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The food enzyme AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) is produced with the non‐genetically modified microorganism Aspergillus sp. strain DEA 56‐111 by Shin Nihon Chemical Co., Ltd. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.005 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The Panel identified a no observed adverse effect level of 1984 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 396,800. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

The food enzyme AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) is produced with the non‐genetically modified Streptomyces murinus strain AE‐DNTS by Amano Enzyme Inc. The food enzyme is free from viable cells. It is intended to be used in yeast processing and the production of mushroom extracts. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.0004 mg TOS/kg body weight (bw) per day in European populations. The food enzyme batches, including the batch used in the toxicological studies, were not fully characterised. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. In the absence of adequate toxicological data, the Panel could not conclude on the safety of the food enzyme AMP deaminase from the non‐genetically modified Streptomyces murinus strain AE‐DNTS. [ABSTRACT FROM AUTHOR]

Systemic branched‐chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long‐Evans‐Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched‐chain α‐keto acid dehydrogenase (BCKDH), a rate‐limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long‐Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH‐E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3‐E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3‐BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3‐BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Lipid biosynthesis is a significant metabolic response to nitrogen starvation in oleaginous fungi. The oleaginous fungus Mucor circinelloides copes with nitrogen stress by degrading AMP through AMP deaminase (AMPD). However, the mechanism of AMPD in regulating lipogenesis remains largely unclear. To elucidate the mechanism of AMPD in lipid synthesis in this M. circinelloides , we identified two genes (ampd1 and ampd2) encoding AMPD and constructed an ampd double knockout mutant. The engineered M. circinelloides strain elevated cell growth and lipid accumulation, as well as the content of oleic acid (OA) and gamma-linolenic acid (GLA). In addition to the expected increase in transcription levels of genes associated with lipid and TAG synthesis, we observed suppression of lipid degradation and reduced amino acid biosynthesis. This suggested that the deletion of AMPD genes induces the redirection of carbon towards lipid synthesis pathways. Moreover, the pathways related to nitrogen metabolism, including nitrogen assimilation and purine metabolism (especially energy level), were also affected in order to maintain homeostasis. Further analysis discovered that the transcription factors (TFs) related to lipid accumulation were also regulated. This study provides new insights into lipid biosynthesis in M. circinelloides , indicating that the trigger for lipid accumulation is not entirely AMPD-dependent and suggest that there may be additional mechanisms involved in the initiation of lipogenesis. [Display omitted] • Deletion of ampd genes increases lipogenesis in Mucor circinelloides. • AMPD plays an essential role in the regulation of lipid and nitrogen metabolism. • Deletion of ampd genes regulates energy metabolism to maintain homeostasis. • TFs involved in lipogenesis are regulated after knockout of ampd genes. [ABSTRACT FROM AUTHOR]

We previously reported that upregulated AMP deaminase (AMPD) contributes to diastolic ventricular dysfunction via depletion of the adenine nucleotide pool in a rat model of type 2 diabetes (T2DM), Otsuka Long-Evans-Tokushima Fatty rats (OLETF). Meanwhile, AMPD promotes the formation of substrates of xanthine oxidoreductase (XOR), which produces ROS as a byproduct. Here, we tested the hypothesis that a functional link between upregulated AMPD and XOR is involved in ventricular dysfunction in T2DM rats. Pressure-volume loop analysis revealed that pressure overloading by phenylephrine infusion induced severer left ventricular diastolic dysfunction (tau: 14.7 ± 0.8 vs 12.5 ± 0.7 msec, left ventricular end-diastolic pressure: 18.3 ± 1.5 vs 12.2 ± 1.3 mmHg, p < 0.05) and ventricular-arterial uncoupling in OLETF than in LETO, non-diabetic rats, though the baseline parameters were comparable in the two groups. While the pressure overload did not affect AMPD activity, it increased XOR activity both in OLETF and LETO, with OLETF showing significantly higher XOR activity than that in LETO (347.2 ± 17.9 vs 243.2 ± 6.1 μg/min/mg). Under the condition of pressure overload, myocardial ATP level was lower, and levels of xanthine and uric acid were higher in OLETF than in LETO. Addition of exogenous inosine, a product of AMP deamination, to the heart homogenates augmented XOR activity. OLETF showed 68% higher tissue ROS levels and 47% reduction in mitochondrial state 3 respiration compared with those in LETO. Overexpression of AMPD3 in H9c2 cells elevated levels of hypoxanthine and ROS and reduced the level of ATP. Inhibition of XOR suppressed the production of tissue ROS and mitochondrial dysfunction and improved ventricular function under the condition of pressure overload in OLETF. The results suggest that increases in the activity of XOR and the formation of XOR substrates by upregulated AMPD contribute to ROS-mediated diastolic ventricular dysfunction at the time of increased cardiac workload in diabetic hearts. [Display omitted] • AMPD is upregulated in type 2 diabetic (T2DM) hearts. • Pressure overload unmasked latent diastolic dysfunction in T2DM hearts. • Activity and substrates of XOR were increased in pressure-overloaded T2DM hearts. • Upregulated AMPD leads to ATP depletion and ROS production. • Inhibition of XOR restored mitochondrial and ventricular functions in T2DM hearts. [ABSTRACT FROM AUTHOR]

Summary: Ascospores generated during sexual reproduction are the primary inoculum for the wheat scab fungus Fusarium graminearum. Purine metabolism is known to play important roles in fungal pathogens but its lifecycle stage‐specific regulation is unclear.By characterizing the genes involved in purine de novo and salvage biosynthesis pathways, we showed that de novo syntheses of inosine, adenosine and guanosine monophosphates (IMP, AMP and GMP) are important for vegetative growth, sexual/asexual reproduction, and infectious growth, whereas purine salvage synthesis is dispensable for these stages in F. graminearum.Addition of GMP rescued the defects of the Fgimd1 mutant in vegetative growth and conidiation but not sexual reproduction, whereas addition of AMP rescued all of these defects of the Fgade12 mutant, suggesting that the function of de novo synthesis of GMP rather than AMP is distinct in sexual stages. Moreover, Acd1, an ortholog of AMP deaminase, is dispensable for growth but essential for ascosporogenesis and pathogenesis, suggesting that AMP catabolism has stage‐specific functions during sexual reproduction and infectious growth. The expression of almost all the genes involved in de novo purine synthesis is downregulated during sexual reproduction and infectious growth relative to vegetative growth.This study revealed that F. graminearum has stage‐specific regulation of purine metabolism during infectious growth and sexual reproduction. [ABSTRACT FROM AUTHOR]

