Mutation of recombinant catalytic subunit {alpha} of the protein kinase CK2 that affects catalytic efficiency and specificity

In order to understand better the structural and functionalrelations between protein kinase CK2 catalytic subunit, thetriphosphate moiety of ATP, the catalytic metal and the peptidicsubstrate, we built a structural model of Yarrowia lipolyticaprotein kinase CK2 catalytic subunit using the recently solvedthree-dimensional structure of the maize enzyme and the structureof cAMP-dependent protein kinase peptidic inhibitor (1CDK) astemplates. The overall structure of the catalytic subunit isclose to the structure solved by Niefind et al. It comprisestwo lobes, which move relative to each other. The peptide usedas substrate is tightly bound to the enzyme, at specific locations.Molecular dynamic calculations in combination with the studyof the structural model led us to identify amino acid residuesclose to the triphosphate moiety of ATP and a residue sufficientlyfar from the peptide that could be mutated so as to modify thespecificity of the enzyme. Site-directed mutagenesis was usedto replace by charged residues both glycine-48, a residue locatedwithin the glycine-rich loop, involved in binding of ATP phosphatemoiety, and glycine-177, a residue close to the active site.Kinetic properties of purified wild-type and mutated subunitswere studied with respect to ATP, MgCl₂ and protein kinase CK2specific peptide substrates. The catalytic efficiency of theG48D mutant increased by factors of 4 for ATP and 17.5 for theRRRADDSDDDDD peptide. The mutant G48K had a low activity withATP and no detectable activity with peptide substrates and wasalso inhibited by magnesium. An increased velocity of ADP releaseby G48D and the building of an electrostatic barrier betweenATP and the peptidic substrate in G48K could explain these results.The kinetic properties of the mutant G177K with ATP were notaffected, but the catalytic efficiency for the RRRADDSDDDDDsubstrate increased sixfold. Lysine 177 could interact withthe lysine-rich cluster involved in the specificity of proteinkinase CK2 towards acidic substrate, thereby increasing itsactivity.     

A protein tyrosine kinase associated with the ATP-dependent inactivation of adipose diacylglycerol acyltransferase

The activity that has been previously reported to reversibly inactivate adipose glycerolphosphate acyltransferase (GPAT) and diacylglycerol acyltransferase (DGAT)in vitro in the presence of ATP is shown here to be partially purified from adipose tissue with an apparent molecular weight of 68 kDa. The activity responsible for inactivating DGAT is associated with a kinase activity as determined by phosphate incorporation both into microsomal proteins and into a synthetic tyrosine-containing peptide as substrate for protein tyrosine kinase. Two microsomal polypeptides of 53 and 69 kDa are major substrates of this kinase. Both DGAT inactivating and kinase activities assayed from the purified sample have been found to be insensitive to the Ser/Thr kinase inhibitor H-7 while being sensitive to genistein and tyrphostin-25. A crude protein phosphatase preparation from liver was capable of reversing the effects of both activities. The purified sample was also shown to inactivate GPAT in the presence of ATP. These results suggest that a protein tyrosine kinase, in concert with a protein tyrosine phosphatase, may regulate the activities of DGAT and GPAT by a phosphorylation-dephosphorylation mechanism.     

X‐ray Structural Analysis of Tau‐Tubulin Kinase 1 and Its Interactions with Small Molecular Inhibitors

Tau‐tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that putatively phosphorylates residues including S422 in tau protein. Hyperphosphorylation of tau protein is the primary cause of tau pathology and neuronal death associated with Alzheimer’s disease. A library of 12 truncation variants comprising the TTBK1 kinase domain was screened for expression in Escherichia coli and insect cells. One variant (residues 14–313) could be purified, but mass spectrometric analysis revealed extensive phosphorylation of the protein. Co‐expression with lambda phosphatase in E. coli resulted in production of homogeneous, nonphosphorylated TTBK1. Binding of ATP and several compounds to TTBK1 was characterized by surface plasmon resonance. Crystal structures of TTBK1 in the unliganded form and in complex with ATP, and two high‐affinity ATP‐competitive inhibitors, 3‐[(6,7‐dimethoxyquinazolin‐4‐yl)amino]phenol ( 1 ) and methyl 2‐bromo‐5‐(7H‐pyrrolo[2,3‐d]pyrimidin‐4‐ylamino)benzoate ( 2 ), were elucidated. The structure revealed two clear basic patches near the ATP pocket providing an explanation of TTBK1 for phosphorylation‐primed substrates. Interestingly, compound 2 displayed slow binding kinetics to TTBK1, the structure of TTBK1 in complex with this compound revealed a reorganization of the L199–D200 peptide backbone conformation together with altered hydrogen bonding with compound 2 . These conformational changes necessary for the binding of compound 2 are likely the basis of the slow kinetics. This first TTBK1 structure can assist the discovery of novel inhibitors for the treatment of Alzheimer’s disease.     

