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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasma membranes have been prepared from porcine thyroid glands using sucrose gradients. The fractions having a density in sucrose of 1.18 g/ml mainly contained plasma membranes and were moderately contaminated with other subcellular components as shown by marker enzyme data. Purified plasma membranes incubated in the presence of [32-P]gamma ATP incorporated 32-P. Kinetics of incorporation of 32-P into endogenous substrates studied in various buffers and with increasing ATP concentration suggest a phosphodephosphorylating system related to cAMP-dependent protein kinase and phosphoprotein phosphatase activities. The two enzymatic activities associated with plasma membranes have been demonstrated using exogenous substrates. cAMP increases and fluoride ions decrease the extent of membrane phosphorylation. The specific activity of protein kinase was 10-12 times higher than in the initial homogenate and was only slightly enhanced in the presence of 0.5% Nonidet as compared to microsomal fraction. cAMP binding to membrane proteins was 3 times higher than to the other particulate fractions. TSH present in the incubating medium or added after 5 min of 32-P labelling induced a rapid stimulation of endogenous phosphorylation followed by a rapid decrease. Phosphorylated membrane substrates were analyzed: high voltage paper electrophoresis after partial hydrolysis indicated that [32-P]phosphate is incorporated into serine and threonine residues as o-phosphate derivatives. SDS-polyacrylamide gel electrophoresis showed several 32--labelled fractions. When enhanced by cAMP, no specific phosphorylation of protein components was observed.
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PMID:Phosphorylation of purified thyroid plasma membranes incubated with [32-P]ATP. 16 13

The protein kinase activities of a transplantable, insulin-producing hamster islet cell tumor were characterized using gel filtration, sucrose density gradient centrifugation and acrylamide gel electrophoresis. The post-microsomal supernatant fluid contains 70-80% of the protein kinase activity present in crude homogenates. A cAMP-dependent protein kinase, PK I (Mr 170,000), represents 25% of the soluble protein kinase activity assayed with protamine as substrate. It dissociates in the presence of cAMP into a cAMP-binding protein, R2 (Mr 90,000) and a catalytic subunit C (Mr 33,000). The dissociation induced by cAMP seems to be facilitated by the addition of Mg2+ and ATP. The regulatory subunit, R2, changes its gel filtration pattern in the presence of 0.5 M NaCl suggesting dissociation into a smaller subunit, R1 (Mr 44,000). By analogy with purified beef heart protein kinase (Erlichman et al., 1973) and skeletal muscle protein kinase, PK I. The presence in crude homogenates of a free cAMP-binding protein indistinguishable from the R2 derived by dissociation of PK I, suggests that PK I is partially dissociated in vivo. A cAMP-independent (casein) kinase (Mr 210,000) elutes with PK I on columns of Sepharose 6B. Another cAMP-independent protein kinase, PK II (Mr 88,000), is the predominatn form of soluble protein kinase accounting for approximately 75% of the soluble protein kinase activity detected using protaimine as substrate. This cAMP-independent protein kinase changes its gel filtration pattern in the presence of 0.5 M NaCl giving rise to a form which appears to have the same Mr (33,000) as the catalytic subunit of PK I. Studies comparing the catalytic subunit C of PK I with PK II and its salt-induced smaller molecular form demonstrate facile association of C with the cAMP-binding protein of purified bovine heart protein kinase to yield a hybrid holoenzyme, whereas PK II and its smaller form fail to recombine in this fashion. The 33,000 dalton forms derived from PK I (by cAMP) and PK II (by salt) also show different substrate specificities. It would appear, therefore, that pK II is a cAMP-independent protein kinase unrelated to PK I.
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PMID:Characterization of the protein kinases in a transplantable islet cell tumor of the Syrian hamster. 17 65

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.
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PMID:Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain. 19 Oct 91

