Vitamin C status of vitamin A-deficient rats

IT was initially suggested that vitamin A-deficiency leads to an interference in the biosynthesis of ascorbic acid, because depletion of vitamin A was found to cause a fall in the tissue-levels of ascorbate and diminished urinary ascorbic acid excretion in animals1-3. Mapson4, however, concluded that lowered ascorbic acid-levels in vitamin A-deficient rats is due to inanition only, because he was able to show that following chloretone treatment vitamin A-deficient and pair-fed vitamin A normal rats excrete comparable amounts of ascorbic acid in their urine and that restriction of food intake reduces the urinary ascorbate even in the chloretone-treated normal rats. Results of our preliminary experiments reported here clearly indicate that the synthesis of ascorbic acid in rats is markedly reduced during vitamin A-deficiency.

Stimulation by thyrotropin, long-acting thyroid stimulator, and dibutyryl 3′, 5′-adenosine monophosphate of protein and ribonucleic acid synthesis and ribonucleic acid polymerase activities in porcine thyroid in Vitro

The in vitro incorporation of [3H]uridine into RNA and [3H]leucine into protein in slices of porcine thyroid was studied. Thyrotropin (10-500 mU/ml of medium), when added with [3H]uridine, inhibited incorporation into RNA, but as little as 10 mU of thyrotropin per ml stimulated incorporation of [3H]orotic acid into RNA. Uridine kinase (EC 2.7.1.48) was found to be inhibited in slices incubated with thyrotropin whereas UMP 5′ nucleotidase (EC 2.1.3.5) was not. Preincubation of slices with thyrotropin (5-50 mU/ml) led to enhanced incorporation of subsequently added [3H]uridine and [3H]leucine. When slices were preincubated with long-acting thyroid stimulator-IgG (2.5 or 5 mg per ml of medium) incorporation of [3H]uridine and [3H]leucine was similarly enhanced, with the smaller concentration being more effective. Without preincubation these stimulatory effects were mimicked by 1 mM dibutyryl 3′,5′-AMP and, to a lesser extent, 1 mM 3′,5′-AMP. AMP and ATP also stimulated [3H]uridine incorporation in this system but only after more prolonged periods of incubation than were required for the other nucleotides. RNA polymerase (EC 2.7.7.6) activity measured in isolated thyroid nuclei had two components, one Mg2+-stimulated and the other requ ring Mn2+ and high salt content [0.4 M (NH4)2SO4]. These activities, and particularly the former, were enhanced if thyroid slices were incubated with thyrotropin (5-100 mU/ml of medium), 2.5 mg or 5.0 mg of long-acting thyroid stimulator-IgG per ml, or 1 mM dibutyryl 3′,5′-AMP, before isolatior of the nuclei and measurement of enzyme activities; 1 mM AMP, ADP, or 2′,3′-GMP had no influence. Added directly to the nuclei, thyrotropin, long-acting thyroid stimulator-IgG, and dibutyryl 3′,5′-AMP had no effect on RNA polymerase activities. These data are seen as affording evidence for mediation by 3′,5′-AMP of effects of thyrotropin and long-acting thyroid stimulator on thyroid RNA and protein synthesis, at least in part through an indirect stimulation of nuclear RNA polymerase activities.

Stimulation by adenosine 3′,5′-cyclic monophosphate of protein synthesis by adenohypophyseal polyribosomes

Addition of dibutyryl 3′,5′-cyclic AMP to slices of bovine pituitary stimulated incorporation of [3H]leucine into protein, whether or not actinomycin D was present; therefore the influence of 3′,5′-cyclic AMP on protein synthesis by bovine pituitary polysomes was studied. If the cyclic nucleotide was added to the complete protein-synthesizing system (including pH 5.0 enzyme), stimulation of [3H]leucine incorporation occurred only with pH 5.0 enzyme from rat liver; there was no stimulation when homologous enzyme, i.e., from bovine pituitary, was used. Addition of 3′,5′-cyclic AMP to the polysomes, before addition of pH 5.0 enzyme, resulted in stimulation of protein synthesis with either source of enzyme, but stimulation was facilitated to a greater degree, over the range 0.5-2 mM 3′,5′-cyclic AMP, when rat liver was the source. The stimulation of protein synthesis was prevented by the addition of cycloheximide. With rat liver pH 5.0 enzyme the product of hydrolysis of 3′,5′-cyclic AMP was mainly 5′-AMP whereas with pituitary pH 5.0 enzyme there was also dephosphorylation and deamination resulting in production of hypoxanthine and other bases. However, using either source of pH 5.0 enzyme and the complete protein-synthesizing system (i.e., including an ATP-regenerating mechanism) most of the 3H from hydrolysis of [3H]3′,5′-cyclic AMP was incorporated into ATP. The data are seen as compatible with a stimulation by 3′,5′-cyclic AMP of translation by pituitary polysomes; the significance of the importance of the source of pH 5.0 enzyme used in the system is obscure.

USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

Chromatographic separation, fractionation and oxidation of selected beta-lactoglobulin peptides

The literature review elucidates the mechanism of oxidation in proteins and amino acids and gives an overview of the detection and analysis of protein oxidation products as well as information about ?-lactoglobulin and studies carried out on modifications of this protein under certain conditions. The experimental research included the fractionation of the tryptic peptides of ?-lactoglobulin using preparative-HPLC-MS and monitoring the oxidation process of these peptides via reverse phase-HPLC-UV. Peptides chosen to be oxidized were selected with respect to their amino acid content which were susceptible to oxidation and fractionated according to their m/z values. These peptides were: IPAVFK (m/z 674), ALPMHIR (m/z 838), LIVTQTMK (m/z 934) and VLVLDTDYK (m/z 1066). Even though it was not possible to solely isolate the target peptides due to co-elution of various fractions, the percentages of target peptides in the samples were satisfactory to carry out the oxidation procedure. IPAVFK and VLVLDTDYK fractions were found to yield the oxidation products reviewed in literature, however, unoxidized peptides were still present in high amounts after 21 days of oxidation. The UV data at 260 and 280 nm enabled to monitor both the main peptides and the oxidation products due to the absorbance of aromatic side-chains these peptides possess. ALPMHIR and LIVTQTMK fractions were oxidatively consumed rapidly and oxidation products of these peptides were observed even on day 0. High rates of depletion of these peptides were acredited to the presence of His (H) and sulfur-containing side-chains of Met (M). In conclusion, selected peptides hold the potential to be utilized as marker peptides in ?-lactoglobulin oxidation.

Kirjallisuuskatsaus: Eturauhassyövän androgeeniresponsiiviset in vivo -mallit; Kokeellinen osa: Bioluminesenssiin perustuvan ortotooppisen eturauhassyöpämallin optimointi

