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)

1. The effects of the protein phosphatase inhibitors okadaic acid and microcystin LR on the regulation by insulin of pyruvate dehydrogenase and acetyl-CoA carboxylase have been studied in rat epididymal fat-pads and isolated cells. These inhibitors both completely blocked the phosphatase activity (against phosphorylase a) present in extracts of epididymal fat-pads, with half-maximal effects in the nanomolar range. 2. Okadaic acid treatment of pads and cells lowered the activity of acetyl-CoA carboxylase assayed in tissue extracts, both before and after treatment of the extracts with the activator, citrate. Further, okadaic acid treatment abolished the 2-3-fold difference in activity observed between extracts from control and insulin-treated tissues, assayed without prior treatment with citrate. 3. Incubation of pads with [32P]Pi, sufficient to label the intracellular pool of ATP, demonstrated that okadaic acid increased the overall phosphorylation of acetyl-CoA carboxylase on a number of distinct sites, as judged by two-dimensional mapping of tryptic peptides. These included the 'I-peptide' [Brownsey & Denton (1982) Biochem. J. 202, 77-86], the phosphorylation of which may be associated with the stimulation of the activity of the enzyme by insulin, as well as inhibitory phosphorylation sites. 4. Incubation with 1 microM-okadaic acid had no effect on the basal level of active pyruvate dehydrogenase apparent after tissue extraction, but abolished the 2-3-fold increase in this parameter which was elicited by insulin in the absence of okadaic acid. However, okadaic acid treatment did not affect the persistent increase in active pyruvate dehydrogenase levels which was apparent in mitochondria subsequently isolated from insulin-treated pads and re-incubated with an oxidizable substrate. It is concluded that the effects of okadaic acid are exerted through changes in metabolite concentrations rather than some direct action on the signalling pathway whereby insulin stimulates pyruvate dehydrogenase. 5. Microcystin LR did not mimic the effects of okadaic acid on intact cells and pads described above.
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PMID:Effects of protein phosphatase inhibitors on the regulation of insulin-sensitive enzymes within rat epididymal fat-pads and cells. 167 87

For use in screening for disorders of pyruvate metabolism, a sensitive assay method was developed for measuring the rate of decarboxylation of [1-14C]pyruvate during in vitro culture of skin fibroblasts with dichloroacetate (DCA). The rate of decarboxylation of [1-14C]pyruvate by skin fibroblasts from control subjects increased from 59.6 +/- 13.2 to 97.3 +/- 12.0 nmol/h/mg protein during in vitro culture in medium supplemented with 10 mM DCA for 3 days. In contrast, the rate hardly increased in cells from four of 20 patients with congenital lactic acidosis of unknown cause during in vitro culture with DCA. On day 3 of culture, the values for the four patients did not overlap those of control cells and so these four patients could be clearly distinguished from control subjects. Measurements of the original activity and the activity of the pyruvate dehydrogenase (PDH) complex after activation with a broad specificity protein phosphatase and DCA suggested that in three of the patients the aberration was a disorder in the mechanism for activation of PDH, including deficiency of PDH phosphatase or a mutation of PDH itself, whereas that in the fourth patient it might be a disorder of the mitochondrial transport system for pyruvate. Thus, measurement of the rate of decarboxylation of [1-14C]pyruvate by skin fibroblasts cultured in medium supplemented with 10 mM DCA for 3 days is a useful method for screening for disorders of pyruvate metabolism in cultured skin fibroblasts.
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PMID:Detection of pyruvate metabolism disorders by culture of skin fibroblasts with dichloroacetate. 283 11

A potent, heat-stable protein inhibitor of branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase has been identified and purified to near homogeneity from bovine kidney mitochondria (Damuni, Z., Humphreys, J. S., and Reed, L. J., Proc. Natl. Acad. Sci. U.S.A., in press). This protein is a noncompetitive inhibitor of BCKDH phosphatase, with a Ki about 0.13 nM. By contrast, this protein inhibitor did not affect the activity of the cytosolic protein phosphatase-1 and phosphatase-2A or the mitochondrial pyruvate dehydrogenase (PDH) phosphatase at concentrations up to 10 nM. The cytosolic protein phosphatase inhibitor-1 and inhibitor-2 had no effect on the activity of BCKDH phosphatase or PDH phosphatase at concentrations up to 50 and 300 nM respectively. These results, together with previous evidence, demonstrate that BCKDH phosphatase and its inhibitor protein are distinct from the cytosolic protein phosphatase-1 and phosphatase-2A and from protein phosphatase inhibitor-1 and inhibitor-2, respectively.
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PMID:Specificity of the heat-stable protein inhibitor of the branched-chain alpha-keto acid dehydrogenase phosphatase. 300 73

