as well as helping to restore what already may have been lost. Administration of calcium and vitamin D and sex hormone replacement, if indicated, should be considered.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Organ transplantation and osteoporosis. 761 20

Potassium transport was measured in equine red blood cells, using 86Rb+ influx as a convenient assay. A significant component of volume- and pH-sensitive K(+)-Cl- cotransport to the overall K+ flux was observed in all blood samples studied, although fluxes were variable between animals, and within individuals when measured at intervals over a period of weeks. The aryloxyacetic acid [(dihydroindenyl)oxy]alkanoic acid (DIOA), at a final concentration of 100 microM, inhibited most (> 95%) of the Cl(-)-dependent K+ flux, and DIOA sensitivity was therefore used to define the activity of the K(+)-Cl- cotransport. K(+)-Cl- cotransport was also sensitive to protein phosphatase inhibition with calyculin A or okadaic acid, with inhibition constants of 9 +/- 1 nM for calyculin and about 100 nM for okadaic acid. Peak fluxes were observed at an external pH of 6.7-7.0, with inhibition at higher and lower values. Volume-sensitive K+ fluxes assayed in autologous plasma, controlled for osmolaity, pH and potassium concentration, were significantly lower (28 +/- 8% of control values, n = 6) than those measured in saline. This inhibition was mimicked by the culture medium RPMI, but disappeared following dialysis of the plasma. Phosphate (5.6 mM) inhibited volume-sensitive K+ fluxes by 48 +/- 2%, n = 3; no significant effect was observed by increasing external magnesium concentrations to 0.5 or 2 mM. Thus, inhibition by RPMI, but not that by plasma, may be due to phosphate. Finally, volume- and pH-sensitive K+ fluxes were sensitive to oxygen tension and were abolished reversibly by equilibrating solutions with nitrogen, as opposed to air. Use of solutions equilibrated with different values of Po2 may account for some of the variability in equine red blood cell KCl fluxes. The importance of these observations to equine red blood cell homeostasis and haemodynamics is discussed.
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PMID:Modulation of K(+)-Cl- cotransport in equine red blood cells. 787 67

alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase b (Ki = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that, in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several alpha- and beta-anhydroglucoheptonic acid derivatives and 1-deoxy-1-thio-beta-D-glucose analogues have been synthesized and tested in a series of crystallographic and kinetic binding studies with glycogen phosphorylase. The structural results of the bound enzyme-ligand complexes have been analyzed, together with the resulting affinities, in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. This work has shown the following: (i) Similar affinities may be obtained through different sets of interactions. Specifically, in the case of the alpha- and beta-glucose-C-amides, similar Ki's (0.37 and 0.44 mM, respectively) are obtained with the alpha-anomer through interactions from the ligand via water molecules to the protein and with the beta-anomer through direct interaction from the ligand to the protein. Thus, hydrogen bonds through water can contribute binding energy similar to that of hydrogen bonds directly to the protein. (ii) Attempts to improve the inhibition by additional groups did not always lead to the expected result. The addition of nonpolar groups to the alpha-carboxamide resulted in a change in conformation of the pyranose ring from a chair to a skew boat and the consequent loss of favorable hydrogen bonds and increase in the Ki. (iii) The addition of polar groups to the alpha-carboxamide led to compounds with the chair conformation, and in the examples studied, it appears that hydration by a water molecule may provide sufficient stabilization to retain the chair conformation. (iv) The best inhibitor was N-methyl-beta-glucose-C-carboxamide (Ki = 0.16 mM), which showed a 46-fold improvement in Ki from the parent beta-D-glucose. The decrease in Ki may be accounted for by a single hydrogen bond from the amide nitrogen to a main-chain carbonyl oxygen, an increase in entropy through displacement of a water molecule, and favorable van der Waals contacts between the methyl substituent and nonpolar protein residues.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Design of inhibitors of glycogen phosphorylase: a study of alpha- and beta-C-glucosides and 1-thio-beta-D-glucose compounds. 818 Feb 1

The effects of oxygen tension (PO2) upon the K influx pathways of equine red cells have been studied using 86Rb+ as congener for K. Equilibration of cells in 100% nitrogen led to a low and Cl-independent K flux. Change to an atmosphere of 100% air led to a rapid sixfold increase in K flux. The oxygen-activated flux was entirely Cl dependent and was maintained for up to 3 h. Oxygenation-evoked activation was dependent upon PO2 over the physiological range with little effect up to 70% saturation of haemoglobin with oxygen but significant effects between 70 and 100%. K flux at low PO2 was unaffected by acidification to pH 7 or by hypotonic cell swelling. By contrast, at high PO2 both manipulations caused a substantial increase in Cl-dependent K flux. N-Ethylmaleimide (NEM; 1 mM) caused a progressive activation of KCl cotransport in cells held under nitrogen. The protein phosphatase inhibitor, calyculin A (100 nM), applied during NEM-evoked activation caused a "clamping" of K influx at that level. This "clamped" activity was unaffected by subsequent oxygenation. We conclude that oxygenation exerts a primary control over cotransport activity and that acidification and cell swelling are secondary modulators. It appears that oxygenation-evoked activation of the Cl-dependent K flux involves a serine/threonine phosphorylation event. Regulating the PO2 of the solution before and during experiments is important in controlling the activity of the KCl cotransporter and cell volume.
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PMID:The effects of oxygenation upon the Cl-dependent K flux pathway in equine red cells. 866 3

