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1.
Biochem Pharmacol ; 76(7): 825-30, 2008 Oct 01.
Article in English | MEDLINE | ID: mdl-18706399

ABSTRACT

Most of the effects described for bisphosphonates (pC(R1)(R2)p) are related, directly or indirectly with a pyrophosphate moiety. Bisphosphonates are (i) analogs of pyrophosphate in the synthesis of ATP derivatives (AppC(R1)(R2)p) catalyzed by ligases and (ii) inhibitors of enzymes of the mevalonate pathway with substrates containing a terminal pyrophosphate. Searching for the role of bisphosphonates on other reactions involving pyrophosphate, we explored their effect on a phosphoribosyltransferase activity, present in Saccharomyces cerevisiae cell extracts, using 5-fluorouracil or uracil as substrates. Unexpectedly, bisphosphonates increased the initial rate of synthesis of 5-FUMP (from 5-fluorouracil and phosphoribosylpyrophosphate): etidronate (2.8+/-0.3 times); pamidronate (2.6+/-0.4 times); alendronate (2.5+/-0.6 times) and clodronate (2.0+/-0.1 times). Similar values for the synthesis of UMP (from uracil and phosphoribosylpyrophosphate) were obtained in the presence of bisphosphonates. The values of the activation constants determined for alendronate and clodronate for the synthesis of UMP were 0.05+/-0.02 mM and 0.32+/-0.22 mM, respectively. These results raise the possibility that bisphosphonates enhance the effect of 5-fluorouracil (or other uracil prodrugs) in the treatment of bone tumors or bone tumor metastases.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Diphosphonates/pharmacology , Fluorouracil/pharmacology , Pentosyltransferases/metabolism , Saccharomyces cerevisiae/drug effects , Bone Density Conservation Agents/pharmacology , Neoplasms/drug therapy , Saccharomyces cerevisiae/enzymology , Uracil/pharmacology
2.
Biochem Pharmacol ; 75(10): 1959-65, 2008 May 15.
Article in English | MEDLINE | ID: mdl-18378215

ABSTRACT

T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.


Subject(s)
Adenosine Triphosphate/chemistry , Diphosphonates/chemistry , Acetate-CoA Ligase/chemistry , Acetate-CoA Ligase/metabolism , Acetyl Coenzyme A/chemistry , Acetyl Coenzyme A/metabolism , Adenosine Triphosphate/metabolism , DNA Ligases/metabolism , Diphosphonates/metabolism , Luciferases/chemistry , Luciferases/metabolism , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Activating Enzymes/metabolism
3.
FEMS Yeast Res ; 8(2): 257-65, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17995957

ABSTRACT

Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1% glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism.


Subject(s)
Antimetabolites/pharmacology , Fluorodeoxyuridylate/analogs & derivatives , Fluorouracil/pharmacology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Uridine Diphosphate Glucose/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/metabolism , Aerobiosis , Alkaline Phosphatase/metabolism , Chromatography, Liquid , Culture Media/chemistry , Fermentation , Fluorodeoxyuridylate/chemistry , Fluorodeoxyuridylate/isolation & purification , Fluorodeoxyuridylate/metabolism , Magnetic Resonance Spectroscopy , Microbial Viability , Phosphodiesterase I/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectrum Analysis , Uracil/analysis , Uridine Diphosphate Glucose/chemistry , Uridine Diphosphate Glucose/isolation & purification , Uridine Diphosphate Glucose/metabolism , Uridine Diphosphate Glucose Dehydrogenase/metabolism , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/isolation & purification
4.
FEBS Lett ; 579(27): 6223-9, 2005 Nov 07.
Article in English | MEDLINE | ID: mdl-16256113

ABSTRACT

Previous work from this laboratory had shown that ligases may catalyze the synthesis of (di)nucleoside polyphosphates. Here, we show that one of the enzymes of the proteasome system (E1 or the ubiquitin (Ub) activating enzyme, EC 6.3.2.19) catalyzes very effectively (k(cat) = 0.29+/-0.05 s(-1)) the transfer of AMP from the E-AMP-ubiquitin complex to tripolyphosphate or tetrapolyphosphate with formation of adenosine tetra- or pentaphosphate (p4A or p5A), respectively. Whereas the concomitant formation of AMP is stimulated by the presence of dithiothreitol in a concentration dependent manner, the synthesis of p4A is only slightly inhibited by this compound. Previous treatment of the enzyme (E1) with iodoacetamide inhibited only partially the synthesis of p4A. p4A can substitute for ATP as substrate of the reaction to generate the ubiquityl adenylate complex. A small amount of diadenosine pentaphosphate (Ap5A) was also synthesized in the presence of p4A.


Subject(s)
Dinucleoside Phosphates/biosynthesis , Ubiquitin-Activating Enzymes/chemistry , Adenosine Monophosphate/biosynthesis , Dithiothreitol/pharmacology , Iodoacetamide/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors
5.
FEBS Lett ; 579(19): 4044-8, 2005 Aug 01.
Article in English | MEDLINE | ID: mdl-16023109

ABSTRACT

Low concentrations of HgCl2 elicited, in Saccharomyces cerevisiae, a transitory increase in the ATP level followed by a decrease of its concentration, until almost disappearance. At 1 microM HgCl2, the increase in ATP lasted for about 30 min, while at 10 microM the increase was only observed in the first 5 min of treatment. The initial burst of ATP was accompanied by a decrease in the level of hexose phosphates, whereas during the decrease of ATP an increase in the inosine and hexose phosphates levels took place. The treatment with HgCl2 inhibited the plasma membrane proton ATPase but not the activities of hexokinase or 6-phosphofructokinase.


