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:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Evidence has been published that L -alanine may, under appropriate conditions, promote insulin secretion in normal rodent islets and various beta cell lines. Previous results utilising the clonal beta-cell line BRIN-BD11, demonstrated that alanine dramatically elevated insulin release by a mechanism requiring oxidative metabolism. We demonstrate in this paper that addition ofL -alanine had an insulinotropic effect in dispersed primary islet cells. Addition of D -glucose increasedL -alanine consumption in both BRIN-BD11 cells and primary islet cells.L -glutamine consumption in the BRIN-BD11 cell line and primary rat islets was also determined. The consumption rate was in line with that previously reported for cells of the immune system and other glutamine-utilising cells or tIssues. However,L -alanine consumption was at least an order of magnitude higher thanL -glutamine consumption. The metabolism ofL -alanine in the beta-cell may result in stimulation of insulin secretion via generation of metabolic stimulus secretion coupling factors such asL -glutamate.
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PMID:A comparative study of amino acid consumption by rat islet cells and the clonal beta-cell line BRIN-BD11 - the functional significance of L-alanine. 1465 14

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.
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PMID:Ischemic preconditioning exaggerates cardiac damage in PKC-delta null mice. 1527 9

In the catalytic mechanism of ATP synthase, phosphate (P(i)) binding and release steps are believed to be correlated to gamma-subunit rotation, and P(i) binding is proposed to be prerequisite for binding ADP in the face of high cellular [ATP]/[ADP] ratios. In x-ray structures, residue betaAsn-243 appears centrally located in the P(i)-binding subdomain of catalytic sites. Here we studied the role of betaAsn-243 in Escherichia coli ATP synthase by mutagenesis to Ala and Asp. Mutation betaN243A caused 30-fold impairment of F(1)-ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and P(i) protected from inhibition. ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory. betaN243D F(1)-ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADP-fluoroscandium. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated betaN243D F(1)-ATPase, and P(i) did not affect activation. We conclude that residue betaAsn-243 is not involved in P(i) binding directly but is necessary for correct organization of the transition state complex through extensive involvement in hydrogen bonding to neighboring residues. It is also probably involved in orientation of the "attacking water" and of an associated second water.
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PMID:Role of betaAsn-243 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F(1)-ATPase. 1532 26

It is known from earlier work that two conserved Glu residues, designated "catalytic carboxylates," are critical for function in P-glycoprotein (Pgp). Here the role of these residues (Glu-552 and Glu-1197 in mouse MDR3 Pgp) was studied further. Mutation E552Q or E1197Q reduced Pgp-ATPase to low but still measurable rates. Two explanations previously offered for effects of these mutations, namely that ADP release is slowed or that a second (drug site-resetting) round of ATP hydrolysis is blocked, were evaluated and appeared unsatisfactory. Thus the study was extended to include E552A, -D, and -K and E1197A, -D, and -K mutants. All reduced ATPase to similar low but measurable rates. Orthovanadate-trapping experiments showed that mutation to Gln, Ala, Asp, or Lys altered characteristics of the transition state but did not eliminate its formation in contrast e.g. with mutation of the analogous catalytic Glu in F1-ATPase. Retention of ATP as well as ADP was seen in Ala, Asp, and Lys mutants. Mutation E552A in nucleotide binding domain 1 (NBD1) was combined with mutation S528A or S1173A in the LSGGQ sequence of NBD1 or NBD2, respectively. Synergistic effects were seen. E552A/S1173A had extremely low turnover rate for ATPase, while E552A/S528A showed zero or close to zero ATPase. Both showed orthovanadate-independent retention of ATP and ADP. We propose that mutations of the catalytic Glu residues interfere with formation and characteristics of a closed conformation, involving an interdigitated NBD dimer interface, which normally occurs immediately following ATP binding and progresses to the transition state.
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PMID:Properties of P-glycoprotein with mutations in the "catalytic carboxylate" glutamate residues. 1532 76