Because metabolism is a complex balanced process involving multiple enzymes, understanding how organisms compensate for transient or permanent metabolic imbalance is a challenging task that can be more easily achieved in simpler unicellular organisms. The metabolic balance results not only from the combination of individual enzymatic properties, regulation of enzyme abundance, but also from the architecture of the metabolic network offering multiple interconversion alternatives. Although metabolic networks are generally highly resilient to perturbations, metabolic imbalance resulting from enzymatic defect and specific environmental conditions can be designed experimentally and studied. Starting with a double amd1 aah1 mutant that severely and conditionally affects yeast growth, we carefully characterized the metabolic shuffle associated with this defect. We established that the GTP decrease resulting in an adenylic/guanylic nucleotide imbalance was responsible for the growth defect. Identification of several gene dosage suppressors revealed that TAT1, encoding an amino acid transporter, is a robust suppressor of the amd1 aah1 growth defect. We show that TAT1 suppression occurs through replenishment of the GTP pool in a process requiring the histidine biosynthesis pathway. Importantly, we establish that a tat1 mutant exhibits synthetic sickness when combined with an amd1 mutant and that both components of this synthetic phenotype can be suppressed by specific gene dosage suppressors. Together our data point to a strong phenotypic connection between amino acid uptake and GTP synthesis, a connection that could open perspectives for future treatment of related human defects, previously reported as etiologically highly conserved. [ABSTRACT FROM AUTHOR]

Highlights • Bioengineering of Y. lipolytica guaranteed lycopene production. • Adjusting crt genes copy numbers led to a high lycopene yield. • AMPD overexpression improved lycopene production. • Fed-batch fermentation showed feasibility for large scale lycopene production. Abstract Lycopene has been broadly studied in recent decades due to its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. To obtain efficient synthesis of lycopene, extensive researches have been conducted in various microbial cells, including Yarrowia lipolytica , to heterologously produce lycopene using various genetic and metabolic engineering methods. In this study, the effects of copy numbers of lycopene synthesis genes, a variety of key central metabolic genes (especially AMP deaminase-encoding gene AMPD), and 5-L fermenter cultivation on lycopene production in Y. lipolytica were investigated and the engineered strains with significantly enhanced lycopene content (46–60 mg/g DCW) were achieved. It is therefore possible to make use of the obtained strains to meet the industrial demand of lycopene production on the basis of further genetic and process optimization. [ABSTRACT FROM AUTHOR]

The AMP deaminase 1 (AMPD1) gene plays an important role in purine nucleotide interconversion and energy metabolism. In this study, two novel single nucleotide polymorphisms (SNPs) (g.-626 G > A and g.-566 A>G) were detected in the proximal promoter region of the AMPD1 gene. The Chinese indigenous pig breeds (Meishan and Tongcheng) had higher frequencies of the G and A alleles than Western meat-type breeds (Landrace and Large White) at the g.-626 G > A and g.-566 A>G loci. The transcriptional activity of the AMPD1 promoter carrying the haplotype H1 (A−626G−566) was significantly (p < 0.05) higher than that of the haplotype H2 (G−626A−566). In addition, pigs with the haplotype combination H1H1 had significantly (p < 0.05) higher mRNA expression levels of the AMPD1 gene than those with haplotype combinations H1H2 and H2H2 in two different skeletal muscles. Association analyses revealed that the pigs with the haplotype combination H1H1 had significantly higher lean meat percentage values but lower average backfat thickness (ABT, cm), buttock fat thickness (cm), and thorax-waist fat thickness (cm) values than the pigs with the haplotype combinations H1H2 and H2H2. These findings demonstrate that the two novel SNPs in the promoter region of the AMPD1 gene have significant associations with pig fat deposition traits. [ABSTRACT FROM AUTHOR]

AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism. Recently we found that upregulated AMPD activity is associated with ATP depletion and contractile dysfunction under the condition of pressure overloading in the heart of a rat model of type 2 diabetes mellitus (T2DM), OLETF. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates by 55% in OLETF compared to those in LETO, a non-diabetic control. In contrast, the mRNA levels of AMPD3 in the myocardium were similar in OLETF and LETO. AMPD3 was comparably ubiquitinated in OLETF and LETO, and its degradation ex vivo was more sensitive to MG-132, a proteasome inhibitor, in OLETF than in LETO. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3’UTR of AMPD3 mRNA. AMPD3 protein level was significantly increased by a miR-301b inhibitor and was decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay confirmed binding of miR-301b to the 3′UTR of AMPD3 mRNA. Transfection of neonatal rat cardiomyocytes with a miR-301b inhibitor increased 90-kDa AMPD3 and reduced ATP level. The results indicate that translational regulation by miR-301b mediates upregulated expression of cardiac AMPD3 protein in OLETF, which potentially reduces the adenine nucleotide pool at the time of increased work load. The miR-301b-AMPD3 axis may be a novel therapeutic target for intervening enegy metabolism in diabetic hearts. [ABSTRACT FROM AUTHOR]

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that is synthesized by parenchymal liver cells. Using Western blot analysis and immunoperoxidase techniques, we have previously shown the presence of HRG in human skeletal muscle. This paper reports the results of immunofluorescence experiments carried out on sections of human normal skeletal muscle biopsies to investigate the subcellular localization of HRG. The HRG localization was also compared with that of skeletal muscle AMP deaminase (AMPD1), since we have previously described an association of the enzyme with the protein. The obtained results give evidence for a preferential localization of HRG at the I-band level, where it shows the same distribution of actin and where AMPD1 is present in major concentration. [ABSTRACT FROM AUTHOR]

