Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Myosin light chain kinase and a fraction of type II cAMP-dependent protein kinase have been partially purified from bovine brain by affinity chromatography on calmodulin-Sepharose. The myosin kinase was purified approximately 3700-fold and has an estimated molecular weight of 130,000 +/- 10,000 by sodium dodecyl sulfate gel electrophoresis. A fraction of soluble cAMP-dependent protein kinase also bound to calmodulin-Sepharose and was purified 2300-fold. A fraction of this cAMP-dependent protein kinase after purification by glycerol gradient centrifugation was shown to contain the two subunits of calcineurin, a major calmodulin-binding protein in brain, and the two subunits of type II cAMP-dependent protein kinase in a ratio of 1:1:2:2. Its sedimentation coefficient was 8.1 S and 9.0 S when centrifuged in the absence or presence of calmodulin, suggesting the formation of a complex between calmodulin and protein kinase. Our results suggest the possibility that calcineurin may be involved in the interaction between the protein kinase and calmodulin. Furthermore, our studies imply that the regulatory subunit of the cAMP-dependent protein kinase, but not the catalytic subunit, is the site of interaction with calmodulin since the catalytic subunit of protein kinase was partially resolved from the complex by cAMP.
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PMID:Interaction of calmodulin with myosin light chain kinase and cAMP-dependent protein kinase in bovine brain. 626 40

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6-8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (less than 5 micrograms/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent Km of 60 microM and Vmax of 20 mumol X min-1 X mg-1, corresponding values with mixed histones were 12 microM and 1.2 mumol X min-1 X mg-1. With both protein substrates the apparent Km for ATP was 11 microM. Concentrations of Mg2+ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7-2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated 32Pi from [gamma-32P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.
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PMID:Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue. 715 5

Cancer cell energy metabolism is characterized by a high glycolytic rate, which is maintained under aerobic conditions. In Ehrlich ascites tumour cells, the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), the powerful activator of 6-phosphofructo-1-kinase, is tenfold increased. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), synthesizing and degrading Fru-2,6-P2, was characterized. The molecular mass is 120 kDa. The dependence of PFK-2 activity on the substrate concentrations is hyperbolic (Km for Fru-6-P = 0.09 mM; Km for ATP = 0.7 mM), while the dependence of the FBPase-2 activity on the concentrations of Fru-2,6-P2 is sigmoidal (K0.5 for Fru-2,6-P2 = 4 microM). The PFK-2/FBPase-2 activity ratio is 1. PFK-2 activity is inhibited by citrate (I0.5 = 0.17 mM) and phosphoenolpyruvate (I0.5 = 0.08 mM) but only weakly by glycerol 3-phosphate (I0.5 = 1.57 mM). In contrast to the liver enzyme, the activity of tumour PFK-2/FBPase-2 is not influenced by the action of cAMP-dependent protein kinase. The kinetic properties as well as ion-exchange chromatography pattern differ from their normal counterparts in liver and muscle. The properties are likely to contribute to the maintenance of the high glycolytic rate in these tumour cells.
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PMID:Fructose 2,6-bisphosphate metabolism in Ehrlich ascites tumour cells. 749 45

The liver isoform of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase of the teleost fish Sparaus aurata has several characteristics similar to the skeletal muscle isoform of mammals. In order to ascertain the relation between muscle and liver isoforms in teleost, 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was purified from skeletal muscle of S. aurata. The muscle isozyme is composed of subunits with a molecular weight of 54 kDa, is bifunctional, and has an activity ratio kinase to bisphosphatase of 2.5. Muscle 6-phosphofructo 2-kinase is not sensitive to glycerol 3-phosphate inhibition and has noncooperative KmATP, higher than the liver isozyme. Thus, the kinetic characteristics of the muscle were distinguishable from the liver isozyme. Furthermore, the muscle isozyme is not a substrate of cAMP-dependent protein kinase. Despite those differences, two polyclonal antibodies raised against purified liver and muscle isozymes from S. aurata are not able to distinguish between them. Both antisera recognize with lower affinity recombinant rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase. A third antibody raised against the rat liver isozyme was also able to immunoprecipitate the teleost enzymes. The close immunological properties found suggest that S. aurata isozymes share epitopes in common. Considering the kinetic and immunological data reported, it is likely that the skeletal muscle/liver isozymes in teleost are products of a differentially spliced transcript of the same gene, as it is in rat. As those species are distant in vertebrate evolution, the similitude suggest that a common ancestral gene is involved in the muscle/liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase system in vertebrates.
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PMID:The muscle isoform of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase of the teleost Sparus aurata: relationship with the liver isoform. 764 54