The heat-stable protein (protein kinase modulator), partially purified from fresh bovine heart, possessed the ability to inhibit and stimulate adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase and guanosine 3':5'-monophosphate (cGMP)-dependent protein kinase activities, respectively. The inhibitory activity of protein kinase modulator on cAMP-dependent protein kinase was abolished almost completely by trypsin treatment, while the ability to stimulate cGMP-dependent protein kinase activity was resistant to trypsin. Fractionation by a linear potassium phosphate gradient on DEAE-cellulose column did not clearly separate both activities. Phosphorylation of cardiac microsomal component, "phospholamban" (molecular weight = 22,000), was inhibited almost completely by the saturating amounts of protein kinase modulator. This inhibition of phospholamban phosphorylation by protein kinase modulator was accompanied by a decreased Ca uptake rate that had been stimulated by cAMP-dependent protein kinase. These findings indicate that protein kinase modulator is functional in controlling the cAMP-dependent protein kinase-catalyzed phosphorylation of phospholamban and the rate of calcium transport, lending further support for the previously proposed mechanism, in which phospholamban is assumed to serve as a regulator of calcium transport in cardiac sarcoplasmic reticulum.
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PMID:Effect of protein kinase modulator on cAMP-dependent protein kinase-catalyzed phosphorylation of phospholamban and stimulation of calcium transport in cardiac sarcoplasmic reticulum. 20 86

Phosphorylation of the rat brain ryanodine receptor was studied using a monoclonal antibody, Ry-1, against the cardiac ryanodine receptor. A large polypeptide with the same SDS-PAGE mobility as that of the canine cardiac receptor was detected in rat brain membranes by immunoblotting. The brain ryanodine receptor was solubilized from the microsomal membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and more than 85% of the solubilized receptor was immunoprecipitated by Ry-1. Immunoprecipitated receptors were phosphorylated by cAMP-dependent protein kinase. The ryanodine receptor was also expressed in cultured fetal rat brain neurons and was phosphorylated by treating the cells with dibutyryl cAMP. The number of cells showing a caffeine-induced Ca2+ transient was increased significantly in the phosphorylating condition. These results suggest that the Ca channel activity of the brain ryanodine receptor is regulated by cAMP-dependent phosphorylation.
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PMID:Cyclic AMP-dependent phosphorylation of the rat brain ryanodine receptor. 131 34

Experiments were carried out to obtain information about the mechanism underlying the fast action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in skeletal muscle. N-2'-o-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP), similarly as 1,25(OH)2D3 (5 x 10(-10) M), rapidly increased 45Ca uptake by soleus muscle from vitamin D-deficient chicks (+25% and +98% at 3 min and 10 min, respectively) in a dose-dependent manner. The effects of the cAMP analog (10 microM) and 1,25(OH)2D3 could be abolished by the Ca(2+)-channel blocker nifedipine and the calmodulin antagonist flufenazine. Calmodulin binding by two muscle microsomal proteins of 28 kDa and 30 kDa was stimulated within 1 min of exposure of the tissue to 1,25(OH)2D3. Direct effects of the sterol on membrane calmodulin binding were shown with isolated microsomes. The 1,25(OH)2D3-mediated rise of [125I]calmodulin binding to microsomal membranes was dependent on the presence of medium ATP. Forskolin (10 microM) and cAMP (10 microM) also increased [125I]calmodulin binding (+75% and +64%, respectively, with respect to controls). Pretreatment of microsomal membranes with cAMP-dependent protein kinase inhibitor (1 microgram/ml) or addition of alkaline phosphates (1 U/ml) after hormonal treatment caused complete inhibition of 1,25(OH)2D3-induced [125I]calmodulin binding to microsomal membrane proteins. These results imply modifications of membrane protein phosphorylation through the cAMP signal pathway and in turn of calmodulin binding in the mechanism by which 1,25(OH)2D3 rapidly stimulates skeletal muscle Ca2+ uptake.
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PMID:Regulation of Ca2+ uptake in skeletal muscle by 1,25-dihydroxyvitamin D3: role of phosphorylation and calmodulin. 132 29