5′-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6 U/g of AMP deaminase at 32 °C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40 °C and 6.0, respectively, but it was partially inhibited by Fe 3+ , Cu 2+ , Al 3+ , and Zn 2+ . With amplification of the AMP deaminase production system, 6 mL of crude enzyme could produce 2.00 mg/g of IMP from 2.04 mg/g of dried yeast with an 84.8% molar yield after 40 min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5′-IMP. [ABSTRACT FROM AUTHOR]

Similar symptoms observed in Myasthenia gravis (MG) can be also detected in the case of skeletal muscle AMP-deaminase deficiency. We compared the activity and expression of AMP- deaminase (AMPD) products in skeletal muscles of MG patients and MG-free individuals. The activity of AMP-deaminase in the muscles of MG patients was significantly higher than in the controls and was 2.05 µmol/min/mg protein (±0.31). The two groups differ in level of AMPD product expression. Furthermore in MG-group molecular size of isoform AMPD1 is 90 kDa in contrast to MG-free group where is present 70 kDa isoform of enzyme. The data suggests that the disturbances in transmission of neuronal signaling, taking place in the skeletal muscles of MG patients, may also change energetic metabolism of the affected muscles by changing molecular mass of isoform. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5′NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5′NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling. [ABSTRACT FROM AUTHOR]

Background: Inflammation plays a major role in the development and progression of atherosclerosis and coronary artery disease (CAD). Inflammation markers, including white blood cell (WBC) count, C-reactive protein (CRP) and interleukin-6 (IL-6), are widely used for cardiovascular risk prediction. The aim of the study was to establish factors associated with WBC, CRP and IL-6 in patients with CAD. Two functional polymorphisms in genes encoding enzymes participating in adenosine metabolism were analyzed (C34T AMPD1, G22A ADA). Methods: Plasma concentrations of IL-6 were measured using high-sensitivity ELISA kits, and the nephelometric method was used for high-sensitivity CRP (hs-CRP) measurement in 167 CAD patients. Results: Presence of metabolic syndrome (MS) and its components, presence of heart failure, severity of CAD symptoms, severe past ventricular arrhythmia (sustained ventricular tachycardia [sVT] or ventricular fibrillation [VF]), lower left ventricle ejection fraction, higher left ventricle mass index, higher end-diastolic volume and higher number of smoking pack-years were significantly associated with higher WBC, CRP and IL-6. Strong associations with arrhythmia were observed for IL-6 (median 3.90 vs 1.89 pg/mL, p<0.00001) and CRP concentration (6.32 vs 1.47 mg/L, p=0.00009), while MS was associated most strongly with IL-6. CRP and IL-6 were independent markers discriminating patients with sVT or VF. There were no associations between AMPD1 or ADA genotypes and inflammation markers. Conclusions: WBC, CRP and IL-6 are strongly associated with components of the metabolic syndrome. Their strong association with life-threatening ventricular arrhythmia emphasizes the proarrhythmic role of inflammation in the increased cardiovascular risk of CAD patients. [ABSTRACT FROM AUTHOR]

Adenosine-5′-monophosphate (AMP) deaminase is an enzyme used to increase concentrations of 5’-inosine monophosphate in certain foods and beverages for flavoring purposes. One commercial source of this enzyme is Aspergillus oryzae , a filamentous fungus with a history of safe use in Asia as a fermentation organism used in the production of miso sauce and sake liquors. Noting the use of the enzyme in food intended for human consumption and potential presence at trace levels in finished goods, a series of safety studies including an in vitro Ames test and chromosome aberration assay with Chinese hamster lung fibroblasts were conducted along with a 90-day oral toxicity study in rats. AMP deaminase showed no evidence of genotoxicity in the in vitro tests. Following gavage administration of Sprague–Dawley rats at dosages of 19.8, 198.4, or 1984 mg total organic solids (TOS)/kg body weight (bw)/day for 90 days, no adverse effects on body weight gain, food consumption, hematology, clinical chemistry, urinalysis, ophthalmological and histopathological examinations were observed. The no-observed-adverse-effect level was considered to be 1984 mg TOS/kg bw/day, the highest dose tested. Results of the genotoxicity studies and subchronic rat study support the safe use of AMP deaminase produced from A. oryzae in food production. [ABSTRACT FROM AUTHOR]

Histidine-proline-rich glycoprotein (HPRG), or histidine-rich glycoprotein (HRG), is a serum protein that is synthesized in the liver and is actively internalised by different cells, including skeletal muscle. The multidomain arrangement of HPRG comprises two modules at the N-terminus that are homologous to cystatin but void of cysteine proteinase inhibitor function, and a second half consisting of a histidine-proline-rich region (HPRR) located between two proline-rich regions (PRR1 and PRR2), and a C-terminus domain. HPRG has been reported to bind various ligands and to modulate angiogenesis via the histidine residues of the HPRR. However, the secondary structure prediction of the HPRR reveals that more than 98% is disordered and the structural basis of the hypothesized functions remains unclear. Comparison of the PRR1 of several mammalian species indicates the presence of a conserved binding site that might coordinate the Zn 2+ ion with an amino acid arrangement compatible with the cysteine-containing site that has been identified experimentally for rabbit HPRG. This observation provides a structural basis to the function of HPRG as an intracellular zinc chaperone which has been suggested by the involvement of the protein in the maintenance of the quaternary structure of skeletal muscle AMP deaminase (AMPD). During Anthropoidea evolution, a change of the primary structure of the PRR1 Zn 2+ binding site took place, giving rise to the sequence M-S-C-S/L-S/R-C that resembles the MxCxxC motif characteristic of metal transporters and metallochaperones. [ABSTRACT FROM AUTHOR]

A number of biologically important enzymes release adenosine 5′-monophosphate (AMP) as a product, including aminoacyl–tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5′-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5′-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD + ) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. [ABSTRACT FROM AUTHOR]