Biotinylated Phosphoproteins from Kinase‐Catalyzed Biotinylation are Stable to Phosphatases: Implications for Phosphoproteomics

Kinase‐catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphorylation is critical to understand cell biology. Our lab recently discovered kinase‐catalyzed biotinylation, where ATP‐biotin is utilized by kinases to label phosphopeptides or phosphoproteins with a biotin tag. To exploit kinase‐catalyzed biotinylation for phosphoprotein purification and identification in a cellular context, the susceptibility of the biotin tag to phosphatases was characterized. We found that the phosphorylbiotin group on peptide and protein substrates was relatively insensitive to protein phosphatases. To understand how phosphatase stability would impact phosphoproteomics research applications, kinase‐catalyzed biotinylation of cell lysates was performed in the presence of kinase or phosphatase inhibitors. We found that biotinylation with ATP‐biotin was sensitive to inhibitors, although with variable effects compared to ATP phosphorylation. The results suggest that kinase‐catalyzed biotinylation is well suited for phosphoproteomics studies, with particular utility towards monitoring low‐abundance phosphoproteins or characterizing the influence of inhibitor drugs on protein phosphorylation.     

The different roles of metal ions and water molecule in the hydrolysis of ATP catalyzed by phenanthroline-based polyamines

The phenanthroline (Phen)-based polyaza ligands can efficiently recognize nucleotides and significantly enhance the rate of ATP hydrolysis under the regulation of metal ions, which act as “messenger” between the substrates and the receptors. The water molecule (or –OH) participates in the catalytic hydrolysis of ATP in different steps with different functions.     

Activators of P‐glycoprotein: Structure–Activity Relationships and Investigation of their Mode of Action

P‐glycoprotein (P‐gp), a 170 kDa plasma membrane protein, is one of the most relevant ABC transporters involved in the development of multidrug resistance (MDR). Understanding its mechanism of transport as well as its interactions with various substrates are basic requirements for the development of adequate therapeutic approaches to overcome this kind of resistance against a broad spectrum of structurally unrelated cytostatic drugs. P‐gp modulators (activators) that exert various effects on the intracellular accumulation of distinct P‐gp substrates are useful tools for investigating the interactions between multiple drug binding sites of this transport protein. In this study, a series of 27 different imidazobenzothiazoles and imidazobenzimidazoles structurally related to the known P‐gp activators QB102 and QB11 was designed, and their modulating properties were investigated. Most of them were able to stimulate P‐gp‐mediated efflux of daunorubicin and rhodamine 123 in a concentration‐dependent manner, but some compounds also displayed weak inhibitory effects. Additionally, P‐gp‐mediated efflux of vinblastine and colchicine was inhibited by several compounds. Therefore, we concluded that the novel compounds bind to the H site of P‐gp and activate the efflux of specific substrates of the R site in a positive cooperative manner, whereas binding of H‐type substrates is inhibited competitively. This hypothesis is confirmed by the observation that the modulators do not influence hydrolysis of ATP or its affinity toward P‐gp.     

The Membrane-Bound Lon Protease from Thermoplasma Displays Unfolding Activity

The membrane-bound Lon protease from Thermoplasma acidophilum (Ta Lon) was shown to unfold and degrade a fusion of the green fluorescent protein with calmodulin (GFP—CaM). Unfolding and degradation were ATP-dependent reactions and could be inhibited by calcium ions, which are known to stabilize calmodulin. Notably, an inverse fusion of the same proteins, i.e., CaM—GFP, as well as GFP or GFP-SsrA, was neither unfolded nor degraded. Thus, Ta Lon seems to unfold and degrade preferentially protein substrates with an extended unstructured C-terminus. A set of Ta Lon variants mutated in critical residues of the AAA⁺ domain, were tested for their respective ATPase and GFP—CaM unfolding activity. This analysis revealed that the rate of ATP hydrolysis correlated with the efficiency of the GFP—CaM unfolding activity. In summary, we show here that the membrane-bound Ta Lon protease displays an unfolding activity, which is correlated with the rate of ATP hydrolysis.     