Subject(s)
Adenosine Triphosphate/metabolism , Mercuric Chloride/pharmacology , Saccharomyces cerevisiae/drug effects , Kinetics , Saccharomyces cerevisiae/metabolism
6.
FEMS Yeast Res ; 5(4-5): 387-98, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15691744

ABSTRACT

Saccharomyces cerevisiae cells (strain W303) grown in a minimal medium (containing 2% or 0.1% glucose) until exponential or stationary phase, were subjected to chronological aging in water, and yeast viability and nucleotide content were analyzed along several days of nutrient starvation. Cells collected in exponential phase (whether grown in the presence of 0.1% or 2% glucose) were viable up to five days and thereafter the viability decreased linearly with a half-survival rate of around eight days. ATP and other nucleoside triphosphates decreased similarly in both cases. Cells collected in stationary phase, and transferred to water, behaved differently whether grown in 0.1% or in 2% glucose, with a half-survival life of around nine and 28 days respectively. A double mutant in glycogen synthase (gsy1delta gsy2delta) and its isogenic wild-type strain, grown to stationary phase in 2% glucose, presented a similar half-survival life of around eight days. The W303 cells grown to stationary phase in the presence of 2% glucose showed a 7-fold increase of UDP-N-acetylglucosamine (UDP-GlcNAc) as compared with the level present in the cells grown in any of the other three metabolic situations. The nature of UDP-GlcNAc was established by MALDI-TOF ionization analysis. It is also worth noting that the rate of decay of NAD+ was lower than that of ATP in any of the situations here considered.


Subject(s)
Nucleotides/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/physiology , Culture Media , Heat-Shock Response , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Time Factors , Uridine Diphosphate N-Acetylglucosamine/metabolism , Water
7.
Arch Microbiol ; 181(3): 231-6, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14735298

ABSTRACT

As has been previously shown, Saccharomyces cerevisiae grown in 2% or 0.025% glucose uses this carbohydrate by the fermentative or oxidative pathways, respectively. Depending on the glucose concentration in the medium, the effect of the addition of H2O2 on the level of ATP and on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity differed. In the presence of 2% glucose, ATP and GAPDH decreased sharply during the first few minutes of treatment, whereas in the presence of 0.025% glucose, GAPDH activity decreased similarly, but the ATP level remained practically unchanged. The addition of 3 mM glutathione to the culture media prevented the depletion of ATP levels and GAPDH activity in the presence of H2O2. Catalase and superoxide dismutase activities did not vary significantly when yeast cells were grown either in 2% or in 0.025% glucose.


Subject(s)
Adenosine Triphosphate/metabolism , Glucose/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Hydrogen Peroxide/pharmacology , Saccharomyces cerevisiae/metabolism , Catalase/analysis , Glutathione/metabolism , Hydrogen Peroxide/metabolism , Inosine/analysis , Kinetics , Nucleotides/analysis , Oxidants/pharmacology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/enzymology , Superoxide Dismutase/analysis
8.
Eur J Biochem ; 270(7): 1578-89, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12654013

ABSTRACT

When Saccharomyces cerevisiae cells, grown in galactose, glucose or mannose, were treated with 1.5 mm hydrogen peroxide (H2O2) for 30 min, an important decrease in the ATP, and a less extensive decrease in the GTP, CTP, UTP and ADP-ribose levels was estimated. Concomitantly a net increase in the inosine levels was observed. Treatment with 83 mm menadione promoted the appearance of a compound similar to adenosine but no appreciable changes in the nucleotide content of yeast cells, grown either in glucose or galactose. Changes in the specific activities of the enzymes involved in the pathway from ATP to inosine, in yeast extracts from (un)treated cells, could not explain the effect of H2O2 on the levels of ATP and inosine. Application of a mathematical model of differential equations previously developed in this laboratory pointed to a potential inhibition of glycolysis as the main reason for that effect. This theoretical consideration was reinforced both by the lack of an appreciable effect of 1.5 mm (or even higher concentrations) H2O2 on yeast grown in the presence of ethanol or glycerol, and by the observed inhibition of the synthesis of ethanol promoted by H2O2. Normal values for the adenylic charge, ATP and inosine levels were reached at 5, 30 and 120 min, respectively, after removal of H2O2 from the culture medium. The strong decrease in the ATP level upon H2O2 treatment is an important factor to be considered for understanding the response of yeast, and probably other cell types, to oxidative stress.


Subject(s)
Adenosine Triphosphate/metabolism , Hydrogen Peroxide/pharmacology , Inosine/metabolism , Oxidants/pharmacology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Vitamin K 3/pharmacology , Ethanol/metabolism , Galactose/metabolism , Glucose/metabolism , Glycerol/metabolism , Glycolysis/drug effects , Glycolysis/physiology , Mannose/metabolism , Nucleotides/metabolism , Oxidative Stress/physiology
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