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G Divita

Publications and source records attributed to G Divita.

53 records · Page 3Linked to original sources
PubMed1995

ASN.1: defining a grammar for the UMLS knowledge sources.

The unified Medical Language System (UMLS) project provides resources on an experimental basis to the research community. In 1995 the four UMLS Knowledge Sources have been provided in an additional data format, Abstract Syntax Notation One (ASN.1). The benefits of ASN.1 are that it provides a standard, formal grammar for complex data and allows exchange of that data in a way which is independent of the particular software and hardware environment in which the data are created and stored. The paper begins with an introduction to the ASN.1 standard itself. It continues with a discussion of the ASN.1 implementation of the UMLS Knowledge Sources and some of the consequences for the newly released UMLS Knowledge Source Server. It concludes with a discussion of some of the benefits of using ASN.1 encoded data.

Computer Communication Networks↗
PubMed1994

Inhibition of human immunodeficiency virus type 1 reverse transcriptase dimerization using synthetic peptides derived from the connection domain.

Based on presently available information on the structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, peptides have been synthesized which correspond to the sequence of a particular region of the protein involved in formation of the active heterodimeric form of the enzyme. Several peptides that are 15-19 amino acids long and that are derived from the so-called connection domain of the reverse transcriptase are able to inhibit dimerization of the enzyme and thus inhibit development of its enzymatic activities. In particular, a tryptophan-rich 19-mer corresponding to residues 389-407 was relatively efficient, showing an apparent dissociation constant in the micromolar range for one or both of the subunits. The sequence of this region is identical for both subunits, since one (molecular mass of 51 kDa) is the proteolytic product of the other (molecular mass of 66 kDa). Dissociation of the preformed heterodimer could not be induced by the peptides, but increasing concentrations reduced the rate of dimerization in a concentration-dependent manner until it became immeasurable at high concentrations. The results suggest that inhibition of dimerization of reverse transcriptase is an attractive approach to chemotherapeutic intervention in HIV infection and that further development of peptide-based inhibition strategies is worth pursuing.

Amino Acid Sequence↗
PubMed1994

Intrinsic tryptophan fluorescence of bovine liver adenosine kinase, characterization of ligand binding sites and conformational changes.

Bovine liver adenosine kinase is a 45-kDa monomeric protein which exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 284 nm and an emission peak centered at 335 nm. A total of three tryptophan residues/molecule has been estimated by using a fluorescence titration method. Low values of Stern-Volmer quenching constants in the presence of either acrylamide or iodide (4.2 M-1 or 1.5 M-1, respectively) indicated that the tryptophan residues are relatively buried in the native molecule. Tryptophan residues also showed a high heterogeneity, with a fractional accessible fluorescence value for iodide of 0.65. The enzyme fluorescence was very sensitive to substrate binding, which induced a marked fluorescence quenching, a lower tryptophan accessibility to acrylamide and iodide, and an increase in the tryptophan heterogeneity. ADP or ATP showed a monophasic saturation curve consistent with the existence of one binding site. In contrast, adenosine and AMP gave biphasic saturation curves, suggesting the existence of at least two binding sites, with a high and a low affinity. The presence of MgCl2 increased the affinity of ATP or ADP, whereas the binding of adenosine or AMP was not affected.

Adenosine↗
PubMed1993

Rapid determination of the affinity of 28- and 14-mer phosphorothioate oligonucleotides for HIV-1 reverse transcriptase by fluorescence spectroscopy.