A polyclonal antibody, CR2, prepared using the C-terminal peptide of the alpha 1 subunit of the rabbit cardiac DHP-sensitive Ca channel, specifically immunoprecipitated the [3H]PN200-110-labeled Ca channel solubilized from cardiac microsomes. The antibody recognized 250 and 200-kDa cardiac microsomal proteins as determined by immunoblotting, and cAMP-dependent protein kinase phosphorylated the 250-kDa, but not the 200-kDa protein in vitro. CHO cells, transfected with the cardiac alpha 1 subunit cDNA carried by an expression vector, synthesized a 250-kDa protein which was recognized by CR2. Adding db-cAMP or forskolin to the transformed CHO cells induced phosphorylation of the 250-kDa protein and stimulated the DHP-sensitive Ba current under patch-clamp conditions. These results suggested that the cardiac DHP-sensitive Ca channel was regulated by cAMP-dependent phosphorylation of the alpha 1 subunit.
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PMID:Cyclic AMP-dependent phosphorylation and regulation of the cardiac dihydropyridine-sensitive Ca channel. 132 77

Rat submandibular and parotid gland exocytosis is primarily controlled by beta-adrenergic receptor stimulation. Although its precise role in the regulation of salivary gland exocytosis is not fully understood, protein phosphorylation, mediated by the activation of cAMP-dependent protein kinase, may be directly involved. Previous studies suggest that analogous 26-kDa integral membrane phosphoproteins may play a direct role in regulating exocytosis. Studies were here undertaken to purify and partially characterize both phosphoproteins. After endogenous phosphorylation with 32P, subcellular fraction and solubilization of the microsomal fraction in n-octyl beta-glucopyranoside, the 26-kDa integral membrane phosphoproteins were purified by high performance liquid chromatography (HPLC), followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroelution of the proteins. Amino acid analysis indicated a significant number of serine amino acids: N-terminal sequence data demonstrated a high level of homology; and trypsin digestion followed by reversed-phase HPLC indicated the possibility of multiple phosphorylation sites.
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PMID:Purification and partial characterization of analogous 26-kDa rat submandibular and parotid gland integral membrane phosphoproteins that may have a role in exocytosis. 152 94

The mitochondria, the microsomes and the cytosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.
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PMID:The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis. 166 Nov 27

The effect of CPT-cAMP and okadaic acid on phosphatidylcholine catabolism in suspension cultures of choline-deficient rat hepatocytes was investigated. Choline-deficient hepatocytes were pulse-labeled for 30 min with [methyl-3H]choline and subsequently chased for up to 60 min with choline in the absence or presence of 0.5 mM CPT-cAMP or 0.5 microM okadaic acid. Radioactivity in phosphatidylcholine and lysophosphatidylcholine were unchanged during the chase. However, the radioactivity incorporated into glycerophosphocholine was significantly increased (P less than 0.05) 59 and 77% after 60 min of chase in hepatocytes incubated with either okadaic acid or CPT-cAMP, respectively. Incubation of choline-deficient hepatocytes with both okadaic acid and CPT-cAMP produced an additive effect on radioactivity incorporated ino glycerophosphocholine. Crude mitochondrial, microsomal, and cytosolic phospholipaselysophospholipase activities, assayed in the presence of exogenously labeled phosphatidylcholine, were unchanged in both CPT-cAMP and okadaic acid treated hepatocytes compared with control. Phospholipase-lysophospholipase activity, assayed with endogenously labeled phosphatidylcholine, was increased 28 and 47% (P less than 0.05) in the crude mitochondrial fraction of hepatocytes treated with either okadaic acid or CPT-cAMP, respectively, compared with the control. Incubation of choline-deficient hepatocytes, labeled with L-[methyl-3H]methionine, with CPT-cAMP or okadaic acid caused a 31 and 20% increase (P less than 0.05) in the radioactivity incorporated into glycerophosphocholine, respectively, compared with the control. We postulate that phosphatidylcholine catabolism in choline-deficient hepatocytes may be regulated by a phosphorylation-dephosphorylation mechanism mediated through cAMP-dependent protein kinase and phosphoprotein phosphatase activities.
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PMID:CPT-cAMP and okadaic acid enhance phosphatidylcholine catabolism in choline-deficient rat hepatocytes. 166 52


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