Objective. In our previous screening of chondrocyte protein profiles, the amount of adenosine monophosphate deaminase (AMPD) 2 was found to be decreased by tofacitinib. Extending the study, here we confirmed the decrease of AMPD2 by tofacitinib and further investigated effects of tofacitinib on purine nucleotide metabolism. Methods. Human articular chondrocytes and a chondrosarcoma cell line: OUMS-27 were stimulated with tofacitinib. Then the levels of AMPD2 and its related enzymes were investigated by Western blot. The levels of AMP and adenosine were assessed by mass spectrometry. Results. We confirmed the significant decrease of AMPD2 by tofacitinib in chondrocytes ( p = 0.025). The levels of adenosine kinase and 5'-nucleotidase were decreased in chondrocytes, although they did not meet statistical significance ( p = 0.067 and p = 0.074, respectively). The results from OUMS-27 were similar to those from the chondrocytes. The cellular adenosine levels were significantly decreased by tofacitinib in OUMS-27 ( p = 0.014). The cellular AMP levels were increased, although they did not meet statistical significance in OUMS-27 ( p = 0.066). Conclusion. Our data indicate that tofacitinib increases the cellular levels of adenosine, which is known to have anti-inflammatory activity, through the downregulation of AMPD2. This would be a novel functional aspect of tofacitinib. [ABSTRACT FROM AUTHOR]

Postmortem energy metabolism drives hydrogen accumulation in muscle and results in a fairly constant ultimate pH. Extended glycolysis results in adverse pork quality and may be possible with greater adenonucleotide availability postmortem. We hypothesized that slowing adenonucleotide removal by reducing AMP deaminase activity would extend glycolysis and lower the ultimate pH of muscle. Longissimus muscle samples were incorporated into an in vitro system that mimics postmortem glycolysis with or without pentostatin, an AMP deaminase inhibitor. Pentostatin lowered ultimate pH and increased lactate and glucose 6-phosphate with time. Based on these results and that AMPK γ3 R200Q mutated pigs (RN − ) produce low ultimate pH pork, we hypothesized AMP deaminase abundance and activity would be lower in RN − muscle than wild-type. RN − muscle contained lower AMP deaminase abundance and activity. These data show that altering adenonucleotide availability postmortem can extend postmortem pH decline and suggest that AMP deaminase activity may, in part, contribute to the low ultimate pH observed in RN − pork. [ABSTRACT FROM AUTHOR]

Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC) and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is characterized clinically by skeletal muscle fatigue following the excessive exercise. Interestingly most of MG patients manifest parallely also some abnormalities of the thymus.AMP-deaminase (AMPD) from human thymus was not a subject of studies up to now. In this paper, mRNA expression and some physico-chemical and immunological properties of AMPD purified from the thymus of MG patients were described. Experiments performed identified the liver isozyme (AMPD2) as the main isoform of AMPD expressed in this organ. The activity of AMPD found in this organ was higher than in other human non-(skeletal) muscle tissues indicating on role the enzyme may play in supplying of guanylates required for the intensive multiplication of thymocytes. [ABSTRACT FROM PUBLISHER]

Background Insulin resistance is one of the hallmark manifestations of obesity and Type II diabetes and reversal of this pathogenic abnormality is an attractive target for new therapies for Type II diabetes. A recent report that metformin, a drug known to reverse insulin resistance, demonstrated in vitro the metformin can inhibit AMP deaminase (AMPD) activity. Skeletal muscle is one of the primary organs contributing to insulin resistance and that the AMPD1 gene is selectively expressed at high levels in skeletal muscle. Methods Recognizing the background above, we asked if genetic disruption of the AMPD1 gene might ameliorate the manifestations of insulin resistance. AMPD1 deficient homozygous mice and control mice fed normal chow diet or a high-fat diet, and blood analysis, glucose tolerance test and insulin tolerance test were performed. Also, skeletal muscle metabolism and gene expression including nucleotide levels and activation of AMP activated protein kinase (AMP kinase) were evaluated in both conditions. Results Disruption of the AMPD1 gene leads to a less severe state of insulin resistance, improved glucose tolerance and enhanced insulin clearance in mice fed a high fat diet. Given the central role of AMP kinase in insulin action, and its response to changes in AMP concentrations in the cell, we examined the skeletal muscle of the AMPD1 deficient mice and found that they have greater AMP kinase activity as evidenced by higher levels of phosphorylated AMP kinase. Conclusions Taken together these data suggest that AMPD may be a new drug target for the reversal of insulin resistance and the treatment of Type II diabetes. [ABSTRACT FROM AUTHOR]

Background: Previously, we identified three loci affecting HDL-cholesterol levels in a screen for ENU-induced mutations in mice and discovered two mutated genes. We sought to identify the third mutated gene and further characterize the mouse phenotype. Methods: We engaged, DNA sequencing, gene expression profiling, western blotting, lipoprotein characterization, metabolomics assessment, histology and electron microscopy in mouse tissues. Results: We identify the third gene as Ampd2, a liver isoform of AMP Deaminase (Ampd), a central component of energy and purine metabolism pathways. The causative mutation was a guanine-to-thymine transversion resulting in an A341S conversion in Ampd2. Ampd2 homozygous mutant mice exhibit a labile hypercholesterolemia phenotype, peaking around 9 weeks of age (251 mg/dL vs. wildtype control at 138 mg/dL), and was evidenced by marked increases in HDL, VLDL and LDL. In an attempt to determine the molecular connection between Ampd2 dysfunction and hypercholesterolemia, we analyzed hepatic gene expression and found the downregulation of Ldlr, Hmgcs and Insig1 and upregulation of Cyp7A1 genes. Metabolomic analysis confirmed an increase in hepatic AMP levels and a decrease in allantoin levels consistent with Ampd2 deficiency, and increases in campesterol and β-sitosterol. Additionally, nephrotic syndrome was observed in the mutant mice, through proteinuria, kidney histology and effacement and blebbing of podocyte foot processes by electron microscopy. Conclusion: In summary we describe the discovery of a novel genetic mouse model of combined transient nephrotic syndrome and hypercholesterolemia, resembling the human disorder. [ABSTRACT FROM AUTHOR]

Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS) performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated. [ABSTRACT FROM AUTHOR]

