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

Intact secretory granules isolated from bovine adrenal medulla express tyrosine hydroxylase (TH) activity. Granule-associated TH sediments on continuous sucrose gradients with dopamine beta-hydroxylase, a marker for granule membranes, indicating that TH is associated with chromaffin granules. Membranes prepared from lysed granules retain TH, whereas granule contents are free of the enzyme. TH immunoreactivity was detected in granule membranes by immunoblot analysis using a polyclonal antiserum against TH. TH immunoreactivity cannot be removed from membranes by washes in high ionic strength buffers and is only partially removed from membranes by treatment with either urea or Na2CO3. TH can be removed from granule membranes by the detergents Nonidet P-40, Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Treatment of membranes with a phosphatidylinositol-specific phospholipase C did not remove TH, ruling out the possibility of a glycosyl phosphatidyl anchor. Fractionation of granule membranes by temperature-induced phase separation in Triton X-114 revealed that TH is recovered in phases in which integral (detergent phase) and hydrophobic (phospholipid phase) membrane proteins are typically found. By contrast, TH from adrenal cytosol fractionated exclusively into the aqueous phase along with other soluble proteins. Digestion of granules with various protease enzymes revealed that TH is resistant to degradation, suggesting that the enzyme is embedded within membranes. TH becomes phosphorylated when intact granules are exposed to the catalytic subunit of the cAMP-dependent protein kinase, indicating that at least the N-terminal region of TH is exposed on the cytoplasmic surface of granules. These results establish that a fraction of TH is an integral component of bovine granule membranes. The association of TH with granule membranes may play a role in coordinating TH activity and catecholamine release.
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PMID:Tyrosine hydroxylase in secretory granules from bovine adrenal medulla. Evidence for an integral membrane form. 196 7

Labeling with [3H]galactose was employed to isolate a glycosylphosphatidylinositol from rat hepatocytes which might be involved in the action of insulin. The polar head group of this glycosylphosphatidylinositol was generated by phosphodiesterase hydrolysis with a phosphatidylinositol-specific phospholipase C from Bacillus cereus. By Dowex AG1 x 8 chromatography the polar head group could be separated into three radioactive peaks eluting at 100 mM (peak I), 200 mM (peak II) and 500 mM (peak III) ammonium formate, respectively. Peak III was the most active as an inhibitor of the cAMP-dependent protein kinase. Treatment of peak III with alkaline phosphatase markedly reduced its activity on cAMP-dependent protein kinase. When peaks I, II or III were treated with alkaline phosphatase and analyzed again by Dowex AG1 x 8 chromatography, the radioactivity eluted with the aqueous fraction. The above results indicate that the polar head group of the insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes exists in three different phosphorylated forms and that the biological activity of this molecule depends on its phosphorylation state.
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PMID:Different phosphorylated forms of an insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes. 304 67

An inositol phosphoglycan that is the polar head group of a glycosyl phosphatidylinositol has been considered as a putative mediator of insulin action. To gain insight into the functions of this hormone during development, the relationships between insulin, insulin receptors, glycosyl phosphatidylinositol, and inositol phosphoglycan were studied. Glycosyl phosphatidylinositol was isolated and characterized in fetal liver as early as day 15 of intrauterine life. In isolated hepatocytes from fetal and adult rats labeled with [3H]glucosamine, [3H]galactose, or [3H]myo-inositol, these molecules were incorporated into glycosyl phosphatidylinositol. In hepatocytes labeled with [3H]glucosamine and then allowed to react with [1-14C]IAI, the [3H]glycosyl phosphatidylinositol was purified as the 14C-labeled amidinated lipid. Glycosyl phosphatidylinositol molecules from fetal and adult cells were sensitive to hydrolysis by a phosphatidylinositol-specific phospholipase C from B. cereus. The product of this hydrolysis inhibits the activity of a cAMP-dependent protein kinase, whereas this effect was abolished by nitrous acid deamination. In isolated hepatocytes from adult animals, an inverse correlation between extracellular insulin and the number of insulin receptors and the cellular content of glycosyl phosphatidylinositol was observed. However, in fetal hepatocytes insulin failed to reduce the glycosyl-phosphatidylinositol content when labeled either with [1-14C]isethionyl acetimidate or [3H]glucosamine, whereas insulin-like growth factor I produced a significant hydrolysis of glycosyl phosphatidylinositol. Fetal and adult hepatocytes were incubated with insulin or inositol phosphoglycan after which glycogen phosphorylase activities were determined. Inositol phosphoglycan mimicked the action of insulin on both forms of the enzyme from adult hepatocytes, whereas in fetal cells insulin did not change, and purified inositol phosphoglycan reduced the activities of glycogen phosphorylase. These findings suggest a dissociation between insulin receptor occupancy and the expected hormonal effects in fetal hepatocytes. This could be related to alterations at a postreceptor level.
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PMID:Insulin does not induce the hydrolysis of a glycosyl phosphatidylinositol in rat fetal hepatocytes. 834 37

