AMP-deaminase from normal and cirrhotic human liver: A comparative study

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.     

AMPD1 C34T Mutation Selectively Affects AMP-Deaminase Activity in the Human Heart

Possession of the nonsense mutation in AMPD1 C34T gene has been linked to improved survival in patients with heart failure, possibly by promoting the formation of adenosine. This mutation is known to decrease the activity of AMP-deaminase in skeletal muscle. We have found that the AMPD1 mutation decreases the activity of AMP-deaminase in the heart without changing the activity of any other enzymes of adenine nucleotide metabolism. Protective mechanism of this mutation may be thus induced by local cardiac metabolic changes.     

Human liver AMP-deaminase – oligomeric forms of the enzyme

AMP-deaminase (EC 3.5.4.6) is a key enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in the liver. Mechanisms regulating activity of the enzyme are not completely elucidated, till now. In this paper experimental data indicating on the potential regulatory significance of changes in oligomeric structure of the enzyme are presented. SDS-PAG electrophoresis of human liver AMP-deaminase revealed the presence of three enzyme fragments. Only largest of them (the protein fragments weighing 68 kDa) reacted immunologically with monoclonal anti- (human liver) AMP-deaminase antibodies. At physiological pH 7.0, in the absence of regulatory ligands, reaction catalysed by human liver AMP-deaminase was strongly dependent on enzyme concentration used, with half-saturation constant (S_0.5) values increasing significantly with the degree of enzyme dilution. Preincubation with activated long-chain fatty acids – substances promoting dissociation of oligomeric enzymes, inhibited the activity of AMP-deaminase studied nearly completely. Gel filtration on Sepharose CL-6B column demonstrated existence of at least three active oligomeric forms of human liver AMP-deaminase. We postulate that oligomeric structure of the enzyme is a factor determining regulatory profile of AMP-deaminase studied.     

Isozymes of AMP-Deaminase in Muscles Myasthenia Gravis Patients

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.     

AMPD1 C34T mutation selectively affects AMP-deaminase activity in the human heart

Possession of the nonsense mutation in AMPD 1 C34T gene has been linked to improved survival in patients with heart failure, possibly by promoting the formation of adenosine. This mutation is known to decrease the activity of AMP-deaminase in skeletal muscle. We have found that the AMPD1 mutation decreases the activity of AMP-deaminase in the heart without changing the activity of any other enzymes of adenine nucleotide metabolism. Protective mechanism of this mutation may be thus induced by local cardiac metabolic changes.     

AMP-deaminase: regulation and physiological role of the enzyme

AMP-deaminase (AMPD) catalyzes the irreversible hydrolysis of AMP to IMP and ammonia. Being the integral enzyme of purine nucleotide cycle, AMPD participates in deamination of amino acids and their involvement into carbohydrate metabolism. This enzyme competes with 5'-nuclease for AMP it is indirectly involved in regulation of adenosine level. The role of AMPD may be supported also by the correlation between its activity and several neuromuscular and immunologic pathologies. The information on the izoforms, gene expression both in the normal and pathological states is given. Activity of AMPD is regulated by the substrate availability, adenylate pool, GTP, product of catalysed reaction IMP, ++inorganical phosphate, etc. Currently the growing scope of data is displaying the possible role of reversible phosphorylation and binding to cellular elements in regulation of the properties of AMPD under change of physiological state of organism.     

AMP-Deaminase from Thymus of Patients with Myasthenia Gravis

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.     

Effect of AMP-Deaminase 3 Knock-Out in Mice on Enzyme Activity in Heart and Other Organs

Recent findings suggest that inhibition of AMP-deaminase (AMPD) could be effective therapeutic strategy in heart disease associated with cardiac ischemia. To establish experimental model to study protective mechanisms of AMPD inhibition we developed conditional, cardiac specific knock-outs in Cre recombinase system. AMPD3 floxed mice were crossed with Mer-Cre-Mer mice. Tamoxifen was injected to induce Cre recombinase. After two weeks, hearts, skeletal muscle, liver, kidney, and blood were collected and activities of AMPD and related enzymes were analyzed using HPLC-based procedure. We demonstrate loss of more than 90% of cardiac AMPD activity in the heart of AMPD3 -/- mice while other enzymes of nucleotide metabolism such as adenosine deaminase, purine nucleoside phosphorylase were not affected. Surprisingly, activity of AMPD was also reduced in the erythrocytes and in the kidney by 20%–30%. No change of AMPD activity was observed in the skeletal muscle and the liver.     