A triacylglycerol lipase, presumably the first enzyme involved in the mobilization of lipid from the insect fat body, has been purified to homogeneity from the fat body of Manduca sexta. The purification procedure involved polyethyleneglycol precipitation, and chromatography on DEAE-cellulose, phenyl-Sepharose, Q-Sepharose and hydroxylapatite. The final product, a protein with an M(r) = 76,000 by SDS-PAGE, was purified nearly 8000-fold from the original homogenate in a yield of about 11%. The enzyme catalyzed the hydrolysis of tri-, di-, and mono-oleoylglycerols, but showed highest affinity for tri- or dioleoylglycerol. Thus, under initial reaction conditions, the end products of trioleoylglycerol hydrolysis were: free fatty acids (66%), sn-2-monooleoylglycerol (24%), sn-1,2(2,3)-dioleoylglycerol (7%), and glycerol (3%). The fat body lipase exhibited a preference for hydrolyzing the primary ester bonds of acylglycerols, and did not show stereoselectivity toward either the sn-1 or sn-3 position of trioleoylglycerol. The enzyme had a pH optimum of 7.9, and was inhibited by diisopropylfluorophosphate, ATP, ADP, Mg2+, and NaF. The enzyme showed a strong tendency to aggregate, but was stable in detergent solutions at high concentration of glycerol. The polypeptide was phosphorylated by the cAMP-dependent protein kinase from bovine heart; however, phosphorylation did not cause activation of the enzyme. It is suggested that this fat body lipase could be analogous to the "hormone-sensitive lipase" of vertebrate adipose tissue.
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PMID:Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect, Manduca sexta. 780 79

The involvement of protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and other phosphorylation mechanisms in the rapid desensitization of the [Ca2+]i response to parathyroid hormone (PTH) stimulation was investigated in osteoblast-like UMR-106 cells. A 5 minute preincubation of the cell suspension with phorbol 12,13-dibutyrate (PDB) decreased the response to PTH in a concentration-dependent manner. 1-Oleoyl-2-acetyl-r-glycerol (OAG) pretreatment likewise decreased the PTH response. Staurosporine, a potent protein kinase inhibitor, completely prevented the desensitization caused by PDB. These PDB and staurosporine effects were also observed in 3 mM EGTA-containing medium ([Ca2+]free < 10(-8) M). A 5 minute pretreatment of cells with 1 microM forskolin had no effect on the calcium response to PTH. Homologous and PDB-induced desensitizations differed in several respects. Staurosporine pretreatment resulted in only a slight restoration of the PTH response under conditions of homologous desensitization. Chronic treatment with phorbol ester prevented the desensitization of the PTH response by acute phorbol treatment but not the homologous desensitization. Both homologous and PDB-induced desensitization were relieved by alkaline phosphatase treatment, consistent with the involvement of phosphorylation in the desensitization. This alkaline phosphatase effect on desensitization was inhibited by L-phenylalanine. These results suggest that PTH receptor homologous desensitization involves phosphorylation process(es) other than or in addition to those of PKC.
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PMID:Studies on the mechanism of desensitization of the parathyroid hormone-stimulated calcium signal in UMR-106 cells: reversal of desensitization by alkaline phosphatase but not by protein kinase C downregulation. 807 54