The role(s) of protein kinases in the regulation of G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha was investigated in the osteoblast cell line MC3T3-E1. We have previously reported the stimulatory effects of tumor necrosis factor-alpha and A1F4-, an activator of G proteins, on this phospholipase pathway documented by a decrease in mass of PI and release of diacylglycerol. In this study, we further explored the mechanism(s) by which the tumor necrosis factor or A1F4(-)-promoted breakdown of phosphatidylinositol and the polyphosphoinositides by phospholipase C is regulated. Tumor necrosis factor-alpha was found to elicit a 4-5-fold increase in the formation of [3H]inositol-1,4-phosphate and [3H]inositol-1,4,5-phosphate; and a 36% increase in [3H]inositol-1-phosphate within 5 min in prelabeled cells. [3H]inositol-4-phosphate, a metabolite of [3H]inositol-1,4-phosphate and [3H]inositol-1,4,5-phosphate, was found to be the predominant phosphoinositol product of tumor necrosis factor-alpha and A1F4(-)-activated phospholipase C hydrolysis after 30 min. In addition, the preincubation of cells with pertussis toxin decreased the tumor necrosis factor-induced release of inositol phosphates by 53%. Inhibitors of protein kinase C, including Et-18-OMe and H-7, dramatically decreased the formation of [3H]inositol phosphates stimulated by either tumor necrosis factor-alpha or A1F4- by 90-100% but did not affect basal formation. The activation of cAMP-dependent protein kinase, or protein kinase A, by the treatment of cells with forskolin or 8-BrcAMP augmented basal, tumor necrosis factor-alpha and A1F4(-)-induced [3H]inositol phosphate formation. Therefore, we report that protein kinases can regulate tumor necrosis factor-alpha-initiated signalling at the cell surface in osteoblasts through effects on the coupling between receptor, G-protein and phosphatidylinositol-specific phospholipase C.
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PMID:Protein kinases A and C positively regulate G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha in MC3T3-E1 osteoblasts. 913 78

Extracts of human liver were found to contain activities which copurified and coeluted with the two major subtypes of mediators (type A and type P) isolated from insulin-stimulated rat liver. The putative type A mediator from human liver inhibited cAMP-dependent protein kinase from bovine heart, decreased phosphoenolypyruvate carboxykinase mRNA levels in rat hepatoma cells, and stimulated lipogenesis in rat adipocytes. The putative type P mediator stimulated bovine heart pyruvate dehydrogenase phosphatase. Both fractions were able to stimulate proliferation of EGFR T17 fibroblasts and the type A was able to support growth in organotypic cultures of chicken embryo cochleovestibular ganglia. Both activities were resistant to Pronase treatment and the presence of carbohydrates, phosphate, and free-amino groups were confirmed in the two fractions. These properties are consistent with the structure/ function characteristics of the type A and P inositolphosphoglycans (IPG) previously characterized from rat liver. Further, the ability of the human-derived mediators to interact with rat adipocytes and bovine-derived metabolic enzymes suggests similarity in structure between the mediators purified from different species. Galactose oxidase-susceptible membrane-associated glycosylphosphatidylinositols (GPI) have been proposed to be the precursors of IPG. GPI was purified from human liver membranes followed by treatment with galactose oxidase and reduction with NaB3H4. Serial t.l.c. revealed three radiolabeled bands which comigrated with the putative GPI precursors found in rat liver. These galactose-oxidase-reactive lipidic compounds, however, were only partially susceptible to hydrolysis with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis and were resistant to glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei. These data indicate that IPG molecules with insulin-like biological activities are present in human liver.
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PMID:Isolation and partial characterisation of insulin-mimetic inositol phosphoglycans from human liver. 925 87