A hypometabolic state can be induced in mice by 5′-AMP administration. Previously we proposed that an underlying mechanism for this hypometabolism is linked to reduced erythrocyte oxygen transport function due to 5′-AMP uptake altering the cellular adenylate equilibrium. To test this hypothesis, we generated mice deficient in adenosine monophosphate deaminase 3 (AMPD3), the key catabolic enzyme for 5′-AMP in erythrocytes. Mice deficient in AMPD3 maintained AMPD activities in all tissues except erythrocytes. Developmentally and morphologically, the Ampd3−/− mice were indistinguishable from their wild type siblings. The levels of ATP, ADP but not 5′-AMP in erythrocytes of Ampd3−/− mice were significantly elevated. Fasting blood glucose levels of the Ampd3−/− mice were comparable to wild type siblings. In comparison to wild type mice, the Ampd3−/− mice displayed a deeper hypometabolism with a significantly delayed average arousal time in response to 5′-AMP administration. Together, these findings demonstrate a central role of AMPD3 in the regulation of 5′-AMP mediated hypometabolism and further implicate erythrocytes in this behavioral response. [ABSTRACT FROM AUTHOR]

Summary: Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Metabolic syndrome represents a collection of abnormalities that includes fatty liver, and it currently affects one-third of the United States population and has become a major health concern worldwide. Fructose intake, primarily from added sugars in soft drinks, can induce fatty liver in animals and is epidemiologically associated with nonalcoholic fatty liver disease in humans. Fructose is considered lipogenic due to its ability to generate triglycerides as a direct consequence of the metabolism of the fructose molecule. Here, we show that fructose also stimulates triglyceride synthesis via a purine-degrading pathway that is triggered from the rapid phosphorylation of fructose by fructokinase. Generated AMP enters into the purine degradation pathway through the activation of AMP deaminase resulting in uric acid production and the generation of mitochondrial oxidants. Mitochondrial oxidative stress results in the inhibition of aconitase in the Krebs cycle, resulting in the accumulation of citrate and the stimulation of ATP citrate lyase and fatty-acid synthase leading to de novo lipogeneis. These studies provide new insights into the pathogenesis of hepatic fat accumulation under normal and diseased states. [ABSTRACT FROM AUTHOR]

AMP deaminase ( AMPD) catalyzes AMP to IMP and plays an important role in energy charge and nucleotide metabolism. Human AMPD3 deficiency is a type of erythrocyte-specific enzyme deficiency found in individuals without clinical symptoms, although an increased level of ATP in erythrocytes has been reported. To better understand the physiological and pathological roles of AMPD3 deficiency, we established a line of AMPD3-deficient [ A3(−/−)] mice. No AMPD activity and a high level of ATP were observed in erythrocytes of these mice, similar to human RBC- AMPD3 deficiency, while other characteristics were unremarkable. Next, we created AMPD3 and pyruvate kinase ( PK) double-deficient [ PKA(−/−,−/−)] mice by mating A3(−/−) mice with CBA- Pk-1slc/ Pk-1slc mice [ PK(−/−)], a spontaneous PK-deficient strain showing hemolytic anemia. In PKA(−/−,−/−) mice, the level of ATP in red blood cells was increased 1.5 times as compared to PK(−/−) mice, although hemolytic anemia in those animals was not improved. In addition, we observed osmotic fragility of erythrocytes in A3(−/−) mice under fasting conditions. In contrast, the ATP level in erythrocytes was elevated in A3(−/−) mice as compared to the control. In conclusion, AMPD3 deficiency increases the level of ATP in erythrocytes, but does not improve anemia due to PK deficiency and leads to erythrocyte dysfunction. [ABSTRACT FROM AUTHOR]

The aims of this study were to determine the distribution of the AMPD1 genotype among groups of high-level Polish power-oriented athletes, and to investigate potential associations between genetic polymorphism in exon 2 of the AMPD1 gene and power-oriented athlete status. Altogether, 158 male Polish power-oriented athletes were genotyped by PCR-RFLP. The genetic control group comprised 160 unrelated male volunteers. We observed significant differences in genotype distribution when all 158 athletes (89.25% CC, 10.75% CT, 0.00% TT; P = 0.0025) were compared with controls (75.00% CC, 23.75% CT, 1.25% TT). A significant deficiency of the T allele was noted in all subgroups (short-distance runners: 5.21%, P = 0.032; short-distance swimmers: 5.56%, P = 0.031; weightlifters: 5.36%, P = 0.024) compared with controls (13.13%), while this trend was even stronger when the frequency of the T allele was compared between controls and all 158 athletes (5.38%, P = 0.0007). Our results indicate a lower frequency of the AMPD1 exon 2 T34 allele in elite Polish power-oriented athletes. Our data suggest that the C allele may help athletes to attain elite status in power-oriented sports. [ABSTRACT FROM PUBLISHER]

The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency of adenosine monophosphate deaminase, and the homozygous C to T mutation at nucleotide 34 of the adenosine monophosphate deaminase-1 gene. This observation indicates the possible existence of a primary adenosine monophosphate deaminase deficiency manifested by congenital muscle weakness and hypotonia. [ABSTRACT FROM AUTHOR]

The mechanism for how metformin activates AMPK (AMP-activated kinase) was investigated in isolated skeletal muscle L6 cells. A widely held notion is that inhibition of the mitochondrial respiratory chain is central to the mechanism. We also considered other proposals for metformin action. As metabolic pathway markers, we focused on glucose transport and fatty acid oxidation. We also confirmed metformin actions on other metabolic processes in L6 cells. Metformin stimulated both glucose transport and fatty acid oxidation. The mitochondrial Complex I inhibitor rotenone also stimulated glucose transport but it inhibited fatty acid oxidation, independently of metformin. The peroxynitrite generator 3-morpholinosydnonimine stimulated glucose transport, but inhibited fatty acid oxidation. Addition of the nitric oxide precursor arginine to cells did not affect glucose transport. These studies differentiate metformin from inhibition of mitochondrial respiration and from active nitrogen species. Knockdown of adenylate kinase also failed to affect metformin stimulation of glucose transport. Hence, any means of increase in ADP appears not to be involved in the metformin mechanism. Knockdown of LKB1, an upstream kinase and AMPK activator, did not affect metformin action. Having ruled out existing proposals, we suggest a new one: metformin might increase AMP through inhibition of AMP deaminase (AMPD). We found that metformin inhibited purified AMP deaminase activity. Furthermore, a known inhibitor of AMPD stimulated glucose uptake and fatty acid oxidation. Both metformin and the AMPD inhibitor suppressed ammonia accumulation by the cells. Knockdown of AMPD obviated metformin stimulation of glucose transport. We conclude that AMPD inhibition is the mechanism of metformin action. [ABSTRACT FROM AUTHOR]

