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)

The time-courses of isoproterenol activation of rat adipocyte particulate low Km cAMP phosphodiesterase (PDE) activity, cAMP-dependent protein kinase (A-kinase), and glycerol production were measured in the presence and absence of insulin. Isoproterenol (100 nM) alone rapidly activated A-kinase 8- to 10-fold and increased particulate cAMP PDE by approximately 100%. A-kinase and PDE activity remained relatively constant for at least 25 to 30 min. Kact values for isoproterenol activation of PDE and lipolysis were similar. In comparison with isoproterenol, insulin (0.1-0.3 nM) alone increased particulate cAMP PDE at a slower rate and to a lesser extent (by approximately 50% within 12 to 16 min) and without any change in A-kinase. With insulin plus isoproterenol there was a rapid, transient, and synergistic activation of particulate cAMP PDE, which temporally correlated with a decrease in A-kinase and reduction in lipolysis. These and other data suggest the following: 1) there is a close concentration-dependent and temporal relationship in isoproterenol activation of adenylate cyclase, of A-kinase, and of particulate cAMP PDE; 2) isoproterenol and insulin activate particulate cAMP PDE by two distinct mechanisms; 3) the temporal changes in PDE and A-kinase in the presence of insulin and isoproterenol suggest that insulin activation of the PDE does not require, but may be enhanced by, elevated cAMP and is important in the antilipolytic action of insulin.
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PMID:Role of hormone-sensitive low Km cAMP phosphodiesterase in regulation of cAMP-dependent protein kinase and lipolysis in rat adipocytes. 253 13

The fluorescent ATP analogue 8-azido-2'-O-[14C]dansyl-ATP ([ 14C]AD-ATP) was used to probe the ATP-binding site in the catalytic (C) subunit of cAMP-dependent protein kinase. AD-ATP was found to inhibit the phosphotransferase activity of C subunit with extremely high specificity. Complete inhibition was observed when each mol of C subunit was covalently labeled with 1 mol of this fluorescent ATP analogue. The labeling can be accelerated by the presence of Mg2+ or Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), whereas high concentrations of ATP can almost completely protect the enzyme from AD-ATP. Detailed studies indicated that AD-ATP competes with ATP for binding to C subunit. Analysis of the kinetic data gave dissociation constants of 2.9 and 13 microM for AD-ATP and ATP bound to C subunit, respectively. AD-ATP has a fluorescence emission peak at 510 nm in pH 7.0 aqueous buffer containing 25% glycerol. After covalent binding to C subunit this emission peak shifts to 455 nm, which suggests that the label at ATP site is in an endogenous hydrophobic environment. Upon the binding of Mg2+ or Kemptide, the fluorescence of AD-ATP-labeled C subunit can be enhanced by 50 and 45%, respectively. This enhancement suggests that the binding of either the peptide substrate or Mg2+ induces conformational change at the active site of C subunit. Analysis of the fluorescence data shows that the values of Kd for Mg2+ and Kemptide bound to AD-ATP-labeled C subunit are 0.2 mM and 2.1 microM, respectively. The normal procedure for the preparation of the C subunit from the bovine heart muscle has been simplified to require only one-fifth of the usual working time to obtain the homogeneous enzyme with 70% yield from the crude extract.
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PMID:A potent fluorescent ATP-like inhibitor of cAMP-dependent protein kinase. 254 58

Microsomal glycerolphosphate acyltransferase from rat adipose tissue is shown to be inactivated with time upon incubation with ATP. The inactivation can be observed in postmitochondrial supernatant as well as in washed microsomes. However, the effect is more pronounced upon addition of the cytosolic fraction. This activity is specific for ATP, is dependent on the nucleotide concentration, and is prevented when ATP is substituted by beta,gamma-methylene-ATP. Some protection is provided by amiloride but not by EGTA or cAMP-protein kinase inhibitor. Also, the level of enzyme inactivation is not modified by addition of cAMP-dependent protein kinase and its substrates. Inactivated glycerol-phosphate acyltransferase from ATP-treated microsomes can be reactivated by incubation with partially purified protein phosphatase from rat liver. These results suggest the existence in adipose tissue of a protein kinase (cAMP independent) that may be involved in the regulation of glycerolphosphate acyltransferase.
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PMID:Reversible ATP-dependent inactivation of glycerolphosphate acyltransferase from rat adipose tissue. 271 27