In order to examine the role of protein kinase A (PKA) in the regulation of arachidonic acid availability, the interaction between cAMP agonists and the G protein activator AIF4- in their effects on phospholipid metabolism were measured in MC3T3-E1 osteoblasts. We show that forskolin and 8-brcAMP, activators of PKA, amplify the AIF4(-)-induced stimulation of phosphatidylinositol-specific phospholipase C (phosphatidylinositol inositolphosphohydrolase; EC 3.1.4.3), measured by the formation of [3H]inositol phosphates in prelabeled cells. However, the AIF4(-)-stimulated production of 1,2-diacylglycerols and the release of [3H]arachidonic acid ([3H]AA) were inhibited 50-75% by forskolin and 8-bromocAMP. Furthermore, pretreatment with PKA activators prevented much of the AIF4(-)-induced loss of [3H]AA from phosphatidylcholine and phosphatidylethanolamine in prelabeled osteoblasts. In addition, in the absence of AIF4-, forskolin was found to stimulate the incorporation of [3H]AA and [32P]orthophosphoric acid selectively into these two major phospholipids and selectively increased their mass. The effects of forskolin and 8-BrcAMP on the levels of free [3H]AA were completely reversed by pretreatment with the PKA inhibitor H-89. Therefore, our findings suggest that the activation of cAMP-dependent protein kinase can reduce the availability of free arachidonic acid for prostaglandin synthesis in osteoblast cells by stimulating its reesterification via phospholipid resynthesis.
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PMID:Activators of protein kinase A decrease the levels of free arachidonic acid in osteoblasts via stimulation of phosphatidylcholine and phosphatidylethanolamine synthesis. 957 54

The nucleus accumbens (NAc) is a forebrain area in the mesocorticolimbic dopamine (DA) system that regulates many aspects of drug addiction. Neuronal activity in the NAc is modulated by different subtypes of DA receptors. Although DA signaling has received considerable attention, the mechanisms underlying D(2)-class receptor (D(2)R) modulation of firing in medium spiny neurons (MSNs) localized within the NAc remain ambiguous. In the present study, we performed whole cell current-clamp recordings in rat brain slices to determine whether and how D(2)R modulation of K(+) channel activity regulates the intrinsic excitability of NAc neurons in the core region. D(2)R stimulation by quinpirole or DA significantly and dose-dependently decreased evoked Na(+) spikes. This D(2)R effect on inhibiting evoked firing was abolished by antagonism of D(2)Rs, reversed by blockade of voltage-sensitive, slowly inactivating A-type K(+) currents (I(As)), or eliminated by holding membrane potentials at levels in which I(As) was inactivated. It was also mimicked by inhibition of cAMP-dependent protein kinase (PKA) activity, but not phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Moreover, D(2)R stimulation also reduced the inward rectification and depolarized the resting membrane potentials (RMPs) by decreasing "leak" K(+) currents. However, the D(2)R effects on inward rectification and RMP were blocked by inhibition of PI-PLC, but not PKA activity. These findings indicate that, with facilitated intracellular Ca(2+) release and activation of the D(2)R/G(q)/PLC/PIP(2) pathway, the D(2)R-modulated changes in the NAc excitability are dynamically regulated and integrated by multiple K(+) currents, including but are not limited to I(As), inwardly rectifying K(+) currents (I(Kir)), and "leak" currents (I(K-2P)).
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PMID:Dopamine D(2) receptor modulation of K(+) channel activity regulates excitability of nucleus accumbens neurons at different membrane potentials. 1688 24