A C34T mutation in the AMPD1 gene is proposed to cause local or systemic augmentations in blood adenosine level and improvement of prognoses in heart diseases like congestive heart failure or heart ischemic disease. This study examines some physico-chemical properties of AMP-deaminase isolated from cardiac muscle of a 10-year-old boy heterozygote for this mutation. [ABSTRACT FROM AUTHOR]

Clinical observation in patients with heart disease indicates that reduced activity of AMP deaminase could be protective in heart failure and ischemic heart disease. This study evaluated the effect of 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol, an AMP deaminase inhibitor (AMPDI) in the mouse heart subjected to hypoxia. ApoE/LDLR knock-out mice were subjected to reduced oxygen tension in breathing air. AMPDI was infused before hypoxia in the treated group. We observed amelioration of elcetrocardiographic changes during hypoxia in the treated group that are consistent with a protective effect. [ABSTRACT FROM AUTHOR]

AMP deaminase could be a potential target for treatment of heart disease but experimental evaluation of this concept is difficult due to limited availability of inhibitors with proven efficiency in biological systems. This study evaluated the effect of 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol, an AMP deaminase inhibitor (AMPDI) on the pathways of nucleotide metabolism in perfused rat heart. We show that AMPDI at 0.3 mM concentration effectively inhibits AMP deaminase in this experimental model. [ABSTRACT FROM AUTHOR]

Abstract: Cytosolic enzymes AMP deaminase and adenosine deaminase (ADA) catalyze AMP and adenosine deamination, constitute rate-limiting steps of adenine nucleotide catabolism and play important roles in cellular energy metabolism. In this study, AMP deaminase and ADA activities of rat liver, neocortex, cerebellum, striatum and hippocampus were investigated in acute ammonia intoxication and subacute CCl4-induced hepatitis. Activities of both AMP deaminase and ADA in the liver were elevated by 2.4–4.2-fold (p <0.0001) in both models of hepatotoxic injury as compared with controls. In acute hyperammonemia activities of AMP, deaminase and ADA increased by 46–59% (p <0.02) in the neocortex and did not change in the striatum. In the hippocampus of hyperammonemiс rats, only AMP deaminase activity was increased by 48% (p =0.0004), and in the cerebellum only ADA activity was increased significantly (by 26%, p <0.05). The adenylate pool size and energy charge were greatly reduced in the neocortex of hyperammonemiс rats. Results suggested that two parallel pathways of AMP breakdown, including AMP deaminase and ADA, respectively, are up-regulated under pathological conditions, probably in order to overcome compensatory synthesis of adenylates, to ensure prompt adenylate pool depletion and reduce the adenylate energy charge in liver and selected brain regions. [Copyright &y& Elsevier]

Tumour necrosis factor alpha (TNF-α) is implicated in post-ischemic myocardial dysfunction. Two distinct TNF-α receptors are shed from cell membranes and circulate in plasma as soluble sTNFR1 and sTNFR2 proteins. The aim of the study was to establish factors associated with plasma concentrations of TNF-α and its receptors in patients with coronary artery disease (CAD). Since adenosine inhibits the expression of TNF-α, two functional polymorphisms in genes encoding enzymes participating in adenosine metabolism, i.e. AMP deaminase-1 ( AMPD1, C34T) and adenosine deaminase ( ADA, G22A), were analyzed. Plasma concentrations of TNF-α, sTNFR1, and sTNFR2 were measured using ELISA in 167 patients with CAD. Common factors significantly associated with higher TNF-α, sTNFR1, and sTNFR2 were lower glomerular filtration rate (GFR), older age, higher BNP, lower blood haemoglobin, and the presence of asthma or chronic obstructive pulmonary disease (COPD). Higher TNF-α and sTNFR1 concentrations were also associated with the presence of heart failure (HF), lower ejection and shortening fraction, the presence of diabetes or metabolic syndrome, lower serum HDL cholesterol, and higher uric acid. In multivariate analysis the common independent predictors of higher TNF-α, sTNFR1, and sTNFR2 were lower GFR, lower HDL cholesterol, higher BNP, and the presence of asthma or COPD. There were no associations between AMPD1 C34T or ADA G22A genotypes and TNF-α or its receptors. In conclusion, the concentrations of TNF-α, sTNFR1, and sTNFR2 reflect the impairment of cardiac and renal function in patients with CAD. Metabolic syndrome and diabetes are associated with higher plasma concentrations of TNF-α and its receptors. [ABSTRACT FROM AUTHOR]

AMP deaminase enzyme showed an optimum temperature at 35°C. It was relatively stable between 30 and 40°C but extremely unstable at higher temperatures. The optimum pH was 6.0 and the enzyme presented an excellent stability with pH between 5.5 and 6.5. The K+ ion acted as a negative effector when used in concentrations higher than 50 mM. The nucleotide ATP acted as a positive effector in concentrations exceeding 2 mM, while ADP was a positive effector between 0 and 2 mM. These results indicated that AMP deaminase is an important enzyme in Dosidicus gigas post-mortem biochemistry, involving the rapid degradation of AMP during iced storage of jumbo squid mantle. [ABSTRACT FROM AUTHOR]

