Adenylate metabolism in the heart. Regulatory properties of rabbit cardiac adenylate deaminase.
Ontology highlight
ABSTRACT:
SUBMITTER: Barsacchi R
PROVIDER: S-EPMC1161315 | biostudies-other | 1979 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Adenylate deaminase (EC 3.5.4.6) may help to regulate the adenine nucleotide catabolism characteristic of such disease states as myocardial ischaemia. We report analysis of the molecular, kinetic and allosteric properties of rabbit heart adenylate deaminase when extracted and purified under phosphate-free conditions (i.e., with Hepes/KOH). The enzyme's subunit molecular mass (approximately 81 kDa), pI (6.5), substrate specificity for 5'-AMP, and activation by K+ were identical in the absence or presence of phosphate. At each chromatographic step during isolation without phosphate, cardiac adenylate deaminase showed a lower apparent activity as compared with the enzyme prepared with phosphate present. Kinetic constants for the phosphate-free rabbit heart adenylate deaminase preparation (Km 0.54 mM AMP; Vmax. 1.4 mumol/min per mg of protein) were approximately 10-fold lower than those of the enzyme isolated with phosphate. The same irreversible decrease in kinetic constants could be achieved by dialysing phosphate from the phosphate-containing enzyme preparation. The relationship between enzyme activity and substrate concentration was sigmoidal in the presence of phosphate, but hyperbolic in its absence. Cardiac adenylate deaminase under phosphate-free conditions was no longer allosterically activated by ATP and ADP, yet remained inhibitable by GTP. Enzyme inhibition by the transition-state mimic coformycin was not influenced by phosphate status. The phosphate-free preparation of rabbit heart adenylate deaminase was markedly labile and extremely susceptible to proteolysis by trypsin or chymotrypsin. The inactivation kinetics and fragmentation pattern in response to controlled proteolysis depended on whether the enzyme had been isolated with or without phosphate present, suggesting a conformational difference between the two enzyme preparations. These data constitute direct evidence that the absence of phosphate irreversibly converts cardiac adenylate deaminase into a pseudo-isoenzyme with distinct kinetic, regulatory and stability properties.
| S-EPMC1138170 | biostudies-other
Project description:Oxidative stress and adenine nucleotide catabolism occur concomitantly in several disease states, such as cardiac ischaemia-reperfusion, and may act as synergistic determinants of tissue injury. However, the mechanisms underlying this potential interaction remain ill-defined. We examined the influence of oxidative stress on the molecular, kinetic and regulatory properties of a ubiquitous AMP-catabolizing enzyme, adenylate deaminase (AMPD) (EC 3.5.4.6). To this intent, rabbit heart AMPD and an H2O2/ascorbate/iron oxidation system were employed. Enzyme exposure to the complete oxidation system acutely impaired its catalytic activity, lowered the Vmax. by 7-fold within 5 min, and rendered the enzyme unresponsive to nucleotide effectors. Irreversible AMPD inactivation resulted within about 15 min of oxidative insult and was not prevented by free-radical scavengers. Oxidative stress did not affect the molecular mass, tetrameric nature, Km, immunoreactivity or trypsinolytic pattern of the enzyme; nor did it induce carbonyl formation, Zn2+ release from the holoenzyme or net AMPD S-thiolation. This injury pattern is inconsistent with a radical-fragmentation mechanism as the basis for the oxidative AMPD inactivation observed. Rather, the sensitivity of the enzyme to both S-thiolation and thiol alkylation and the significant (3 of 9/mol of denatured enzyme) net loss of DTNB-reactive thiols on exposure to oxidant strongly implicate the conversion of essential thiol moieties into stable higher-oxidation states in the oxidative inactivation of cardiac AMPD. The altered thiol status of the enzyme on oxidative insult may prohibit a catalytically permissible conformation and, in so doing, increase AMP availability to 5'-nucleotidase in vivo.
