Muscle FBPase in a complex with muscle aldolase is insensitive to AMP inhibition

Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism inListeria monocytogenesby inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition ofLactococcus lactisPC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. InL. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool inL. lactisis negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP–insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

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Roles of Cyclic Nucleotides in the Inhibition of Platelet Function by Physiolocic and Pharmacological Agents

10.1055/s-0039-1684475 ◽

1979 ◽

Author(s):

R.J. Haslam

Keyword(s):

Platelet Aggregation ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Platelet Function ◽

Inhibitory Effects ◽

Pharmacological Agents ◽

Arterial Blood ◽

Cyclic Cmp ◽

Amp Inhibition

Cyclic AMP mediates the inhibitions of platelet aggregation caused by PCI2, PGE1 and PGD2. Thus, these compounds activate platelet adenylate cyclase and Increase platelet cyclic AMP; their inhibitory effects are blockod by inhibitor? of adenylate cyclase, are potentiated by inhibitors of cyclic AKP phosphodiesterase and are mimicked hy N6 ,2'-0-dibutyryl cyclic AMP. Inhibition of adenylate cyclase does not potentiate platelet aggregation in the absence of inhibitory prostaglandins, indicating that platelet cyclic AMP is too low to affect aggregation under these conditions. To determine whether platelets in the circulation are exposed to agents that increase platelet cyclic AMP, washed rabbi platelets labelled with [3H] adenine were incubated with rabbit arterial blood under various conditions; any increases in cyclic [3H]AMP were measured. These experiments showed that freshly taken rabbit arterial blood does not normally contain any factors that can increase platelet cyclic AMP sufficiently to affect platelet function; specifically, circulating PGI2 was less than 0.1 pmol/ml of blood. It follows that increases in cyclic AMP in circulating rabbit platelets must occur only locally or under special conditions. The role of the moderate increases in platelet cyclic CMP caused by aggregating agents remains uncertain, but the inhibition of aggregation by compounds such as sodium nitroprusside that increase cyclic CMP up to 100-fold suggests that cyclic CMP may, like cyclic AMP, be an inhibitory mediator.

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Fatty acids reverse the cyclic AMP inhibition of triacylglycerol and phosphatidylcholine synthesis in rat hepatocytes

Biochemical Journal ◽

10.1042/bj2160129 ◽

1983 ◽

Vol 216 (1) ◽

pp. 129-136 ◽

Cited By ~ 32

Author(s):

S L Pelech ◽

P H Pritchard ◽

D N Brindley ◽

D E Vance

Keyword(s):

Fatty Acids ◽

Cyclic Amp ◽

Rat Hepatocytes ◽

Phosphocholine Cytidylyltransferase ◽

Triacylglycerol Synthesis ◽

Monolayer Cultures ◽

Membrane Bound ◽

Amp Inhibition ◽

Phosphatidylcholine Synthesis

The influence of cyclic AMP analogues and fatty acids on glycerolipid biosynthesis in monolayer cultures of rat hepatocytes was investigated. Chlorophenylthio-cyclic AMP and adenosine 3′:5′-cyclic phosphorothioate inhibited the rate of triacylglycerol synthesis from [1(3)-3H]glycerol, and phosphatidylcholine synthesis from [Me-3H]-choline. Supplementation of the hepatocytes with palmitate (1 mM) reversed chlorophenylthio-cyclic AMP inhibition of triacylglycerol synthesis. Similarly, cyclic AMP analogue-inhibition of phosphatidylcholine synthesis was abolished when the cells were simultaneously incubated with oleate (3 mM). Reactivation of phosphatidylcholine synthesis in chlorophenylthio-cyclic AMP-supplemented cells with oleate was accompanied by conversion of CTP: phosphocholine cytidylyltransferase into the membrane-bound form, since these cells released the enzyme more slowly after treatment with digitonin. The opposing actions of cyclic AMP and fatty acids are discussed in relation to the regulation of glycerolipid biosynthesis during starvation, diabetes and stress.

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Calcium-mediated cyclic AMP inhibition of Na-H exchange in small intestine

AJP Cell Physiology ◽

10.1152/ajpcell.1987.252.3.c315 ◽

1987 ◽

Vol 252 (3) ◽

pp. C315-C322 ◽

Cited By ~ 54

Author(s):

C. E. Semrad ◽

E. B. Chang

Keyword(s):

Small Intestine ◽

Cyclic Amp ◽

Ionophore A23187 ◽

Isolated Enterocytes ◽

Direct Measurements ◽

Intracellular Ca ◽

Buffering Agent ◽

20 Nm ◽

Amp Inhibition ◽

Na Uptake

8-Bromo cyclic AMP (cAMP) (10(-4) M) inhibits Na absorption in isolated chicken enterocytes as has been reported previously. Direct measurements of intracellular pH (pHi) using 5,6-carboxyfluorescein diacetate showed that both 8-bromo cAMP and the diuretic amiloride (10(-3) M) stimulated a persistent decrease in pHi of approximately 0.1 pH units, effects that were Na dependent and were not additive when cells were stimulated with both agents. These results suggest inhibition of an amiloride-sensitive Na/H exchange by cAMP. Direct measurements of intracellular Ca [Ca]i were also made using quin 2. 8-Bromo cAMP (10(-4) M) stimulated an immediate and persistent (greater than 10 min) increase in [Ca]i of approximately 20 nM, an effect that was not dependent on extracellular Ca. Pretreatment of cells with the specific calmodulin inhibitor calmidazolium (10(-7) M) and the intracellular Ca-buffering agent MAPTAM blocked cAMP's effects on pH and Na uptake, but did not interfere with amiloride's effects. An increase in [Ca]i stimulated by the Ca ionophore A23187 (10(-6) M) was sufficient by itself to decrease pHi and inhibit amiloride-sensitive Na influx in isolated enterocytes. We conclude that cAMP stimulates the release of endogenous Ca in isolated enterocytes. This increase in [Ca]i appears to be essential for inhibition of amiloride-sensitive Na-H exchange by this cyclic nucleotide.

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Different Sensitivities of Mutants and Chimeric Forms of Human Muscle and Liver Fructose- 1,6-Bisphosphatases towards AMP

Biological Chemistry ◽

10.1515/bc.2003.006 ◽

2003 ◽

Vol 384 (1) ◽

pp. 51-58 ◽

Cited By ~ 18

Author(s):

D. Rakus ◽

H. Tillmann ◽

R. Wysocki ◽

S. Ulaszewski ◽

K. Eschrich ◽

...

Keyword(s):

Liver Enzyme ◽

Human Liver ◽

Human Muscle ◽

Wild Type ◽

Muscle Enzyme ◽

Double Mutation ◽

Binding Domains ◽

Fructose 1,6 Bisphosphatase ◽

Amp Inhibition ◽

Acid Exchange

Abstract AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 uM and 4.8 uM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the Nterminal region of the muscle enzyme towards liver FBPase (Lys20→Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20→Lys and double mutation Glu19→Asp/Glu20→Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 uM). The decrease of sensitivity for AMP of the muscle mutant Lys20→Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20→Lys and Glu19→Asp/Glu20→Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.

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