A fluoride-insensitive, non-metal-requiring pyruvate dehydrogenase phosphatase has been purified 730-fold from pigeon liver acetone powder and proven to be a convenient reagent for studies of pyruvate dehydrogenase complex and its activation (phosphorylation) state in brain and other tissues. This phosphatase is a cytoplasmic enzyme (Mr = 80,000), and fits the functional definition of a type 1 phosphoprotein phosphatase. The pigeon liver phosphatase can be used to activate pyruvate dehydrogenase complex in vitro in brain and other crude tissue homogenates. Addition of the cytoplasmic pigeon liver phosphatase to a homogenate from rat or mouse brain frozen in situ activated pyruvate dehydrogenase to levels comparable to that found in ischemic brain. The fluoride insensitivity of this phosphatase was used to develop a convenient technique for stopping the pyruvate dehydrogenase activation state in situ in cultured skin fibroblasts and then fully activating the complex in vitro in 5 min. The use of this phosphatase as a reagent can facilitate the study of pyruvate dehydrogenase activation defects in mammalian tissues including cultured cells in normal and disease states.
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PMID:Pigeon liver phosphoprotein phosphatase: an effective activator of pyruvate dehydrogenase in tissue homogenates. 300 58

Branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase in isolated rat pancreatic islets were shown to be regulated by a phosphorylation/dephosphorylation mechanism. Broad-specificity phosphoprotein phosphatase treatment stimulated and ATP addition inhibited their activities. The kinases responsible for inactivating these complexes were shown to be sensitive to inhibition by known inhibitors, alpha-chloroisocaproate and dichloroacetate. Total activity (nmol/min/islet / 37 degrees C) of branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase was 0.86 and 5.09, with a % active form (activity before phosphatase treatment divided by activity after phosphatase treatment X 100) of 36% and 94%, respectively. Incubation of intact isolated islets with alpha-chloroisocaproate affected neither insulin release nor flux through branched-chain alpha-ketoacid dehydrogenase.
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PMID:Branched chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase activity in isolated rat pancreatic islets. 304 32

Stimulation of bovine chromaffin cell in culture changed (increased or decreased) the phosphorylation state of several proteins as examined by 32P incorporation. Enhanced phosphorylation of 22 protein bands as well as increased dephosphorylation of a 20.4 kilodaltons protein band was observed when extracts of cultured chromaffin cells stimulated by either acetylcholine or high K+ were subjected to mono-dimensional gel electrophoresis. For several protein bands, the degree of phosphorylation was larger in cells stimulated by acetylcholine than in those challenged by a depolarizing concentration of K+. The most affected phosphoproteins have apparent molecular weights of 14,800, 29,000, 33,000, 57,000 (tubulin subunit), 63,000 (tyrosine hydroxylase subunit) and 94,000. The presence of a low extracellular calcium concentration (0.5 mM Ca2+ plus 15 mM Mg2+) in the incubation medium inhibited (38-100%) the acetylcholine-evoked increases in protein phosphorylation observed previously for 18 protein bands. Trifluoperazine at the concentration required for 50% inhibition of acetylcholine-induced catecholamine release decreases (33-100%) the stimulation-induced phosphorylation in all polypeptides, with the exception of the 14.8 kilodaltons and the dephosphorylated 20.4 kilodaltons components which were not affected. Two-dimensional gel electrophoresis analysis revealed that exposure of chromaffin cells to acetylcholine produced two types of effect on protein phosphorylation: activation of protein kinase activities affecting about 30 polypeptides; activation of protein phosphatase activities resulting in the dephosphorylation of about 40 polypeptides, most of them appearing as minor phosphoproteins, with the exception of the alpha-subunit of pyruvate dehydrogenase and the 20.4 kilodaltons polypeptide. On the basis of their molecular properties (molecular weight and pI) and their abundance in chromaffin cells, the 80 kilodaltons phosphoprotein which focused at pI 4.8 and the 117.5 kilodaltons phosphoprotein which focused at pI 5.0 were identified as chromogranins A and B, respectively. The relationship between acetylcholine-induced protein phosphorylation (or dephosphorylation) and catecholamine secretion was also investigated. The time course of protein phosphorylation (or dephosphorylation) paralleled or preceded [3H]noradrenaline release for 16 phosphoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Phosphorylation and dephosphorylation of chromaffin cell proteins in response to stimulation. 377 57