pH-stat methodology in continuous monitoring of the kinetics of hydrolysis of phosphate esters catalysed by alkaline phosphatase from human placenta: II. Kinetic aspects

The adaptation of the pH-stat to continuous monitoring of the in-vitro hydrolase activity of alkaline phosphatase in solution, an activity which until now has only been analysed by means of spectrophotometric methods in a continuous or static state, is described. To control the reliability of the method in the monitoring of these enzymic reactions, a series of kinetics of hydrolysis of chromogenic (p-nitrophenyl and o-carboxyphenyl phosphates) and non-chromogenic (ATP) substrates was carried out, comparing the results obtained via continuous spectrophotometric analysis of the corresponding phenol released or via a discontinuous technique by the phosphate produced with those results obtained via pH-stat analysis of the H⁺ released or utilized by the enzymic reaction of hydrolysis. It can be concluded that kinetic studies carried out on the pH-stat of in-vitro alkaline phosphatase activity offer results analogous to those obtained for the same system through classic spectrophotometric methods, offering as well notable advantages of speed and simplicity in the kinetic assays since it is always possible to monitor the enzymic activity continuously, with chromogenic or non-chromogenic substrates. The pH-stat methodology affords additional information on the kinetics of action of alkaline phosphatase from placenta acting on non-chromogenic biological substrates. In this sense, kinetic studies with the pH-stat on the enzymic hydrolysis of ATP were begun, determining their rate versus pH profile (optimum pH at 9.1) and proposing possible ‘non-Michaelian’ type kinetic behaviour of the enzyme in solution as deduced from the graphical analysis of the data v([ATP]).     

K‐BILDS: A Kinase Substrate Discovery Tool

Kinases catalyze protein phosphorylation to regulate cell signaling events. However, identifying kinase substrates is challenging due to the often low abundance and dynamic nature of protein phosphorylation. Development of novel techniques to identify kinase substrates is necessary. Here, we report kinase‐catalyzed biotinylation with inactivated lysates for discovery of substrates (K‐BILDS) as a tool to identify direct substrates of a kinase. As a proof of concept, K‐BILDS was applied to cAMP‐dependent protein kinase A (PKA) with HeLa cell lysates. Subsequent enrichment and MS/MS analysis identified 279 candidate PKA substrates, including 56 previously known PKA substrates. Of the candidate substrates, nuclear autoantigenic sperm protein (NASP), BCL2‐associated athanogene 3 (BAG3), and 14‐3‐3 protein Tau (YWHAQ) were validated as novel PKA substrates. K‐BILDS provides a valuable tool to identify direct substrates of any protein kinase.     

Reversible ATP-dependent inactivation of adipose diacylglycerol acyltransferase

Diacylglycerol acyltransferase from rat adipose tissue is shown to be inactivated by 30 to 40% upon incubation with adenosine 5′-triphosphate (ATP) and Mg²⁺. The activity responsible for this inactivation is associated with the cytosolic fraction, specific for ATP, prevented when ATP is substituted by β,γ-methylene-ATP, and partially blocked by 1 mM ethylenediaminetetraacetate or 40 mM NaF, but not by inhibitors of adenosine 3′,5′-cyclic-monophosphate (cAMP)-dependent protein kinase and/or protein kinase C (PKC). The cytosolic activity cannot be mimicked by (cAMP)-protein kinase nor by PKC. Inactivated diacylglycerol acyltransferase from ATP/cytosol-treated microsomes can be reactivated by incubation with partially purified protein phosphate from rat liver, and can be inactivated again by further addition of ATP in the presence of cytosol. The results suggest the existence in adipose tissue of a protein kinase other than cAMP-protein kinase or PKC, which may be involved in the regulation of triacylglycerol synthesis.     

Comparison of conservation within and between the Ser/Thr and Tyr protein kinase family: proposed model for the catalytic domain of the epidermal growth factor receptor