| S-EPMC1136536 | biostudies-other
Project description:We examined the kinetic and regulatory properties of the two isoenzymes of red muscle AMP deaminase, forms A and B, corresponding respectively to the single isoenzymes present in the heart and white skeletal muscle. At the optimal pH value, 6.5, both enzymes show hyperbolic substrate-velocity curves and are inhibited by GTP, inducing sigmoid kinetics. An effect similar to that of GTP is exerted on form B by ATP, whereas form A is almost insensitive to this nucleotide. At pH 7.1 both enzymes follow sigmoid kinetics. ATP enhances the sigmoidicity of the substrate-velocity curve of form B, but it stimulates form A, reverting sigmoidal to hyperbolic kinetics shown by the enzyme at optimal pH. At pH 7.1, form A is also less sensitive to the inhibitory action of Pi and GTP. These results suggest that, owing to the presence of form A, AMP deamination occurs in red muscle also at moderate work intensity. A possible role of this process in counteracting the production of adenosine by 5'-nucleotidase is hypothesized.
| S-EPMC1147790 | biostudies-other
Project description:Adenylate deaminase (AMP deaminase, EC 3.5.4.6) of a high substrate specificity was purified from pig heart by chromatography on cellulose phosphate. The enzyme shows a co-operative binding of AMP [h (Hill coefficient) 2.35, with SO.5 (half-saturating substrate concentration) 5mM]. ATP and ADP act as positive effectors, lowering h to 1.55 and SO.5 to 1 mM. The addition of liposomes (phospholipid bilayers) to ATP-activated or ADP-activated enzyme causes a further shift of the h value to 1.04 and SO.5 to 0.5 mM. For ATP-activated enzyme the addition of liposomes increases Vmax. by about 100%, and for ADP-activated enzyme by 50%. Liposomes have no effect on the kinetics of AMP deaminase in the absence of ATP and ADP, and neither do they influence the inhibitory effect of orthophosphate on heart muscle AMP deaminase. Metabolic implications of these findings are discussed.
| S-EPMC1186110 | biostudies-other
Project description:Heart failure (HF) is associated with metabolic changes that cause a progressive impairment of cardiac and skeletal muscle high-energy phosphate production. As a consequence of the impaired cardiac metabolism, other processes are activated in the failing heart that further exacerbate the progression of HF. The reduced production of high-energy phosphates has important implications for both systole and diastole in HF with both preserved and reduced left ventricular function. The aim of this review is to summarise the state-of-the-art on metabolic therapy in HF with a particular focus on trimetazidine. Metabolic agents optimise cardiac substrate metabolism without exerting negative haemodynamic effects. In particular, as studies with metabolic agents modulating cardiac metabolism have consistently demonstrated, this approach is effective in improving symptoms, functional capacity and prognosis in people with HF when added to optimal medical therapy. Therefore, the modulation of cardiac metabolism is an important therapeutic approach to the treatment of HF, especially in patients where it is of ischaemic or metabolic origin. Although further studies are needed, metabolic agents might be a new, effective strategy for the treatment of HF.
| S-EPMC6125709 | biostudies-literature
Project description:These studies examined the structural specificity for guanine nucleotide-facilitated hormonal activation and guanine nucleotide stabilization of cardiac adenylate cyclase. 1. The phosphonate analogues of GTP, p[CH(2)]ppG (guanosine 5'-[betagamma-methylene]-triphosphate) and pp[CH(2)]pG (guanosine 5'-[alphabeta-methylene]triphosphate), were the most effective activators of adenylate cyclase. Other nucleotides producing significant activation (P<0.01) were, in decreasing order of activation: ITP, GDP, GMP, GTP, XTP, CTP, p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate), dGTP and 2'-O-methyl-GTP. Guanosine, cyclic GMP, UTP and ppppG (guanosine tetraphosphate) had no effect, and 7-methyl-GTP caused a decrease in the activity. 2. Preincubation of membranes at 37 degrees C for 15min before assay at 24 degrees C produced an 80% decrease in adenylate cyclase activity, and preincubation with p[CH(2)]ppG and pp[CH(2)]pG protected and resulted in a net increase in activity. Other nucleotides that completely or partially preserved activity in decreasing order of effectiveness were p[NH]ppG, GDP, GTP, dGTP, ITP, ppppG, 2'-O-methyl-GTP, GMP, CTP and XTP. Several compounds had no effect, including guanosine, cyclic GMP and UTP, whereas preincubation with 7-methyl-GTP produced a further decrease (P<0.05) in activity. 3. The concentration-dependence for activation and stabilization by the naturally occurring guanine nucleotides was examined in the absence of a regenerating system and revealed GMP to have no stabilizing effect and to be less potent than either GDP or GTP in activating adenylate cyclase. 4. A significant correlation (r=0.90) was found between the properties of activation and stabilization for the compounds examined. These findings are consistent with there being a single nucleotide site through which both the activation and stabilization of adenylate cyclase are mediated.