Although the renal medulla is rich in triacylglycerols, the lipolysis of these intracellular triacylglycerols by a renomedullary triacylglycerol lipase has not been directly demonstrated. The present study demonstrates triacylglycerol lipase activity localized in the particulate subcellular fractions of rabbit renal medullae. Renomedullary triacylglycerol lipase activity, as determined by the hydrolysis of [14C]triolein to [14C]oleic acid, was observed to have a pH optimum of 5.8. Addition of cAMP/ATP/magnesium acetate resulted in an 80% activation of crude homogenate triacylglycerol lipase activity; addition of exogenous cAMP-dependent protein kinase resulted in a further activation of lipolysis. 3 mM CaCl2 had no effect on basal triacylglycerol lipase activity. 1 M NaCl did not inhibit lipolysis, suggesting that the lipase activity measured was not due to lipoprotein lipase. Endogenous renomedullary triacylglycerols were hydrolysed by a lipase in the 100,000 X g pellet of renomedullary homogenates, resulting in the release of free fatty acids including arachidonic and adrenic acids. Dispersed renomedullary cells were prepared to monitor hormone-sensitive triacylglycerol lipase activity in intact cells. Addition of 10 microM forskolin and 10 microM epinephrine resulted in 8-fold and 50-fold increases in triacylglycerol lipase activity, respectively, as defined by release of free glycerol from the cells. These studies demonstrate that a cAMP-dependent hormone-sensitive triacylglycerol lipase is present in the renal medulla, and is responsible for the hydrolysis of renomedullary triacylglycerols.
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PMID:Triacylglycerol lipase activity in the rabbit renal medulla. 282 29

Activation of glycolysis by insulin in cultured adult rat hepatocytes is accompanied by an activation of phosphofructokinase 2 (PFK 2). PFK 2 activation might be caused by insulin-dependent changes of (a) metabolite levels, (b) basal and (c) Br8cAMP-stimulated cAMP-dependent protein kinase activity; this problem was investigated. 1. Cells cultured with 0.1 nM insulin for 48 h exhibited a low glycolytic rate and low fructose 2,6-bisphosphate [Fru(2,6)P2] levels. Addition of insulin increased Fru(2,6)P2 and Fru(1,6)P2 levels sequentially which points to PFK 2 as first target enzyme of insulin action. 2. Concentrations of Glc6P, Fru6P, phosphoenolpyruvate, glycerol 3-phosphate and citrate, which modulate PFK 2/fructose 2,6-bisphosphatase 2 activity, were not altered by insulin. 3. Activation of PFK 2 by insulin occurred without changes in the levels of total and protein-bound cAMP. Bound cAMP amounted to about 14% of total cAMP. 4. Insulin neither decreased the basal dissociation state of the cAMP-dependent protein kinase nor lowered the sensitivity of the kinase towards cAMP in cell extracts. 5. Addition of the phosphodiesterase-resistant Br8cAMP to the cultures increased cAMP levels 3-4-fold, elevated the protein kinase activity ratio from 0.14 to 0.6 and decreased the Fru(2,6)P2 level and the rate of glycolysis. When Br8cAMP and insulin were given together, insulin was capable of counteracting Br8cAMP in that it activated glycolysis and PFK 2 and elevated the Fru(2,6)P2 level; however, it did not decrease the elevated protein kinase activity ratio. It is concluded that insulin presumably does not activate PFK 2 through changes in cAMP and effector levels or through inhibition of cAMP-dependent protein kinase dissociation. The data support the hypothesis that insulin may act via activation of PFK 2 phosphatase.
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PMID:Activation of phosphofructokinase 2 by insulin in cultured hepatocytes without accompanying changes of effector levels or cAMP-stimulated protein kinase activity ratios. 284 74