Evidence is presented for defective pyruvate dehydrogenase (EC 4.1.1.1) in leukocytes and muscle tissue from a 10-year old child with persistent lactic acidosis, suffering from myasthenia and growth retardation. The defect is expressed in vitro by a depressed stimulation of pyruvate dehydrogenase catalytic activity by exogenous phosphoprotein phosphatase, and in vivo by a lack of response to muscle work, in comparison with healthy controls. Pyruvate dehydrogenase activity is in the normal range when measured without addition of phosphoprotein phosphatase in cells obtained from the resting patient. The defect reported here represents a new, hitherto undescribed form of a pyruvate dehydrogenase deficiency. The insufficient catalytic activity explains the observed accumulation of pyruvate, lactate, oxaloacetate and alanine and the decrease of citrate concentration in the blood of this patient. Electron microscope studies of the muscle tissue show an enhanced number of enlarged mitochondria with bizarre shapes and high densities of cristae.
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PMID:[Pyruvate dehydrogenase deficiency in a child with persistent lactic acidosis]. 392 41

We have measured the total activity of pyruvate dehydrogenase (PDH) complex, by in vitro activation with a broad specificity protein phosphatase, and the basal activity, supposed to be present in vivo, in biopsied muscles from three patients with PDH complex deficiency and 11 patients with lactic acidemia. Results showed that the total PDH complex activity must be determined in biopsied muscles for the diagnosis, because the basal activities of two of three patients with PDH complex deficiency overlapped those of two patients with lactic acidemia whose total activities were within normal range.
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PMID:Diagnosis of partial deficiency of the pyruvate dehydrogenase complex in biopsied muscle. 393 99

The phosphorylation of the alpha-subunit of mitochondrial pyruvate dehydrogenase may be involved in the development of long-term potentiation in the hippocampus. Study of this hypothesis is hampered by variability in the incorporation of 32P into pyruvate dehydrogenase of hippocampal subcellular preparations, in vitro. 32P from [gamma-32P]ATP was incorporated into pyruvate dehydrogenase present in mitochondria and in a membrane-enriched synaptic particulate fraction from hippocampus. However, the presence of the synaptic fraction decreased isotopic labeling of the mitochondrial protein. This effect was not due to inhibition of the protein kinase or activation of a protein phosphatase, but the rate of ATP hydrolysis was found to be higher in the synaptic fraction than in the mitochondria (34 nmol/mg protein/min vs 14 nmol/mg protein/min). These data raise a variety of questions about the interpretation of the in vitro phosphorylation assay. It is concluded that variability in in vitro labeling can be minimized if the effect of ATP hydrolysis is diminished by use of a higher concentration of ATP. In addition, these data indicate that quantitative comparisons of the in vitro phosphorylation of diverse subcellular preparations must take into account differential rates of ATP hydrolysis.
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PMID:Factors affecting the phosphorylation of a 41,000 dalton protein of hippocampal subcellular fractions. 673 99

Brief treatment of rat adipocytes with low concentration of trypsin activated both cell membrane and intracellular insulin-sensitive functions in marked contrast H2O2 (1), increase in pH, and oxidized glutathione (papers I and II). Glucose oxidation was activated maximally by trypsin in 30 s and preceded maximal activation of glycogen synthase, which occurred in 60s. Trypsin action to activate glycogen synthase was further enhanced by insulin. Mitochondrial pyruvate dehydrogenase was also rapidly activated by trypsin. With both insulin and trypsin action, mediator generation was directly demonstrated by glycogen synthase phosphoprotein phosphatase activation. Trypsin is thus the most insulin-like of these four agents studied since it acts by the formation of chemical mediator peptide(s) which are similar but not identical to those produced by insulin.
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PMID:Independent control of selected insulin-sensitive cell membrane and intracellular functions-the linkage of cell membrane and intracellular events controlled by insulin. III. The influence of trypsin on cell membrane hexose transport and on glycogen synthase and mitochondrial pyruvate dehydrogenase activation. 679 3


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