The protein kinase family can be subdivided into two main groupsbased on their ability to phosphorylate Ser/Thr or Tyr substrates.In order to understand the basis of this functional difference,we have carried out a comparative analysis of sequence conservationwithin and between the Ser/Thr and Tyr protein kinases. A multiplesequence alignment of 86 protein kinase sequences was generated.For each position in the alignment we have computed the conservationof residue type in the Ser/Thr, in the Tyr and in both of thekinase subfamilies. To understand the structural and/or functionalbasis for the conservation, we have mapped these conservationproperties onto the backbone of the recently determined structureof the cAMP–dependent Ser/Thr kinase. The results showthat the kinase structure can be roughly segregated, based uponconservation, into three zones. The inner zone contains residueshighly conserved in all the kinase family and describes thehydrophobic core of the enzyme together with residues essentialfor substrate and ATP binding and catalysis. The outer zonecontains residues highly variable in all kinases and representsthe solvent–exposed surface of the protein. The thirdzone is comprised of residues conserved in either the Ser/Thror Tyr kinases or in both, but which are not conserved betweenthem. These are sandwiched between the hydrophobic core andthe solvent-exposed surface. In addition to analyzing overallconservation hi the kinase family, we have also looked at conservationof its substrate and ATP binding sites. The ATP site is highlyconserved throughout the kinases, whereas the substrate bindingsite is more variable. The active site contains several positionswhich differ between the Ser/Thr and Tyr kinases and may beresponsible for discriminating between hydroxyl bearing sidechains. Using this information we propose a model for Tyr substratebinding to the catalytic domain of the epidermal growth factorreceptor (EGFR).     

Insight into the inhibition of human choline kinase: homology modeling and molecular dynamics simulations

A homology model of human choline kinase (CK-alpha) based on the X-ray crystallographic structure of C. elegans choline kinase (CKA-2) is presented. Molecular dynamics simulations performed on CK-alpha confirm the quality of the model, and also support the putative ATP and choline binding sites. A good correlation between the MD results and reported CKA-2 mutagenesis assays has been found for the main residues involved in catalytic activity. Preliminary docking studies performed on the CK-alpha model indicate that inhibitors can bind to the binding sites of both substrates (ATP and choline). A possible reason for inhibition of choline kinase by Ca(2+) ion is also proposed.     

Imparting affinity sites for adenosine triphosphate on the surface of polyurethane through molecular imprinting

A simple methodology for imparting affinity sites for adenosine triphosphate (ATP) on the surface of polyurethane (PU) film is discussed. It is widely known that, through oxidation, a thin layer conjugated polymer can be coated onto many polymeric substrates. This possibility is explored in this report. 3‐Amino phenyl boronic acid was polymerized in the presence of ATP as template using ammonium per sulfate. A thin layer of polyamino phenyl boronic acid was coated onto the surface of PU film immersed in the solution during the reaction. The coated layer showed remarkably high affinity toward ATP and equilibrium adsorption was attained rapidly in contrast to free standing molecularly imprinted polymers in which equilibrium adsorption is normally attained after several hours. The imprinted layer also showed a high degree of selectivity toward ATP compared to adenosine diphosphate (ADP). The approach is simple and affinity sites for any water soluble molecules can virtually be created on the surface of polymers of interest. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 94: 2088–2090, 2004     

Arylamino Esters As P‐Glycoprotein Modulators: SAR Studies to Establish Requirements for Potency and Selectivity

A set of basic aryl‐group‐containing compounds was synthesized with the aim of developing potent and selective P‐glycoprotein (P‐gp) modulators that are able to reverse multidrug resistance (MDR). The natures of the spacer (dicyclohexylamine or dialkylamine) and the aryl moieties were modified to investigate selectivity and the mechanism of P‐gp interaction. The inhibitory activities of the compounds toward P‐gp, multidrug resistance‐associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), the most relevant ATP binding cassette (ABC) transporters for MDR, were evaluated. The mechanism of P‐gp interaction for each compound was investigated with three biological assays: apparent permeability (P_app) determination (B→A/A→B) in Caco‐2 cell monolayers, ATP cell depletion, and inhibition of Calcein‐AM transport in MDCK‐MDR1 cells. These assays allowed us to estimate the selectivity of the compounds for the three efflux pumps and to identify the structural requirements that define the P‐gp‐interaction profile. All dicyclohexylamine derivatives were found to be P‐gp substrates, whereas one dialkylamine derivative was shown to be a P‐gp inhibitor. The good MRP1 activity of one cis/cis isomer highlighted this as a lead candidate for the development of MRP1 ligands.     