We designed a simple procedure for the purification of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) from rabbit brain, using affinity chromatography with a new affinity adsorbent. The adsorbent was synthesized by attaching the amino residue of N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) to cyanogen bromide-activated Sepharose. H-9 is a potent competitive inhibitor of protein kinase C, cGMP-, and cAMP-dependent protein kinase with respect to ATP and exhibits inhibition constants of 18, 0.87, and 1.9 microM, respectively (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry, 23, 5036). A 960-fold purification was achieved in the two-step procedure, which entailed DEAE-cellulose and the affinity chromatography. The resultant preparation was essentially homogeneous, as indicated by polyacrylamide gel electrophoresis under conditions of denaturation with sodium dodecyl sulfate. The affinity of protein kinase C for the H-9-Sepharose was high, and the enzyme could not be eluted either by a high concentration of sodium chloride or by 40% glycerol. The protein kinase C could be eluted from H-9-Sepharose by the buffer containing both 0.2 M NaCl and 20% glycerol, thereby suggesting that the binding between protein kinase C and H-9-Sepharose was due to both hydrophobic and electrostatic interactions. H-9 coupled to Sepharose retained both cyclic nucleotide-dependent protein kinases and protein kinase C, and these enzymes could be eluted separately by the buffer containing L-arginine, a potent inhibitor of these three kinases. The novel aspects of these three multifunctional protein kinases can thus be investigated using isoquinolinesulfonamide derivatives.
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PMID:N-(2-Aminoethyl)-5-isoquinolinesulfonamide, a newly synthesized protein kinase inhibitor, functions as a ligand in affinity chromatography. Purification of Ca2+-activated, phospholipid-dependent and other protein kinases. 298 42

Addition of the commonly used anesthetic pentobarbital to hepatocytes from fed rats resulted in a dose-dependent decrease in the level of fructose 2,6-bisphosphate. At a concentration of pentobarbital (0.4 mM) that lowered fructose 2,6-bisphosphate by 60%, there was no significant change in the level of fructose 6-phosphate, ATP, or L-glycerol 3-phosphate. Higher concentrations of pentobarbital (2 mM) enhanced both glycolysis and glycogenolysis and fructose 2,6-bisphosphate levels were reduced to less than 10% of the control. Concomitant with these changes there was a decrease in ATP, glucose 6-phosphate, and fructose 6-phosphate and a two- and fivefold increase in ADP and AMP, respectively. In hepatocytes from starved rats pentobarbital also lowered ATP levels and inhibited gluconeogenesis but had no effect on either lactate production or the already low level of sugar diphosphate. However, in the fasted case pentobarbital completely prevented the 10-fold elevation of fructose 2,6-bisphosphate brought about by 30 mM glucose. The anesthetic had no effect on cAMP-dependent protein kinase activity or on pyruvate kinase activity in hepatocytes from fed or starved rats but caused reciprocal changes in the activities of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Kinase activity was decreased and bisphosphatase activity was increased. These results suggest that the effects of pentobarbital on gluconeogenesis and glycolysis are due to inhibition of energy metabolism with elevated AMP levels causing activation of 6-phosphofructo-1-kinase and inhibition of fructose 1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of pentobarbital on fructose 2,6-bisphosphate metabolism in isolated rat hepatocytes. 299 99

Although insulin effectively blocked hormone-stimulated glycerol output in adipocytes or phosphorylase activation in hepatocytes, the inhibitory effect of insulin on cAMP analog-stimulated cells depended on the cAMP analog used. Of the 20 analogs tested in adipocytes and 13 tested in hepatocytes, the effects of about half of them were effectively blocked by insulin, whereas the effects of many of them were not inhibited at all. In order to approach the explanation for this discriminative insulin action, the inhibitory effects of insulin on the responses to the analogs in the intact cells were correlated with the in vitro cAMP analog specificity for the hepatocyte cAMP-dependent protein kinase isozymes and the low Km, hormone-sensitive phosphodiesterases from both cell types. No correlation was found between insulin resistance of analog-stimulated hepatocyte phosphorylase and the concentration of analog required in vitro for half-maximal activation of either type I or type II cAMP-dependent protein kinase from hepatocytes. However, a good correlation was found between insulin resistance of cAMP analog-stimulated responses and the analog I50 values for the phosphodiesterase from both cell types. Using a new method capable of measuring hydrolysis at low analog concentrations, several of those analogs which had relatively low, but not high, phosphodiesterase I50 values were shown to be directly hydrolyzed by the low Km adipocyte phosphodiesterase. The insulin inhibition of cell responses when stimulated by hydrolyzable analogs, but not by poorly hydrolyzable analogs, is best explained by insulin stimulation of the low Km phosphodiesterases from both cell types.
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PMID:Discriminative insulin antagonism of stimulatory effects of various cAMP analogs on adipocyte lipolysis and hepatocyte glycogenolysis. 299 37