Starvation for nitrogen in the absence of a fermentable carbon source causes diploid Saccharomyces cerevisiae cells to leave vegetative growth, enter meiosis, and sporulare; the former nutritional condition also induces expression of the YVH1 gene that encodes a protein phosphatase. This correlation prompted us to determine whether the Yvh1p phosphatase was a participant in the network that controls the onset of meiosis and sporulation. We found that expression of the IME2 gene, encoding a protein kinase homologue required for meiosis- and sporulation-specific gene expression, is decreased in a yvh1 disrupted strain. We also observed a decrease, albeit a smaller one, in the expression of IME1 which encodes an activator protein required for IME2 expression. Under identical experimental conditions, expression of the MCKI and IME4 genes (which promote sporulation but do not require Ime1p for expression) was not affected. These results demonstrate the specificity of the yvh1 disruption phenotype. They suggest that decreased steady-state levels of IME1 and IME2 mRNA were not merely the result of non-specific adverse affects on nucleic acid metabolism caused by the yvh1 disruption. Sporulation of a homozygous yvh1 disruption mutant was delayed and less efficient overall compared to an isogenic wild-type strain, a result which correlates with decreased IME1 and IME2 gene expression. We also observed that expression of the PTP2 tyrosine phosphatase gene (a negative regulator of the osmosensing MAP kinase cascade), but not the PTP1 gene (also encoding a tyrosine phosphatase) was induced by nitrogen-starvation. Although disruption of PTP2 alone did not demonstrably affect sporulation or IME2 gene expression, sporulation was decreased more in a yvh1, ptp2 double mutant than in a yvh1 single mutant; it was nearly abolished in the double mutant. These data suggest that the YVH1 and PTP2 encoded phosphatases likely participate in the control network regulating meiosis and sporulation. Expression of YVH1 and PTP2 was not affected by nitrogen source quality (asparagine compared to proline) suggesting that nitrogen starvation-induced YVH1 and PTP2 expression and sensitivity to nitrogen catabolite repression are on two different branches of the nitrogen regulatory network.
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PMID:The S. cerevisiae nitrogen starvation-induced Yvh1p and Ptp2p phosphatases play a role in control of sporulation. 889 80

A Chinese hamster ovary cell line resistant to okadaic acid (OA), OAR2-3 has a mutation of the protein phosphatase (PP) 2A alpha gene and expresses a multi-drug resistance (MDR) phenotype. In the present work, we isolated two additional OA-resistant variants, also showing MDR with a cross-resistance profile similar to that of OAR2-3, and with increased and decreased expressions of the P-glycoprotein (Pgp) and DNA topoisomerase (topo) II protein, respectively. Unlike OAR2-3, however, they had no mutation in the same region of the PP2A alpha gene. Except for OA-resistance in OAR2-3, the MDR was found to decrease in the absence of OA, and this decrease was again associated with changes in topo II- and Pgp-expressions. Thus, we conclude that 1) OA regulates the expressions of Pgp and topo II positively and negatively, respectively, resulting in reversible expression of MDR irrespective of genetic changes and 2) in OAR2-3, the mutation in the PP2A alpha gene confers stable resistance to OA. The MDR was also linked with collateral sensitivity to some drugs, like cisplatin and nitrogen mustard.
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PMID:Unstable expression of the multi-drug-resistant phenotype in Chinese hamster ovary cells resistant to okadaic acid. 912 89

The hydrolysis of p-nitrophenyl phosphate (pNPP) catalyzed by calcineurin has been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)nonbridge], at the position of bond cleavage in the bridging oxygen atom [18(V/K)bridge], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The isotope effects increased in magnitude upon moving from the pH optimum of 7.0 to 8.5; 18(V/K)bridge increased from 1.0072 to 1.0115, and 15(V/K) from 1.0006 to 1.0014. The value for 18(V/K)nonbridge is 0.9942 at pH 8.5. These data are consistent with P-O bond cleavage being partially rate-limiting at the pH optimum and more so at the higher pH. The 18(V/K)nonbridge isotope effect indicates that the dianion is the substrate for catalysis, and a dissociative transition state is operative for phosphoryl transfer. Increasing the concentration of the activating metal ion Mn2+ at pH 7.0 from 1 mM to 5 mM increases the magnitude of the isotope effects by an amount similar to that observed with the shift in pH from 7.0 to 8.5, indicative of a change in the commitment factor in the kinetic mechanism so as to make the chemical step more rate-limiting.
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PMID:Isotope effect studies on the calcineurin phosphoryl-transfer reaction: transition state structure and effect of calmodulin and Mn2+. 925 16


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