Specific amino acids are now known to acutely and chronically regulate insulin secretion from pancreatic beta-cells in vivo and in vitro. Understanding the molecular mechanisms by which amino acids regulate insulin secretion may identify novel targets for future diabetes therapies. Mitochondrial metabolism is crucial for the coupling of amino acid and glucose recognition to the exocytosis of the insulin granules. This is illustrated by in vitro and in vivo observations discussed in the present review. Mitochondria generate ATP, which is the main coupling factor in insulin secretion; however, the subsequent Ca2+ signal in the cytosol is necessary, but not sufficient, for full development of sustained insulin secretion. Hence mitochondria generate ATP and other coupling factors serving as fuel sensors for the control of the exocytotic process. Numerous studies have sought to identify the factors that mediate the amplifying pathway over the Ca2+ signal in nutrient-stimulated insulin secretion. Predominantly, these factors are nucleotides (GTP, ATP, cAMP and NADPH), although metabolites have also been proposed, such as long-chain acyl-CoA derivatives and the key amino acid glutamate. This scenario highlights further the importance of the key enzymes or transporters, glutamate dehydrogenase, the aspartate and alanine aminotransferases and the malate/aspartate shuttle, in the control of insulin secretion. Therefore amino acids may play a direct or indirect (via generation of putative messengers of mitochondrial origin) role in insulin secretion.
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PMID:New insights into amino acid metabolism, beta-cell function and diabetes. 1554 73

The conserved residue Gly47 of the chloroplast ATP synthase beta subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants epsilon G47L, epsilon G47R, epsilon G47A and epsilon G47E, respectively. All the beta variants showed lower inhibitory effects on the soluble CF1(-epsilon) Ca2+-ATPase compared with wild-type epsilon. In reduced conditions, epsilon G47E and epsilon G47R had a lower inhibitory effect on the oxidized CF1(-epsilon) Ca2+-ATPase compared with wild-type epsilon. In contrast, epsilon G47L and epsilon G47A increased the Ca2+-ATPase activity of soluble oxidized CF1(-epsilon). The replacement of Gly47 significantly impaired the interaction between the subunit epsilon and gamma in an in vitro binding assay? Further study showed that all epsilon variants were more effective in blocking proton leakage from the thylakoid membranes. This enhanced ATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to a level that was more efficient than that achieved by wild-type epsilon. These results indicate that the conserved Gly47 residue of the epsilon subunit is very important for maintaining the structure and function of the epsilon subunit and may affect the interaction between the epsilon subunit, beta subunit of CF1 and subunit III of CFo, thereby regulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit III of CFo.
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PMID:Substitutions of the conserved Gly47 affect the CF1 inhibitor and proton gate functions of the chloroplast ATP synthase epsilon subunit. 1599 6

ATP synthesis from ADP, P(i), and Mg2+ takes place in mitochondria on the catalytic F1 unit (alpha3beta3gammedeltaepsilon) of the ATP synthase complex (F0F1), a remarkable nanomachine that interconverts electrochemical and mechanical energy, producing the high energy terminal bond of ATP. In currently available structural models of F1, the P-loop (amino acid residues 156GGAGVGKT163) contributes to substrate binding at the subunit catalytic sites. Here, we report the first transition state-like structure of F1 (ADP.V(i).Mg.F1) from rat liver that was crystallized with the phosphate (P(i)) analog vanadate (VO(3-)4 or V(i)). Compared with earlier "ground state" structures, this new F1 structure reveals that the active site region has undergone significant remodeling. P-loop residue alanine 158 is located much closer to V(i) than it is to P(i) in a previous structural model. No significant movements of P-loop residues of the subunit were observed at its analogous but noncatalytic sites. Under physiological conditions, such active site remodeling involving the small hydrophobic alanine residue may promote ATP synthesis by lowering the local dielectric constant, thus facilitating the dehydration of ADP and P(i). This new crystallographic study provides strong support for the catalytic mechanism of ATP synthesis deduced from earlier biochemical studies of liver F1 conducted in the presence of V(i) (Ko, Y. H., Bianchet, M., Amzel, L. M., and Pedersen, P. L. (1997) J. Biol. Chem. 272, 18875-18881; Ko, Y. H., Hong, S., and Pedersen, P. L. (1999) J. Biol. Chem. 274, 28853-28856).
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PMID:Mitochondrial ATP synthase. Crystal structure of the catalytic F1 unit in a vanadate-induced transition-like state and implications for mechanism. 1653 9