Eturauhassyöpä on yksi yleisimmistä syövistä länsimaissa. Eturauhassyöpä on yleensä hitaasti kehittyvä tauti. Edetessään se voi kuitenkin muuntua aggressiivisemmaksi ja aiheuttaa metastaaseja, jotka ovat pääasiallisena syynä taudin kuolleisuuteen. Androgeenit ovat merkittäviä tekijöitä eturauhassyövän patogeneesissä ja eturauhassyöpäkudos on useimmiten riippuvainen androgeeneista. Tämän vuoksi hoidon tavoitteena on estää niiden eritys kirurgisella tai kemiallisella kastraatiolla ja/tai estää androgeenien vaikutus antiandrogeeneilla. Eturauhassyöpää sekä sen hoitoon tarkoitettuja uusia lääkehoitomahdollisuuksia tutkitaan kiivaasti. Eturauhassyövän tutkimiseen on kehitetty lukematon määrä erilaisia in vivo -malleja. Koska eturauhassyöpä on yleensä androgeeneille herkkä, kuvaavat androgeeniresponsiiviset eläinmallit ihmisen tautia parhaiten. Eturauhassyövän mallintamiseen in vivo voidaan käyttää eri eläinlajeja, mutta hiiri on ylivoimaisesti käytetyin mallieläin. Immuunipuutteisiin hiiriin voidaan aiheuttaa kasvaimia inokuloimalla ihmisen kasvainsoluja tai osia ihmisen kasvaimista. Ortotooppisesti eturauhaseen inokuloitavat kasvainmallit mallintavat eturauhassyövässä esiintyvää syöpäsolujen ja stroomasolujen välistä epänormaalia vuorovaikutusta. Muuntogeeniset hiirimallit ovat yhä yleisempiä eturauhassyövän tutkimuksessa. Muuntogeenisilla malleilla voidaan mallintaa taudin kehittymistä ja sen etenemistä kokonaisuudessaan parhaiten. Eturauhasessa olevaa kasvainta ja sen kasvua on vaikea seurata ilman prostataspesifisen antigeenin (PSA) pitoisuuden mittausta tai erityisiä kuvantamistekniikoita. Tällaisia menetelmiä, kuten optista kuvantamista, käytetään yhä enemmän hyödyksi erilaisissa eturauhassyövän in vivo -malleissa. Tutkielman kokeellisen osan tavoitteena oli optimoida bioluminesenssiin perustuva optinen kuvantamismenetelmä androgeeniresponsiivisessa LNCaP-luc2-solulinjassa ortotooppisessa eturauhassyöpämallissa. Bioluminesenssikuvantaminen perustuu kasvainsolujen ilmentämän lusiferaasin katalysoimaan reaktioon, jossa entsyymin substraatti, lusiferiini, hapettuu ja tuottaa näkyvää valoa. Lisäksi tavoitteena oli tutkia lääkehoitojen ja kastraation vasteita mallissa. Bioluminesenssiin perustuvalla kuvantamisella oli mahdollista seurata eturauhaskasvainten kasvua noninvasiivisesti, reaaliaikaisesti ja toistuvasti. Bioluminesenssikuvantamisen avulla kasvainten kvantitointi oli nopeampaa kuin ultraäänikuvantamisen avulla, ja kasvainten kasvua oli myös mahdollista seurata useammin kuin seerumin PSA-mittausten avulla. Bioluminesenssikuvantamisen todettiin korreloivan paremmin PSA-pitoisuuden kanssa kuin kasvaimen todelliseen kokoon lopetushetkellä. Seerumin PSA-pitoisuus korreloi kuitenkin bioluminesenssimittausta paremmin eturauhaskasvaimen kokoon tässä kokeessa. Kasvainten oletettua suurempaa kokoa voidaan pitää todennäköisimpänä syynä sille, ettei lääkehoitojen tai kastraation todettu vaikuttavan kasvainten kasvuun bioluminesenssikuvantamisella mitattuna. Bioluminesenssikuvantaminen ei sovellu suurille eikä nekroottisille kasvaimille, sillä kuvantamismenetelmä toimii vain elävillä soluilla. Bioluminesenssikuvantamisen hyödyntämisen kannalta oleellista tässä mallissa on myös lusiferiini-injektion onnistuminen. Jatkotutkimuksia tarvitaan edelleen mallin validoimiseksi mm. lääkehoitojen vasteiden osoittamiseksi.

Sex steroids in Sjögren's syndrome

Sjögren s syndrome (SS) is a strongly female dominant autoimmune disease. SS targets mainly salivary and lacrimal glands and leads to loss of the secreting acinar cells of these glands. Accordingly, secretion of the affected glands is diminished and the main symptoms of SS, dryness of mouth and eyes, follow. In addition to these sicca symptoms, SS patients suffer from severe fatigue and can have various extraglandular symptoms. To date, the etiology of SS still remains unknown. Female dominance and the late onset of the disease simultaneously with remarkable hormonal changes in the body (menopause, adrenopause) encouraged us to hypothesize that sex steroids, especially androgens, are involved in the onset and progression of SS. We confirmed our hypothesis and showed that patients with SS suffer from androgen depletion both systemically and locally in the target tissue of SS, salivary glands. We especially focused on the local androgen environment in salivary glands and demonstrated that healthy salivary glands contain a complete enzymatic machinery for local synthesis of androgens and estrogens from pro-hormone dehydroepiandrosterone (DHEA). However, in SS salivary glands the enzymes catalyzing the local androgen synthesis are defective and, in a subgroup of patients, practically non-functional. Probably due to this local defect in DHEA processing, therapy with DHEA was found unbeneficial for SS patients in the treatment of fatigue. We also studied the effect of the local androgen depletion on salivary glands. We found that in salivary gland cells and healthy labial salivary glands androgens upregulate integrin subunits α1 and α2, which are important for the communication, differentiation and function of the acinar cells. On the contrary, in SS salivary glands DHEA failed to upregulate these signaling molecules, again probably due to defective processing of DHEA into active androgens. Our finding highlights the importance of the local androgen environment and local DHEA processing for the function and welfare of salivary glands. In conclusion, this study showed that patients with SS are androgen depleted both systemically and locally in salivary glands. SS patients also have a defective local sex steroid synthesizing enzymatic machinery further impairing the local androgen depletion. We also showed that the local androgen defect leads to decreased expression of acinar cell specific integrin molecules, which impairs the signaling between the acinar cells and basement membrane and might thus explain the acinar cell loss seen in SS salivary glands. By showing the importance of the local sex steroid imbalance in SS we have clarified some etiopathogenetic mechanisms of SS, which have thus far remained unknown.