The induction of granulosa cell differentiation by follicle-stimulating hormone (FSH) is characterized by cellular aggregation, expression of luteinizing hormone (LH) receptors, and biosynthesis of steroidogenic enzymes. These actions of FSH are mediated by activation of adenylate cyclase and cAMP-dependent protein kinase and can be mimicked by choleragen, forskolin, and cAMP analogs. Gonadotropin releasing hormone (GnRH) agonists inhibit these maturation responses in a calcium-dependent manner and promote phosphoinositide turnover. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also prevented FSH-induced cell aggregation and suppressed cAMP formation, LH receptor expression, and progesterone production, with an ID50 of 0.2 nM. In FSH-treated cells, PMA did not reduce the initial increase in cAMP formation during the first 24 hr of culture but prevented its secondary increase from 24 to 48 hr. PMA also inhibited LH receptor induction by cholera toxin, forskolin, and 8-bromo-cAMP, but it did not impair cAMP responses to the former two agents, indicating that the site of action of the phorbol ester is distal to adenylate cyclase. The early stimulation of cAMP-dependent protein kinase activity by FSH was also unaffected by PMA, consistent with its lack of effect on the initial cAMP response to FSH. However, PMA caused a marked decrease in cytosolic protein kinase C activity within 1 min of its addition to the cells. The permeant diacylglycerols, 1-oleoyl-2-acetoyl-sn-glycerol and sn-1,2-dioctanoyl glycerol, also inhibited LH receptor formation, while the nonpermeant diacylglycerol, diolein, was inactive. These results indicate that in situ activation of protein kinase C by PMA or permeant diacylglycerols inhibits cAMP-dependent granulosa cell differentiation, and suggest that the inhibitory actions of GnRH agonists on granulosa cell maturation are also mediated by protein kinase C.
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PMID:Inhibition of gonadotropin-induced granulosa cell differentiation by activation of protein kinase C. 300 7

The bisphosphatase reaction sequence of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase involves a phosphoenzyme intermediate. Catalysis is activated in vitro by cAMP-dependent protein kinase-catalyzed phosphorylation. We investigated the mechanism of this activation by studying the effect of protein kinase-catalyzed phosphorylation on the formation and breakdown of the phosphoenzyme intermediate. The significant findings were as follows. 1) Phosphorylation decreased the rate of phosphoenzyme formation. 2) More importantly, phosphorylation increased the much slower rate of phosphoenzyme breakdown both in the absence and presence of the regulatory ligands, inorganic phosphate and alpha-glycerol phosphate. The increase in the rate of phosphoenzyme breakdown correlated with the degree of activation of the bisphosphatase; both were increased about 2-fold. 3) The potent inhibition of phosphoenzyme breakdown by fructose 6-phosphate indicates that, in the catalytic sequence, the release of nascent fructose 6-phosphate from the active site precedes phosphoenzyme breakdown and Pi release. 4) Phosphorylation reduced the fructose 6-phosphate inhibition of phosphoenzyme breakdown both in the absence and presence of phosphate and alpha-glycerol phosphate. 5) Phosphorylation decreased the potent substrate inhibition which occurs at physiological substrate concentrations. It appears that protein kinase-catalyzed phosphorylation activates fructose-2,6-bisphosphatase by promoting the dissociation of fructose 6-phosphate and fructose 2,6-bisphosphate from the same phosphoenzyme intermediate, hastening its exposure to water and thereby relieving both product and substrate inhibitions.
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PMID:Mechanism of activation of fructose-2,6-bisphosphatase by cAMP-dependent protein kinase. 301 63


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