The b subunit dimer of Escherichia coli ATP synthase serves essential roles as an assembly factor for the enzyme and as a stator during rotational catalysis. To investigate the functional importance of its coiled coil dimerization domain, a series of internal deletions including each individual residue between Lys-100 and Ala-105 (b(deltaK100)-b(deltaA105)), b(deltaK100-A103), and b(deltaK100-Q106) as well as a control b(K100A) missense mutation were prepared. All of the mutants supported assembly of ATP synthase, but all single-residue deletions failed to support growth on acetate, indicating a severe defect in oxidative phosphorylation, and b(deltaK100-Q106) displayed moderately reduced growth. The membrane-bound ATPase activities of these strains showed a related reduction in sensitivity to dicyclohexylcarbodiimide, indicative of uncoupling. Analysis of dimerization of the soluble constructs of b(deltaK100) and the multiple-residue deletions by sedimentation equilibrium revealed reduced dimerization compared with wild type for all deletions, with b(deltaK100-Q106) most severely affected. In cross-linking studies it was found that F1-ATPase can mediate the dimerization of some soluble b constructs but did not mediate dimerization of b(deltaK100) and b(deltaK100-Q106); these two forms also were defective in F1 binding analyses. We conclude that defective dimerization of soluble b constructs severely affects F1 binding in vitro, yet allows assembly of ATP synthase in vivo. The highly uncoupled nature of enzymes with single-residue deletions in b indicates that the b subunit serves an active function in energy coupling rather than just holding on to the F1 sector. This function is proposed to depend on proper, specific interactions between the b subunits and F1.
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PMID:Mutations in the dimerization domain of the b subunit from the Escherichia coli ATP synthase. Deletions disrupt function but not enzyme assembly. 1653 10

Elevated plasma homocysteine has been reported in individuals with diseases of the metabolic syndrome including vascular disease and insulin resistance. As homocysteine exerts detrimental effects on endothelial and neuronal cells, this study investigated effects of acute homocysteine exposure on beta-cell function and insulin secretion using clonal BRIN-BD11 beta-cells. Acute insulin release studies in the presence of various test reagents were performed using monolayers of BRIN-BD11 cells and samples assayed by insulin radioimmunoassay. Cellular glucose metabolism was assessed by nuclear magnetic resonance (NMR) analysis following 60-min exposure of BRIN-BD11 cell monolayers to glucose in either the absence or presence of homocysteine. Homocysteine dose-dependently inhibited insulin release at moderate and stimulatory glucose concentrations. This inhibitory effect was reversible at all but the highest concentration of homocysteine. 13C-glucose NMR demonstrated decreased labelling of glutamate from glucose at positions C2, C3 and C4, indicating that the tricarboxylic acid (TCA) cycle-dependent glucose metabolism was reduced in the presence of homocysteine. Homocysteine also dose-dependently inhibited insulinotropic responses to a range of glucose-dependent secretagogues including nutrients (alanine, arginine, 2-ketoisocaproate), hormones (glucagon-like peptide-1 (7-36)amide, gastric inhibitory polypeptide and cholecystokinin-8), neurotransmitter (carbachol), drug (tolbutamide) as well as a depolarising concentration of KCl or elevated Ca2+. Insulin secretion induced by activation of adenylate cyclase and protein kinase C pathways with forskolin and phorbol 12-myristate 13-acetate were also inhibited by homocysteine. These effects were not associated with any adverse action on cellular insulin content or cell viability, and there was no increase in apoptosis/necrosis following exposure to homocysteine. These data indicate that homocysteine impairs insulin secretion through alterations in beta-cell glucose metabolism and generation of key stimulus-secretion coupling factors. The participation of homocysteine in possible beta-cell demise merits further investigation.
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PMID:Detrimental actions of metabolic syndrome risk factor, homocysteine, on pancreatic beta-cell glucose metabolism and insulin secretion. 1664 97

The F(1)F(o)-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F(1) moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F(1) complexes from this thermoalkaliphile. Like the native F(1)F(o)-ATP synthase, the recombinant TA2F(1) was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the epsilon subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F(1) mutant with either a truncated epsilon subunit [i.e., TA2F(1)(epsilon(DeltaC))] or substitution of basic residues in the second alpha-helix of epsilon with nonpolar alanines [i.e., TA2F(1)(epsilon(6A))] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F(1)F(o)-ATP synthase and TA2F(1)(epsilon(WT)) complex with proteases revealed that the epsilon subunit was resistant to proteolytic digestion. In contrast, the epsilon subunit of TA2F(1)(epsilon(6A)) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that epsilon was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the epsilon subunit in the entire holoenzyme, we reconstituted recombinant TA2F(1) complexes with F(1)-stripped native membranes of strain TA2.A1. The reconstituted TA2F(o)F(1)(epsilon(WT)) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F(1)F(o)-ATP synthase in native membranes. Reconstituted TA2F(o)F(1)(epsilon(6A)) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the epsilon subunit also inhibits ATPase activity of TA2F(o)F(1).
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PMID:Inhibition of ATP hydrolysis by thermoalkaliphilic F1Fo-ATP synthase is controlled by the C terminus of the epsilon subunit. 1670 72


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