The lipolytic action of the beta 3-adrenoceptor-selective agonist 4-[2-[(2-hydroxy-2-(3-chlorophenyl)ethyl)-amino]propyl]-phenoxyacetic acid (BRL 37344) was compared to that of isoprenaline in adipocytes derived from rat white adipose tissue. Concentration-response curves for activation of lipolysis by each agonist correlated well with the dose-response curves for activation of cAMP-dependent protein kinase (A-Kinase). Addition of propranolol at a concentration (0.1 microM) sufficient to block beta 1- and beta 2-adrenoceptors did not affect the stimulation of either parameter by BRL 37344 or isoprenaline, indicating that lipolysis was predominantly dependent on beta 3-adrenoceptor stimulation. Blockade of beta 3-adrenoceptors by 3 microM propranolol antagonized both A-Kinase activation and glycerol release. Activation of lipolysis by BRL 37344 was blocked by treatment of the cells with N-[2-p-(bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide (H89) a potent and selective isoquinolinesulphonamide inhibitor of A-Kinase activity. Taken together, these results indicate that lipolysis in rat white adipocytes is primarily controlled by beta 3-adrenoceptors, and that cyclic AMP generation alone is responsible for activation of lipolysis in this tissue.
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PMID:Correlation of beta 3-adrenoceptor-induced activation of cyclic AMP-dependent protein kinase with activation of lipolysis in rat white adipocytes. 810 24

The aim of this study was to evaluate the rapid regulation of cell-cell communication by using the microinjection of purified cAMP-dependent protein kinase (protein kinase A), the Ca(2+)-phospholipid-dependent protein kinase (protein kinase C), or the inhibitor proteins (PKI and CKI) that are, respectively, specific for each of these enzymes. Gap junction phenotypes of myometrial tissue and cells were studied by means of immunocytochemistry with antibody to connexin 43 (alpha 1; Cx43). Cells were enzymatically disaggregated from myometrium of nonpregnant, mid-pregnant (Day 14), and late-pregnant (Day 29) rabbit uteri (n = 8 per group) and seeded at high density such that after 4 days, cultures had the appearance of a cross-sectioned myometrium. Purified proteins and their subunits were microinjected, and intercellular communication was evaluated by monitoring Lucifer Yellow dye transfer. Cultures were treated with 0.5 mM 8Br-cAMP (8-bromo adenosine 3',5' cyclic monophosphate) or 10 microM OAG (1-oleoyl-2-acetyl-sn-glycerol), which, respectively, activate protein kinase A and protein kinase C. Immunoreactive Cx43 and cell-cell communication were examined 5 min to 2 h later. Cx43 was detected in myometrial cryosections and cultured cells by indirect immunofluorescence, and its expression increased with gestation. Exposure to 8Br-cAMP increased the amount of immunoreactive Cx43. Basal dye transfer was minimal in nonpregnant cells, increased in cells of mid-pregnant uteri, and was maximal in late-pregnant cells. Treatment with 8Br-cAMP enhanced transfer in mid- and late-pregnant cells but had no obvious effect on cells from nonpregnant animals. OAG treatment inhibited dye transfer in greater than 95% of the cells tested irrespective of pregnancy status. PKI inhibited cell-cell communication within 2 min and up to 40 min. Injection of free catalytic subunit of protein kinase A following PKI inhibition restored communication within 2-3 min, with maximal transfer in 4-5 min. Protein kinase C inhibited communication, which resumed in < 3 min after injection of CKI. We conclude that rabbit myometrial cells engage in Cx43-mediated cell-cell communication and that this process increases during pregnancy. Further, activators of protein kinase A or injected free catalytic subunit rapidly enhances cell-cell communication, whereas activators of protein kinase C or the enzyme itself diminishes this process.
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PMID:Regulation of cell-cell communication mediated by connexin 43 in rabbit myometrial cells. 814 55