AMP-deaminase from hen stomach smooth muscle--physico-chemical properties of the enzyme

AMP-deaminase from hen stomach smooth muscle was isolated and physico-chemical properties of the purified enzyme were investigated. The enzyme had an activity optimum at pH 6.5, and poorly deaminated the substrate analogues tested. At optimum pH (6.5), in the absence of regulatory ligands (control conditions), the enzyme manifested hyperbolic substrate-saturation kinetics with half-saturation constant (S(0.5)) of about 4.5 mM. Additions of adenine nucleotide effectors (ATP, ADP) activated the enzyme strongly at all the concentrations tested, diminishing significantly the value of S(0.5) constant. In contrast, the regulatory effect of orthophosphate was variable, and depended on the orthophosphate concentration used. The molecular mass of the enzyme subunit determined in SDS/PAG electrophoresis was about of 37kDa. The obtained results suggest that in different types of hen muscle, similarly as in humans and rats, expression of AMP-deaminase is under the control of independent genes.     

AMP-deaminase from human term placenta

AMP-deaminase from human term placenta was chromatographed on a phosphocellulose column and physico-chemical and immunological properties of the purified enzyme were investigated. At physiological pH7.0, in the absence of regulatory ligands (control conditions) studied AMP-deaminase manifested sigmoid-shaped substrate saturation kinetics, with half-saturation parameter (S_0.5) value of about 7 mM. Addition of important allosteric effectors (ATP, ADP or orthophosphate) modified kinetic properties of studied AMP-deaminase, influencing mainly the value of S_0.5 parameter. Micromolar concentrations of stearylo-CoA inhibited potently the enzyme making it no longer sensitive towards 1 mM ATP-induced activation. SDS-PAGE electrophoresis of the purified enzyme revealed presence of 68 kDa protein fragment, reacting with anti-(human) liver AMP-deaminase antibodies. Experimental results presented indicate that liver type of AMP-deaminase is an enzyme form present in human term placenta.     

Activities of purine converting enzymes in heart,liver and kidney mice LDLR−/− and Apo E−/−

Nucleotide metabolism plays a major role in a number of vital cellular processes such as energetics. This, in turn, is important in pathologies such as atherosclerosis.

Three month old atherosclerotic mice with knock outs for LDLR and apolipoprotein E (ApoE) were used for the experiments. Activities of AMP-deaminase (AMPD), ecto5′-nucleotidase (e5NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) were measured in heart, liver and kidney cortex and medulla by analysing conversion of substrates into products using HPLC.

The activity of ecto5′-nucleotidase differ in hearts of LDLR^?/? and ApoE^?/? mice with no differences in ADA and AMPD activity. We noticed highest activity of e5NT in kidney medulla of the models.

This model of atherosclerosis characterize with an inhibition of enzyme responsible for production of protective adenosine in heart but not in other organs and different metabolism of nucleotides in kidney medulla.     

Modulation of AMP deaminase in rat hearts subjected to ischemia and reperfusion by purine riboside

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 micro 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 micro 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.     

Modulation of AMP Deaminase in Rat Hearts Subjected to Ischemia and Reperfusion by Purine Riboside

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.     

Pharmacological Inhibition of AMP-Deaminase in Rat Cardiac Myocytes

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 IC ₅₀ = 0.5 μ M. AMPDI was much less effective with isolated cardiomyocytes (IC ₅₀ = 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.     

Molecular Characterization of Adenosine 5′-monophosphate Deaminase—The Key Enzyme Responsible for the Umami Taste of Nori (<Emphasis Type="Italic">Porphyra yezoensis</Emphasis> Ueda,Rhodophyta)

The enzyme adenosine 5′-monophosphate deaminase (AMPD, EC 3.5.4.6) catalyzes the conversion of adenosine 5′-monophosphate to inosine 5′-mononucleotide (IMP). IMP is generally known as the compound responsible for the umami taste of the edible red alga Porphyra yezoensis Ueda that is known in Japan as nori. Therefore, we suspect that AMPD plays a key role in providing a favorable nori taste. In this study, we undertake the molecular characterization of nori-derived AMPD. The nori AMPD protein has a molecular mass of 55 kDa as estimated from both gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The calculated molecular mass from the amino acid sequence deduced from cDNA is 57.1 kDa. The isoelectric point is 5.71. The coding region of AMPD consists of 1,566 bp encoding 522 amino acids and possesses a transmembrane domain and two N-glycosylation sites. The sequence identity of nori AMPD in human and yeast AMPDs was found to be less than 50% and 20% in DNA and amino acid sequences, respectively. Proline in the conserved motif of [SA]-[LIVM]-[NGS]-[STA]-D-D-P was found to be converted to glutamate. These results indicate that nori AMPD is a novel type of AMPD.     