CREMOFAC- a database of chromatin remodeling factors

Motivation: Chromatin-remodeling is an important event in the eukaryotic nucleus rendering nucleosomal DNA accessible for various transaction processes. Remodeling Factors facilitate the dynamic nature of chromatin through participation of the collective action of (i) ATP and (ii) Non-ATP dependent factors. Considering the importance of these factors in eukaryotes, we have developed, CREMOFAC, a dedicated and frequently updated web-database for chromatin-remodeling factors.Results: The database harbors factors from 49 different organisms reported in literature and facilitates a comprehensive search for them. In addition, it also provides in-depth information for the factors reported in the three widely studied mammals namely, human, mouse and rat. Further, information on literature, pathways and phylogenetic relationships has also been covered. The development of CREMOFAC as a central repository for chromatin-remodeling factors and the absence of such a pre-existing database heighten its utility thus making its presence indispensable.

M.tuberculosis Pantothenate Kinase: Dual Substrate Specificity and Unusual Changes in Ligand Locations

Kinetic measurements of enzyme activity indicate that type I pantothenate kinase from Mycobacterium tuberculosis has dual substrate specificity for ATP and GTP, unlike the enzyme from Escherichia coli, which shows a higher specificity for ATP. A molecular explanation for the difference in the specificities of the two homologous enzymes is provided by the crystal structures of the complexes of the M. tuberculosis enzyme with (1) GMPPCP and pantothenate, (2) GDP and phosphopantothenate, (3) GDP, (4) GDP and pantothenate, (5) AMPPCP, and (6) GMPPCP, reported here, and the structures of the complexes of the two enzymes involving coenzyme A and different adenyl nucleotides reported earlier. The explanation is substantially based on two critical substitutions in the amino acid sequence and the local conformational change resulting from them. The structures also provide a rationale for the movement of ligands during the action of the mycobacterial enzyme. Dual specificity of the type exhibited by this enzyme is rare. The change in locations of ligands during action,observed in the case of the M. tuberculosis enzyme, is unusual, so is the striking difference between two homologous enzymes in the geometryof the binding site, locations of ligands, and specificity. Furthermore, the dual specificity of the mycobacterial enzyme appears to have been caused by a biological necessity. (C) 2010 Elsevier Ltd.All rights reserved.

Asynchronous behaviour in some three-atom linear concerted radical-exchange reactions

A few simple three-atom thermoneutral radical exchange reactions (i.e. A + BC --> AB + C) are examined by ab initio SCF methods. Emphasis is laid on the detailed analysis of density matrices rather than on energetics. Results reveal that the sum of the bond orders of the breaking and forming bonds is not conserved to unity, due to development of free valence on the migrating atom 'B' in the transition state. Bond orders, free valence and spin densities on the atoms are calculated. The present analysis shows that the bond-cleavage process is always more advanced than the bond-formation process in the transition state. Further analysis shows a development of the negative spin density on the migrating atom 'B' in the transition state. The depletion of the alpha-spin density on the radical site "A" in the reactant during the reaction lags behind the growth of the alpha-spin density on the terminal atom "C" of the reactant bond, 'B-C' in the transition state. But all these processes are completed simultaneously at the end of the reaction. Hence, the reactions are asynchronous but kinetically concerted in most cases.

Varistors based on polycrystalline ZnO:Cu

Sintered, polycrystalline ZnO ceramics with copper as the only additive exhibit highly nonlinear current‐voltage characteristics. Increasing nonlinearity index (α=4–45) with Cu concentration of 0.01–1 mol % is also variable with respect to ceramic processing methods. Incorporation of Cu in the ZnO lattice is indicated from the electron probe microanalysis and the photoluminescence spectra. Cu acceptors are compensated by holes in the grain boundary layers, whereas the concentration of intrinsic donors is higher in the grain interior. The presence of positive charges leads to thinning of the depletion region, resulting in nonlinear characteristics.

Ab initio studies on the tri- and diphosphate fragments of adenosine triphosphate

Pyrophosphate prototypes such as methyl triphosphate and methyl diphosphate molecules in their different protonation states have been investigated at high levels of quantum chemical calculations. The optimized geometries, the thermochemistry of the hydrolysis and the molecular orbitals contributing to the high energy of these compounds have been analyzed. These investigations provide insights into the "high energy" character of ATP molecule. Further, the dependence of vibrational frequencies on the number of phosphate groups and the charged states has also been presented. These results can aid the interpretation of spectra obtained by experiments on complexes containing pyrophosphate prototypes.