Skeletal muscle AMP deaminase (AMPD1) regulates the concentration of adenine nucleotides during muscle contraction. We previously provided evidence that rabbit AMPD1 is composed by two HPRG 73 kDa subunits and two 85 kDa catalytic subunits with a dinuclear zinc site with an average of two histidine residues at each metal site. AMPD1 is mainly expressed in fast twitching fibers and is inhibited by ATP. The limited trypsinization of the 95-residue N-terminal domain of rabbit AMPD1 desensitizes the enzyme towards ATP inhibition at the optimal pH 6.5, but not at pH 7.1. The modified residues of rabbit AMPD1 after incubation with radioactive diethyl pyrocarbonate ([14C]DEP) causing the desensitization to inhibition by ATP at pH 7.1 have been identified by sequence analysis and MS analysis of the radioactive peptides liberated from the carbethoxylated enzyme by limited proteolysis with trypsin. The study confirms the presence of a dinuclear zinc site in rabbit AMPD1 and shows that carbethoxylation of His-51 at the N-terminus of the catalytic subunit removes the inhibition of the enzyme by ATP at pH 7.1. The desensitization to ATP is due to the modification of His-51 of the Zn 2 coordination sphere which is transduced in a conformational change of the enzyme C-terminus, where an ATP-binding site has been localized. The progress in the study of the complex regulation of rabbit AMPD1 that shares an identical amino acid sequence with the human enzyme is important in relation to the role of the enzyme during mammalian evolution. • Skeletal muscle AMP deaminase (AMPD1) is a key player in muscle contraction. • Positive homotropic cooperativity behaviour of rabbit AMPD1 is inhibited by ATP. • Proteolysis of the 95-residue N-terminal domain of AMPD1 removes the ATP inhibition. • Carbethoxylation of the N-terminus of the catalytic subunit removes the ATP inhibition. • MALDI-MS-peptide mapping identified a change at the C-terminal ATP-binding site. [ABSTRACT FROM AUTHOR]

Ca2+-calmodulin (Ca2+-CaM) activates erythrocyte adenosine monophosphate deaminase (AMPD) in conditions of disturbed calcium homeostasis, prompting us to investigate adenine nucleotide metabolic dysregulation in sickle cell disease (SCD). However, higher ATP concentrations in reticulocytes, compared to erythrocytes, confound a comparative evaluation of SCD and normal RBCs. Therefore, a combination of centrifugation and antiCD71-labelled magnetic bead selection was used to prepare reticulocyte-poor fractions (reticulocytes <4% of total RBCs) of SCD RBCs. ATP and total adenine nucleotide concentrations were 12% lower in sickle erythrocytes compared to normal erythrocytes and inosine monophosphate (IMP) concentrations were threefold elevated (all P < 0·05). Furthermore, preincubation with a diffusible CaM antagonist slowed IMP accumulation in sickle erythrocytes during an experimental period of energy imbalance, thus showing that Ca2+-CaM activates AMPD in SCD. Finally, adenine treatment (100 μmol/l) of ex vivo SCD RBCs significantly expanded ATP levels (16% higher) and reduced phosphatidylserine (PS)-exposure, specifically those cells with the highest levels of PS externalization (46% fewer events) (both P-values <0·05 compared to untreated samples). We conclude that Ca2+-CaM activation of AMPD contributes to increased turnover of the adenine nucleotide pool in sickle erythrocytes and that this metabolic dysregulation promotes PS exposure that may contribute to the pathogenesis of SCD. [ABSTRACT FROM AUTHOR]

Objective. The common C34T polymorphism in the AMP deaminase-1 (AMPD1) gene results in an inactive enzyme in homozygotes for the mutated T allele. Some studies have shown an association of T allele with longer survival in heart failure (HF) and/or coronary artery disease (CAD). The aim of this study was to assess genotype-phenotype correlations in such patients, with emphasis on components of the metabolic syndrome. Methods. Ninety-seven patients with CAD without HF (CAD+ HF-) and 104 with HF (HF+) were genotyped by PCR-RFLP. The genetic control group comprised 200 newborns. Results. No significant differences were found in the frequency of AMPD1 genotypes between the groups. In the CAD+ HF- group, the carriers of T allele compared to CC homozygotes had significantly lower values of waist circumference (89.5±8.5 versus 97.7±11.2 cm; p = 0.00029), waist/hip ratio (p = 0.0059) and BMI (p = 0.045). There was no diabetes or fasting glycaemia ≥126 mg/dL in T carriers, while these features were present in 25 % of CC homozygotes (p = 0.0024). In the HF+ group, a tendency towards a lower prevalence of diabetes (20 % versus 41 %; p = 0.068) and significantly lower systolic blood pressure (p = 0.048) were observed in T allele carriers. Conclusions. C34T AMPD1 polymorphism may be associated with reduced frequency of obesity in CAD patients and of hyperglycaemia and diabetes in both CAD and HF patients. Morphometric parameters associated with adipose tissue distribution and parameters of glucose metabolism should be analysed as potential confounders in further studies on the role of polymorphisms of AMPD1 and other genes associated with AMP and adenosine metabolism in cardiovascular disease. [ABSTRACT FROM AUTHOR]

Glacier ice worms, Mesenchytraeus solifugus and related species, are the largest glacially obligate metazoans. As one component of cold temperature adaptation, ice worms maintain atypically high energy levels in an apparent mechanism to offset cold temperature-induced lethargy and death. To explore this observation at a mechanistic level, we considered the putative contribution of 5′ adenosine monophosphate deaminase (AMPD), a key regulator of energy metabolism in eukaryotes.We cloned cDNAs encoding ice worm AMPD, generating a fragment encoding 543 amino acids that included a short N-terminal region and complete C-terminal catalytic domain. The predicted ice worm AMPD amino acid sequence displayed conservation with homologues from other mesophilic eukaryotes with notable exceptions. In particular, an ice worm-specific K188E substitution proximal to the AMP binding site likely alters the architecture of the active site and negatively affects the enzyme's activity. Paradoxically, this would contribute to elevated intracellular ATP levels, which appears to be a signature of cold adapted taxa. [ABSTRACT FROM AUTHOR]