Purine metabolism in Leishmania donovani and Leishmania braziliensis

We have studied purine metabolism in the culture forms of Leishmania donovani and Leishmania braziliensis. These organisms are incapable of synthesizing purines de novo from glycine, serine, or formate and require an exogenous purine for growth. This requirement is better satisfied by adenosine or hypoxanthine than by guanosine. Bothe adenine and inosine are converted to a common intermediate, hypoxanthine, before transformation to nucleotides. This is due to the activity of an adenine aminohydrolase (EC 3.5.4.2), a rather unusual finding in a eukaryotic cell. There is a preferential synthesis of adenine nucleotides, even when guanine or xanthine are used as precursors.The pathways of purine nucleotide interconversions in these Leishmania resemble those found in mammalian cells except for the absence of de novo purine biosynthesis and the presence of an adenine-deaminating activity.     

Regulation of skeletal muscle AMP deaminase. Carbethoxylation of His-51 belonging to the zinc coordination sphere of the rabbit enzyme promotes its desensitization towards the inhibition by ATP

BackgroundSkeletal 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.MethodsThe 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.ResultsThe 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.ConclusionsThe desensitization to ATP is due to the modification of His-51 of the Zn₂ coordination sphere which is transduced in a conformational change of the enzyme C-terminus, where an ATP-binding site has been localized.General significanceThe 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.     

Myc-Induced Liver Tumors in Transgenic Zebrafish Can Regress in tp53 Null Mutation

Hepatocellular carcinoma (HCC) is currently one of the top lethal cancers with an increasing trend. Deregulation of MYC in HCC is frequently detected and always correlated with poor prognosis. As the zebrafish genome contains two differentially expressed zebrafish myc orthologs, myca and mycb, it remains unclear about the oncogenicity of the two zebrafish myc genes. In the present study, we developed two transgenic zebrafish lines to over-express myca and mycb respectively in the liver using a mifepristone-inducible system and found that both myc genes were oncogenic. Moreover, the transgenic expression of myca in hepatocytes caused robust liver tumors with several distinct phenotypes of variable severity. ~5% of myca transgenic fish developing multinodular HCC with cirrhosis after 8 months of induced myca expression. Apoptosis was also observed with myca expression; introduction of homozygous tp53^-/- mutation into the myca transgenic fish reduced apoptosis and accelerated tumor progression. The malignant status of hepatocytes was dependent on continued expression of myca; withdrawal of the mifepristone inducer resulted in a rapid regression of liver tumors, and the tumor regression occurred even in the tp53^-/- mutation background. Thus, our data demonstrated the robust oncogenicity of zebrafish myca and the requirement of sustained Myc overexpression for maintenance of the liver tumor phenotype in this transgenic model. Furthermore, tumor regression is independent of the function of Tp53.     

ZIP4, a Novel Determinant of Tumor Invasion in Hepatocellular Carcinoma,Contributes to Tumor Recurrence after Liver Transplantation

Background and purpose: Recently, evidence that Zinc transporter ZRT/IRT-like protein 4 (ZIP4) is involved in invasiveness and apoptosis has emerged in pancreatic cancer and prostate cancer. Our aim was to assess the role of ZIP4 in invasiveness, migration and apoptosis of hepatocellular carcinoma (HCC). The prognostic value of ZIP4 in HCC after liver transplantation was evaluated.Methods: The role of ZIP4 in HCC was investigated by overexpressing ZIP4 in BEL7402 and HepG2 cells and inhibiting ZIP4 in HuH-7 and HepG2 cells, using overexpression and shRNA plasmids in vitro studies. Immunohistochemical analysis was used to evaluate ZIP4 expression in HCC tissues from 60 patients undergoing liver transplantation, 36 cirrhotic tissue samples, and 6 normal tissue samples. Prognostic significance was assessed using the Kaplan-Meier method and the log-rank test.Results: Specific suppression of ZIP4 reduced cell migration and invasiveness, whereas ZIP4 overexpression caused increases in cell migration and invasiveness. Furthermore, overexpression of ZIP4 resulted in increased expression of pro-metastatic genes (MMP-2, MMP-9) and decreased expression of pro-apoptotic genes (caspase-3, caspase-9, Bax). In contrast, suppression of ZIP4 resulted in an opposite effect. ZIP4 was more highly expressed in tumor tissues than non-tumor tissues (P < 0.0001). ZIP4 expression was significantly associated with tumor recurrence (P = 0.002), tumor node metastasis stage (P = 0.044), Child-Turcotte-Pugh score (P = 0.042), and tumor size (P = 0.022). Univariate analysis showed that ZIP4 expression was significantly associated with overall survival (P = 0.020) and tumor-free survival (P = 0.049). Multivariate analysis revealed that ZIP4 was an independent predictor of overall survival (P = 0.037) after liver transplantation.Conclusions: ZIP4 could promote migration, invasiveness, and suppress apoptosis in hepatocellular carcinoma, and represent a novel predictor of poor prognosis and therapeutic target for patients with HCC who undergo liver transplantation.