Heme is a positive regulator of cytochrome P-450 gene transcription

Inhibitors of heme biosynthesis such as CoCl2, 3-amino-1,2,4-triazole, and thioacetamide block the 3-methylcholanthrene-mediated induction of cytochrome P-450 (c + d) messenger RNAs and their transcription in rat liver. This effect is specific, since the messenger RNA levels for albumin and glutathione transferase (Ya + Yc) and their transcription are not significantly influenced under conditions of heme depletion. Exogenous administration of heme at very low doses (50 μg/100 g body wt) is able to completely counteract the effects of the heme biosynthetic inhibitors on cytochrome P-450 (c + d) messenger RNA levels and their transcription. This constitutes a direct proof for the role of heme as a positive regulator of cytochrome P-450 gene transcription.

Diabetic cardiomyopathy and post-infarct ventricular remodelling : Effects of levosimendan in a rodent model of type II diabetes

Type 2 diabetes is a risk factor for the development of cardiovascular disease. Recently, the term diabetic cardiomyopathy has been proposed to describe the changes in the heart that occur in response to chronic hyperglycemia and insulin resistance. Ventricular remodelling in diabetic cardiomyopathy includes left ventricular hypertrophy, increased interstitial fibrosis, apoptosis and diastolic dysfunction. Mechanisms behind these changes are increased oxidative stress and renin-angiotensin system activation. The diabetic Goto-Kakizaki rat is a non-obese model of type 2 diabetes that exhibits defective insulin signalling. Recently two interconnected stress response pathways have been discovered that link insulin signalling, longevity, apoptosis and cardiomyocyte hypertrophy. The insulin-receptor PI3K/Ak pathway inhibits proapoptotic FOXO3a in response to insulin signalling and the nuclear Sirt1 deacetylase inhibits proapoptotic p53 and modulates FOXO3a in favour of survival and growth. --- Levosimendan is a calcium sensitizing agent used for the management of acute decompensated heart failure. Levosimendan acts as a positive inotrope by sensitizing cardiac troponin C to calcium and exerts vasodilation by opening mitochondrial and sarcolemmal ATP-sensitive potassium channels. Levosimendan has been described to have beneficial effects in ventricular remodelling after myocardial infarction. The aims of the study were to characterize whether diabetic cardiomyopathy associates with cardiac dysfunction, cardiomyocyte apoptosis, hypertrophy and fibrosis in spontaneously diabetic Goto-Kakizaki (GK) rats, which were used to model type 2 diabetes. Protein expression and activation of the Akt FOXO3a and Sirt1 p53 pathways were examined in the development of ventricular remodelling in GK rats with and without myocardial infarction (MI). The third and fourth studies examined the effects of levosimendan on ventricular remodelling and gene expression in post-MI GK rats. The results demonstrated that diabetic GK rats develop both modest hypertension and features similar to diabetic cardiomyopathy including cardiac dysfunction, LV hypertrophy and fibrosis and increased apoptotic signalling. MI induced a sustained increase in cardiomyocyte apoptosis in GK rats together with aggravated LV hypertrophy and fibrosis. The GK rat myocardium exhibited decreased Akt- FOXO3a phosphorylation and increased nuclear translocation of FOXO3a and overproduction of the Sirt1 protein. Treatment with levosimendan decreased cardiomyocyte apoptosis, senescence and LV hypertrophy and altered the gene expression profile in GK rat myocardium. The findings indicate that impaired cardioprotection via Akt FOXO3a and p38 MAPK is associated with increased apoptosis, whereas Sirt1 functions in counteracting apoptosis and the development of LV hypertrophy in the GK rat myocardium. Overall, levosimendan treatment protects against post-MI ventricular remodelling and alters the gene expression profile in the GK rat myocardium.

Ab initio studies on the tri- and diphosphate fragments of adenosine triphosphate

Pyrophosphate prototypes such as methyl triphosphate and methyl diphosphate molecules in their different protonation states have been investigated at high levels of quantum chemical calculations. The optimized geometries, the thermochemistry of the hydrolysis and the molecular orbitals contributing to the high energy of these compounds have been analyzed. These investigations provide insights into the ``high energy'' character of ATP molecule. Further, the dependence of vibrational frequencies on the number of phosphate groups and the charged states has also been presented. These results can aid the interpretation of spectra obtained by experiments on complexes containing pyrophosphate prototypes. (c) 2005 Elsevier B.V. All rights reserved.