Abstract: Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5′-AMP sepharose. Specific activity of 2.5U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS–PAGE showed a single band with 87kDa molecular mass, native PAGE proved a band of 178kDa, whereas gel filtration detected a 180kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with V max of 1.16μM/min/mg, K m of 13mM, K cat of 3.48μM.s−1 and a K cat/K m of 267 (mol/L)−1.s−1. The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms. [Copyright &y& Elsevier]

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC50 = 0.5 μ M. AMPDI was much less effective with isolated cardiomyocytes (IC50 = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies. [ABSTRACT FROM AUTHOR]

Changes in AMP deaminase (AMPD) activity influence heart function and progression of heart disease, but the underlying mechanism is unknown. We evaluated the effect of purine riboside (Purr) on the activity of AMPD in perfused rat hearts and in isolated rat cardiomyocytes. Brief perfusion of the pre-ischemic heart with 200 μ M Purr resulted in activation of AMPD, more pronounced degradation of the adenine nucleotides, and reduced recovery of the adenine nucleotide pool during reperfusion. Brief incubation of rat cardiomyocytes with 200 μ M Purr also activated AMPD, while prolonged exposure resulted in enzyme inhibition. We conclude that Purr activates AMPD, whereas metabolites of this compound may inhibit the enzyme. [ABSTRACT FROM AUTHOR]

Muscle-type phosphofructokinase deficiency (PFKD) causes a hemolytic disorder and exertional myopathy in humans and dogs. In humans, PFKD is accompanied by a disturbed calcium homeostasis and associated adenine nucleotide dysregulation, which may potentiate the erythroenzymopathy associated with this inherited disorder. This study shows that canine PFKD also manifests these erythrocyte abnormalities. Compared to normal, healthy red cells, PFK-deficient erythrocytes contain lower concentrations of ATP and higher concentrations of IMP and calcium, the latter as per a calcium indicator dye. Adenosine monophosphate deaminase (AMPD) enriched 5000-fold from canine erythrocytes adsorbs to immobilized calcium–calmodulin and the interaction between these two proteins activates AMPD through a K mapp effect. This behavior is similar to that of the human erythrocyte enzyme and provides a potential contributing mechanism for accelerated adenine nucleotide turnover in canine PFKD. We propose that adenine nucleotide replacement strategies could benefit the erythroenzymopathy in human and canine PFKD and that the dog model of this disorder is an appropriate vehicle for further elucidating this hypothesis. [ABSTRACT FROM AUTHOR]

The purpose of this study was to assess if there exists an association between C34T muscle adenosine monophosphate deaminase (AMPD1) genotypes (i.e., normal homyzygotes [CC] vs. heterozygotes [CT]) and directly measured indices of exercise capacity (peak oxygen uptake [VO2peak], ventilatory threshold [VT], gross mechanical efficiency [GE], etc.) in 44 Caucasian McArdle patients (23 males, 21 females). All patients performed a graded cycle ergometer test until exhaustion (for VO2peak and VT determination) and a 12-min constant-load test at the power output eliciting the VT (for GE determination). We found no significant difference in indices of exercise capacity between CC (n = 18) and CT genotypes (n = 5) in the group of male patients (p >0.05). In contrast, the VO2 at the VT was significantly lower (p <0.05) in CT(n = 4; 7.9 ± 0.4 ml/kg/min) than in CC female patients (n=17; 11.0±0.9 ml/kg/min). In summary, heterozigosity for the C34T allele of the AMPD gene is associated with reduced submaximal aerobic capacity in female patients with McArdle disease and might partly account, in this gender, for the variability that exists in the phenotypic manifestation of the disease. [ABSTRACT FROM AUTHOR]

Metalloproteinases (MMPs, also called matrixins) are extracellular proteolytic enzymes involved in the degradation of both matrix and nonmatrix proteins. Currently, 25 MMPs have been identified in humans, and the overexpression of one or more MMPs has been implicated in several pathologies, spanning from cancer to rheumathoid arthritis to cardiovascular disease. While research over the past 20 years has focused on understanding MMP biology and selectively inhibiting MMP activity, key issues that remain to be addressed include MMP roles in the context of normal versus pathological conditions and whether globally inhibiting MMPs improves or deteriorates overall organ function. In terms of cardiovascular disease, increased MMP expression has been demonstrated in the setting of myocardial ischemia, reperfusion injury, and during the progression to congestive heart failure. MMPs are also major contributors to the progression of atherosclerotic lesions. In this review, we focus on cardiovascular effects produced by PD 166793, a wide-broad spectrum MMP inhibitor, originally developed by Parke-Davis (now Pfizer). We will briefly review its structure, mechanism of action, and inhibitory capacity. Finally, we will illustrate the cardiac contexts, both in vivo and in vitro, in which PD166793 administration has proven beneficial. [ABSTRACT FROM AUTHOR]

Abstract: We have previously provided evidence for a dinuclear zinc site in rabbit skeletal muscle AMPD compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site. XAS of the zinc binding site of the enzyme in the presence of PRN favors a model where PRN is added to the coordination sphere of one of the two zinc ions increasing its coordination number to five. The uncompetitive nature of the inhibition of AMPD by fluoride reveals that the anion probably displaces the nucleophile water molecule terminally coordinated to the catalytic Zn1 ion at the enzyme C-terminus, following the binding of AMP at the Zn2 ion located at N-terminus of the enzyme. Thus, the two Zn ions in the AMPD metallocenter operate together as a single catalytic unit, but have independent function, one of them (Zn1) acting to polarize the nucleophile water molecule, whilst the other (Zn2) acts transiently as a receptor for an activating substrate molecule. The addition of fluoride to AMPD also abolishes the cooperative behaviour induced in the enzyme by the inhibitory effect of ATP at acidic pH that probably resides in the competition with the substrate for an adenine nucleotide specific regulatory site located in the Zn2 ion binding region and which is responsible for the positive homotropic cooperativity behaviour of AMPD. [Copyright &y& Elsevier]

Abstract: XAS of Zn–peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP deaminase (AMPD) strongly suggest that the region 48–61 of the enzyme contains a zinc binding site, whilst the region 360–372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn–Zn distance of about 3.3 Å. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD. [Copyright &y& Elsevier]