| S-EPMC1161640 | biostudies-other
Project description:Adenylate kinase catalyses the equilibrium 2ADP = ATP + AMP. There are two isoenzymes of adenylate kinase in bovine ventricular tissue, one cytoplasmic, the other mitochondrial. Mitochondrial subfractionation locates this isoenzyme between the mitochondrial membranes with fatty acid-CoA ligase. The cytoplasmic and mitochondrial isoenzymes are distributed in ratio 3:2, and both forms were purified to homogeneity. They differ principally by charge, Km values for ATP, ADP and AMP, pH-stability and -activity profiles, and susceptibility to the inhibitor adenosine pentaphosphoadenosine. The forward and reverse reactions show similar energies of activation for the cytoplasmic enzyme, but differ for the mitochondrial enzyme. The molecular weights are indistinguishable. An integrated mechanism is formulated whereby one isoenzyme suppresses the activation of fatty acid and the other enhances carbohydrate utilization in hypoxic myocytes.
| S-EPMC1158238 | biostudies-other
Project description:Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine.
| S-EPMC2812975 | biostudies-literature
Project description:Aim of this work is to verify if pericytes (Pc) residing in ischemic failing human hearts display altered mechano-transduction properties and to assess which alterations of the mechano-sensing machinery are associated with the observed impaired response to mechanical cues. Results: microvascular rarefaction and defects of YAP/TAZ activation characterize failing human hearts. Although both donor (D-) and explanted (E-) heart derived cardiac pericytes (CPc) support angiogenesis, D-CPc exert this effect significantly better than E-CPc. The latter are characterized by reduced focal adhesion density, decreased activation of the focal adhesion kinase (FAK)/ Crk-associated substrate(CAS) pathway, low expression of caveolin-1, and defective transduction of extracellular stiffness into cytoskeletal stiffening, together with an impaired response to both fibronectin and lysophosphatidic acid. Importantly, Mitogen-activated protein kinase kinase inhibition restores YAP/TAZ nuclear translocation.
2020-11-16 | GSE157205 | GEO
Project description:AimsAdenylate kinase 1 (AK1) catalyses the reaction 2ADP ↔ ATP + AMP, extracting extra energy under metabolic stress and promoting energetic homeostasis. We hypothesised that increased AK1 activity would have negligible effects at rest, but protect against ischaemia/reperfusion (I/R) injury.Methods and resultsCardiac-specific AK1 overexpressing mice (AK1-OE) had 31% higher AK1 activity (P = 0.009), with unchanged total creatine kinase and citrate synthase activities. Male AK1-OE exhibited mild in vivo dysfunction at baseline with lower LV pressure, impaired relaxation, and contractile reserve. LV weight was 19% higher in AK1-OE males due to higher tissue water content in the absence of hypertrophy or fibrosis. AK1-OE hearts had significantly raised creatine, unaltered total adenine nucleotides, and 20% higher AMP levels (P = 0.05), but AMP-activated protein kinase was not activated (P = 0.85). 1H-NMR revealed significant differences in LV metabolite levels compared to wild-type, with aspartate, tyrosine, sphingomyelin, cholesterol all elevated, whereas taurine and triglycerides were significantly lower. Ex vivo global no-flow I/R, caused four-of-seven AK1-OE hearts to develop terminal arrhythmia (cf. zero WT), yet surviving AK1-OE hearts had improved functional recovery. However, AK1-OE did not influence infarct size in vivo and arrhythmias were only observed ex vivo, probably as an artefact of adenine nucleotide loss during cannulation.ConclusionModest elevation of AK1 may improve functional recovery following I/R, but has unexpected impact on LV weight, function and metabolite levels under basal resting conditions, suggesting a more nuanced role for AK1 underpinning myocardial energy homeostasis and not just as a response to stress.
| S-EPMC8044416 | biostudies-literature