Cardiac rH1 Na+ channel alpha subunits were expressed in cells of the Chinese hamster lung 1610 cell line by transfection, and a stable cell line expressing cardiac Na+ channels (SNa-rH1) was isolated. Mean Na+ currents of 2.2 +/- 1.0 nA were recorded, which corresponds to a cell surface density of approximately 1-2 channels active at the peak of the Na+ current per micron2. The expressed cardiac Na+ current was tetrodotoxin resistant (Kd = 1.8 microM) and had voltage-dependent properties similar to those of the Na+ current in neonatal ventricular myocytes. Activation of protein kinase C by 1-oleoyl-2-acetyl-sn-glycerol (OAG) (10 microM) decreased this current approximately 33% at a holding potential of -114 mV and 56% at -94 mV. This reduction in peak current was caused in part by an 8- to 14-mV shift of steady-state inactivation in the hyperpolarized direction. Na+ channel activation was unchanged. Effects of OAG in SNa-rH1 cells and in neonatal rat cardiac myocytes were similar, except that the time course of inactivation was slowed either transiently or persistently when protein kinase C was activated in myocytes bathed in low-Ca2+ (1 microM) or Ca(2+)-free solution but was unaffected in SNa-rH1 cells. The effects of OAG on cardiac Na+ current were blocked in cells that had been previously microinjected with a peptide inhibitor of protein kinase C but not with a peptide inhibitor of cAMP-dependent protein kinase, indicating that protein kinase C is responsible for the effect of OAG. Single-channel recordings from SNa-rH1 cells showed that the probability of channel opening was reduced by OAG, but the conductance was unaffected. OAG did not induce the late Na+ channel openings observed with PKC modulation of neuronal and skeletal muscle Na+ channels. Thus, the substantial reduction in Na+ current at normal diastolic depolarizations with 10 microM OAG is due to failure of channel opening in response to depolarization. Such Na+ current reductions may have profound effects on cardiac cell excitability.
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PMID:Modulation of cardiac Na+ channels expressed in a mammalian cell line and in ventricular myocytes by protein kinase C. 815 41

Lipid synthesis and secretion was measured in primary rat mammary epithelial cells cultured on basement matrix in medium supplemented with lactogenic hormones. The cells grew and differentiated to form alveolar-like structures reminiscent of lactating mammary gland. They synthesized abundant triacylglycerol, containing fatty acids characteristic of rat milk (C10:0-C14:0), using 14C-glucose, 14C-oleic acid or 14C-glycerol as precursors. Basal levels of triacylglycerol secretion were measured using 14C-oleic acid labeling; 1.3 +/- 0.3% of the labeled cellular triacylglycerol was secreted into the medium in 24 hours. Secreted lipid droplets were surrounded by a bilayer membrane with an electron-dense inner coat characteristic of fat globules secreted by the mammary gland. The rate of triglycerol secretion was increased to 998 +/- 98% of control (P < 0.01) by the addition of phorbol 12-myristate 13-acetate (PMA) in combination with staurosporine, a protein kinase inhibitor. Several other protein kinase inhibitors, when combined with PMA, also markedly stimulated secretion. Effective protein kinase inhibitors included sphingosine (has diverse cellular effects including the inhibition of protein kinase C; 13-fold increase in secretion), and KT5823 (a cGMP dependent protein kinase inhibitor; 5-fold increase). KT5720 (a cAMP-dependent protein kinase inhibitor) did not alter secretion. Kinase inhibitors were effective only in the presence of a phorbol ester. 4 alpha-phorbol-12,13-didecanoate, a phorbol ester which does not activate protein kinase C (PKC), could substitute for PMA. Lipid release was not mediated by disruption of cell-cell tight junctions, as EGTA did not release lipid. Based on these observations we suggest that two signals are needed to enable or stimulate lipid secretion in cultured rat mammary epithelial cells: 1) inhibition of a protein kinase and 2) a PKC-independent effect of phorbol ester. We have, for the first time, characterized a cell culture model suitable for studying lipid synthesis and secretion by mammary epithelial cells.
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PMID:Lipid synthesis and secretion by primary cultures of rat mammary epithelial cells. 825 58


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