Intrinsic fluorescence of human immunodeficiency virus type 1 reverse transcriptase (E.C. 2.7.7.49) and displacement experiments of a fluorescent template.primer probe were used to study the interaction of the enzyme with several types of 28- and 14-mer normal or phosphorothioate oligodeoxycytidinylates and their duplexes with poly(rI). The two methods gave convergent results and allowed in each case fast determinations of ligand affinities for the enzyme. The dissociation constants (Kd) obtained from intrinsic fluorescence changes were slightly lower than those determined from the less direct competitive displacement experiments. In all cases, the enzyme displayed better recognition of the hybrid than of the unannealed oligonucleotide. The Kd values of phosphorothioate oligomers and their hybrids were lower than those of the corresponding normal oligomers and hybrids, but the difference was not as significant as in the case of the Ki constants for (dC)28 and S(dC)28 (Majumdar et al. (1989) Biochemistry 28, 1340). The affinities of the annealed phosphorothioate oligodeoxycytidinylates for the enzyme were found to be larger than for any other compounds in this series (Kd of poly(rI).S(dC)28: 0.28 nM at 25 degrees C). Changing the beta stereochemistry of the oligomer bases to alpha did not alter the affinity of the oligodeoxycytidinylate and its hybrids for the enzyme.

Base Sequence↗
PubMed1993

Functional nucleotide-binding domain in the F0F1-ATPsynthase alpha subunit from the yeast Schizosaccharomyces pombe.

The segment R165-T330 of the alpha subunit of Schizosaccharomyces pombe F1-ATPase, corresponding to a putative nucleotide-binding domain by comparison with related nucleotide-binding proteins, has been overexpressed in Escherichia coli. Produced as a nonsoluble material, it was purified in a nonnative form, using a rapid procedure that includes one reversed-phase chromatography step. Refolding of the domain, called DN alpha 19, was achieved quantitatively by using a high-dilution step and monitored by circular dichroism and intrinsic fluorescence. Once folded, DN alpha 19 was highly soluble and stable. It bound 1 mol/mol either of adenine or guanine di- or triphosphate nucleotide, with a Kd ranging from 2.3 to 5.4 microM, or of methylanthraniloyl derivatives of the same nucleotides, with a Kd ranging from 0.2 to 0.6 microM. Interesting, DN alpha 19 was able to hydrolyze nucleoside triphosphates at a low but significant rate. The distance between one tryptophan residue located in the nucleotide-binding site and the ribose-linked methylanthraniloyl group of di- or triphosphate nucleotides was estimated by fluorescence resonance energy transfer to be 13 or 11 A, respectively, suggesting that the tryptophan is close to the polyphosphate moiety of the nucleotide. This tryptophan residue was tentatively assigned to W190 by a hydrophobic cluster comparison with the H-ras p21 protein, suggesting that the putative loop of DN alpha 19 containing W190 could play a functional role in nucleotide binding.

Adenosine Triphosphate↗
PubMed1993

Glutamine 170 to tyrosine substitution in yeast mitochondrial F1 beta-subunit increases catalytic site interaction with GDP and IDP and produces negative cooperativity of GTP and ITP hydrolysis.

Glutamine 170 to tyrosine mutation in the beta-subunit from Schizosaccharomyces pombe mitochondrial F1 was found to increase both affinity for ADP, apparent negative cooperativity of ATPase activity, and sensitivity to azide inhibition (Falson, P., Di Pietro, A., Jault, J.-M., Gautheron, D.C., and Boutry, M. (1989) Biochim. Biophys. Acta 975, 119-126). The mutation is shown here to increase the affinity for GDP, IDP, and guanosine 5'-(beta,gamma-imidotriphosphate), which are competitive inhibitors of GTPase and ITPase activities. Various fluorescence approaches also reveal an increased affinity of the catalytic site in mutant as compared with wild-type enzyme for GDP, IDP, and 2'(3')-N-methylanthraniloyl GDP. The mutation alters the maximal rates and pH dependence of GTPase and ITPase activities, whereas wild-type F1 exhibits single optima at pH 7.5-8.0. The pH activity profiles of the mutant enzyme for these substrates are biphasic, with optima at pH 8.5-9.0 and below 6.5. The mutation increases the sensitivity of GTPase and ITPase activities to azide inhibition, which increases with decreasing pH. At pH 6.0-7.0, an apparent negative cooperativity is observed when mutant F1 hydrolyzes GTP or ITP, whereas the wild-type enzyme shows Michaelian kinetics. Addition of bicarbonate at pH 7.0 substantially stimulates GTP or ITP hydrolysis and abolishes the apparent negative cooperativity by the mutant enzyme; on the contrary, the anion produces a slight inhibition of these activities catalyzed by wild-type F1. The overall results suggest that apparent negative cooperativity can be observed with GTP or ITP hydrolysis provided that the release of the respective diphosphate is a rate-limiting step.