NS5ATP9 Contributes to Inhibition of Cell Proliferation by Hepatitis C Virus (HCV) Nonstructural Protein 5A (NS5A) via MEK/Extracellular Signal Regulated Kinase (ERK) Pathway

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions and viral pathogenesis. However, on the impact of cell growth, there have been different study results. NS5ATP9, also known as KIAA0101, p15PAF, L5, and OEACT-1, was first identified as a proliferating cell nuclear antigen-binding protein. Earlier studies have shown that NS5ATP9 might play an important role in HCV infection. The aim of this study is to investigate the function of NS5ATP9 on hepatocellular carcinoma (HCC) cell lines proliferation under HCV NS5A expression. The results showed that overexpression of NS5ATP9 inhibited the proliferation of Bel7402 cells, whereas knockdown of NS5ATP9 by interfering RNA promoted the growth of HepG2 cells. Under HCV NS5A expression, RNA interference (RNAi) targeting of NS5ATP9 could reverse the inhibition of HepG2 cell proliferation, suggesting that NS5ATP9 might be an anti-proliferation gene that plays an important role in the suppression of cell growth mediated by HCV NS5A via MEK/ERK signaling pathway. These findings might provide new insights into HCV NS5A and NS5ATP9.     

Mechanical Disturbance of Osteoclasts Induces ATP Release That Leads to Protein Synthesis in Skeletal Muscle through an Akt-mTOR Signaling Pathway

Muscle and bone are tightly integrated through mechanical and biochemical signals. Osteoclasts are cells mostly related to pathological bone loss; however, they also start physiological bone remodeling. Therefore, osteoclast signals released during bone remodeling could improve both bone and skeletal muscle mass. Extracellular ATP is an autocrine/paracrine signaling molecule released by bone and muscle cells. Then, in the present work, it was hypothesized that ATP is a paracrine mediator released by osteoclasts and leads to skeletal muscle protein synthesis. RAW264.7-derived osteoclasts were co-cultured in Transwell^® chambers with flexor digitorum brevis (FDB) muscle isolated from adult BalbC mice. The osteoclasts at the upper chamber were mechanically stimulated by controlled culture medium perturbation, resulting in a two-fold increase in protein synthesis in FDB muscle at the lower chamber. Osteoclasts released ATP to the extracellular medium in response to mechanical stimulation, proportional to the magnitude of the stimulus and partly dependent on the P2X₇ receptor. On the other hand, exogenous ATP promoted Akt phosphorylation (S473) in isolated FDB muscle in a time- and concentration-dependent manner. ATP also induced phosphorylation of proteins downstream Akt: mTOR (S2448), p70S6K (T389) and 4E-BP1 (T37/46). Exogenous ATP increased the protein synthesis rate in FDB muscle 2.2-fold; this effect was blocked by Suramin (general P2X/P2Y antagonist), LY294002 (phosphatidylinositol 3 kinase inhibitor) and Rapamycin (mTOR inhibitor). These blockers, as well as apyrase (ATP metabolizing enzyme), also abolished the induction of FDB protein synthesis evoked by mechanical stimulation of osteoclasts in the co-culture model. Therefore, the present findings suggest that mechanically stimulated osteoclasts release ATP, leading to protein synthesis in isolated FDB muscle, by activating the P2-PI3K-Akt-mTOR pathway. These results open a new area for research and clinical interest in bone-to-muscle crosstalk in adaptive processes related to muscle use/disuse or in musculoskeletal pathologies.     

Free-energy simulations and experiments reveal long-range electrostatic interactions and substrate-assisted specificity in an aminoacyl-tRNA synthetase

Specific recognition of their cognate amino acid substrates by the aminoacyl-tRNA synthetase enzymes is essential for the correct translation of the genetic code. For aspartyl-tRNA synthetase (AspRS), electrostatic interactions are expected to play an important role, since its three substrates (aspartate, ATP, tRNA) are all electrically charged. We used molecular-dynamics free-energy simulations and experiments to compare the binding of the substrate Asp and its electrically neutral analogue Asn to AspRS. The preference for Asp is found to be very strong, with good agreement between simulations and experiment. The simulations reveal long-range interactions that electrostatically couple the amino acid ligand, ATP, and its associated Mg2+ cations, a histidine side chain (His448) next to the amino acid ligand and a flexible loop that closes over the active site in response to amino acid binding. Closing this loop brings a negatively charged glutamate into the active site; this causes His448 to recruit a labile proton, which interacts favorably with Asp and accounts for most of the Asp/Asn discrimination. Cobinding of the second substrate, ATP, increases specificity for Asp further and makes the system robust towards removal of His448, which is mutated to a neutral amino acid in many organisms. Thus, AspRS specificity is assisted by a labile proton and a cosubstrate, and ATP acts as a mobile discriminator for specific Asp binding to AspRS. In asparaginyl-tRNA synthetase, a close homologue of AspRS, a few binding-pocket differences modify the charge balance so that asparagine binding predominates.     

Identification of putative binding sites of P-glycoprotein based on its homology model

A homology model of P-glycoprotein based on the crystal structure of the multidrug transporter Sav1866 is developed, incorporated into a membrane environment, and optimized. The resulting model is analyzed in relation to the functional state and potential binding sites. The comparison of modeled distances to distances reported in experimental studies between particular residues suggests that the model corresponds most closely to the first ATP hydrolysis step of the protein transport cycle. Comparison to the protein 3D structure confirms this suggestion. Using SiteID and Site Finder programs three membrane related binding regions are identified: a region at the interface between the membrane and cytosol and two regions located in the transmembrane domains. The regions contain binding pockets of different size, orientation, and amino acids. A binding pocket located inside the membrane cavity is also identified. The pockets are analyzed in relation to amino acids shown experimentally to influence the protein function. The results suggest that the protein has multiple binding sites and may bind and/or release substrates in multiple pathways.     

Azotobacter vinelandii metal storage protein: "classical" inorganic chemistry involved in Mo/W uptake and release processes

The release of Mo (as molybdate) from the Mo storage protein (MoSto), which is unique among all existing metalloproteins, is strongly influenced by temperature and pH value; other factors (incubation time, protein concentration, degree of purity) have minor, though significant effects. A detailed pH titration at 12 degrees C revealed that three different steps can be distinguished for the Mo-release process. A proportion of approximately 15% at pH 6.8-7.0, an additional 25% at pH 7.2-7.5 and ca. 50% (up to 90% in total) at pH 7.6-7.8. This triphasic process supports the assumption of the presence of different types of molybdenum-oxide-based clusters that exhibit different pH lability. The complete release of Mo was achieved by increasing the temperature to 30 degrees C and the pH value to >7.5. The Mo-release process does not require ATP; on the contrary, ATP prevents, or at least reduces the degree of metal release, depending on the concentration of the nucleotide. From this point of view, the intracellular ATP concentration is suggested to play-in addition to the pH value-an indirect but crucial role in controlling the extent of Mo release in the cell. The binding of molybdenum to the apoprotein (reconstitution process) was confirmed to be directly dependent on the presence of a nucleotide (preferably ATP) and MgCl2. Maximal reincorporation of Mo required 1 mM ATP, which could partly be replaced by GTP. When the storage protein was purified in the presence of ATP and MgCl2 (1 mM each), the final preparation contained 80 Mo atoms per protein molecule. Maximal metal loading (110-115 atoms/MoSto molecule) was only achieved, if Mo was first completely released from the native protein and subsequently (re-) bound under optimal reconstitution conditions: 1 h incubation at pH 6.5 and 12 degrees C in the presence of ATP, MgCl2 and excess molybdate. A corresponding tungsten-containing storage protein ("WSto") could not only be synthesized in vivo by growing cells, but could also be constructed in vitro by a metalate-ion exchange procedure by using the isolated MoSto protein. The high W content of the isolated cell-made WSto (approximately 110 atoms/protein molecule) and the relatively low amount of tungstate that was released from the protein under optimal "release conditions", demonstrates that the W-oxide-based clusters are more stable inside the protein cavity than the Mo-oxide analogues, as expected from the corresponding findings in polyoxometalate chemistry. The optimized isolation of the W-loaded protein form allowed us to get single crystals, and to determine the crystal X-ray structure. This proved that the protein contains remarkably different types of polyoxotungstates, the formation of which is templated in an unprecedented process by the different protein pockets. (Angew. Chem. Int. Ed. 2007, 46, 2408-2413).     

Energy metabolism during placental development in rats--effect of chronic maternal malnutrition

The effect of maternal malnutrition on the energy metabolism of the developing rat placenta, related to fetal growth, was studied. Female virgin rats of the Wistar strain were fed restricted amounts of 25% casein diet, from puberty and throughout pregnancy. According to results, a significant decrease occurred in the activity of adenylate kinase, as well as a significant increase in the energy charge of the adenylate system (ATP, ADP), per gram of DNA of placental tissue near term, in the malnourished group. The data suggest that the significant increase of energy charge (ATP + 1/2 ADP:/ATP + ADP + AMP) of the placenta in the malnourished group, is the consequence of an inhibition of the reactions controlling ATP-consuming process, such as macromolecular synthesis pathways and active transport of substrates near term. This coincides with the simultaneous and significant decrease in fetal growth in this group.