Azides↗
PubMed1993

Kinetics of interaction of HIV reverse transcriptase with primer/template.

Intrinsic protein fluorescence of reverse transcriptases from HIV-1 and HIV-2 provides a sensitive signal for monitoring the interaction of the enzymes with primer/template duplex molecules. Kd values for 18/36-mer DNA/DNA duplexes were found to be in the range of a few nanomolar (about 3 times higher for the enzyme from HIV-2 than for that from HIV-1). The quenching of protein fluorescence induced on binding primer/template, together with an increase in extrinsic fluorescence on interaction with primer/template containing a fluorescent nucleotide at the 3'-end of the primer, was used to investigate the kinetics of interaction with reverse transcriptase from HIV-1. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial association (k(ass) = ca. 5 x 10(8) M-1 s-1) followed by a slow isomerization step (k = ca. 0.5 s-1). These (forward) rate constants are increased in the presence of a non-nucleoside inhibitor (S-TIBO) of HIV-1 reverse transcriptase, while the reverse rate constant for the second step is decreased, leading to an increase in affinity between the enzyme and primer/template by a factor of at least 10 when S-TIBO is bound. The results are discussed in terms of present knowledge of the structure of reverse transcriptase.

Base Sequence↗
PubMed1993

Structural mapping of catalytic site with respect to alpha-subunit and noncatalytic site in yeast mitochondrial F1-ATPase using fluorescence resonance energy transfer.

The intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1 is a very sensitive probe to differentiate nucleotide binding to catalytic and noncatalytic sites (Divita, G., Di Pietro, A., Roux, B., and Gautheron, D. C. (1992) Biochemistry 31, 5791-5798), the catalytic site saturation producing quenching of Trp-257 fluorescence (Divita, G., Jault, J.-M., Gautheron, D. C., and Di Pietro, A. (1993) Biochemistry 32, 1017-1024). The present results indicate that two types of fluorescent nucleotide analogues, bearing either 2'(3')N-methylanthraniloyl (mant) or 2',3'-O-(2,4,6-trinitrophenyl) (TNP) group, exhibit high-affinity binding and behave similarly to the corresponding unmodified nucleotides. Selective binding of mant GDP to the catalytic site produces a marked quenching of intrinsic fluorescence which is due to resonance energy transfer between Trp-257 and the mant group. The high efficiency of the transfer allows the determination of a short distance, 10.5 A, indicating the close proximity of catalytic site and alpha-subunit Trp-257. Selective saturation of the noncatalytic site by TNP-ADP produces a marked quenching of the extrinsic fluorescence of mant GDP bound to the catalytic site, which is correlated to an important resonance energy transfer between the two fluorescent groups. A rather short distance of 17.5 A is calculated, indicating vicinity of catalytic and noncatalytic sites.

Adenosine Diphosphate↗
PubMed1993

Characterization of the dimerization process of HIV-1 reverse transcriptase heterodimer using intrinsic protein fluorescence.

Intrinsic protein fluorescence has been used to study dimerization of the HIV-1 reverse transcriptase (RT). We observed a 25% increase of the tryptophan fluorescence of the enzyme during dissociation of the subunits induced by the addition of acetonitrile. Upon reassociation of the separated subunits, the original fluorescence emission of the heterodimer is restored. A two-state transition model for the RT dimerization process in which the dimers are in equilibrium with folded monomers is proposed. The free energy of dissociation was determined to be 12.2 (+/- 0.2) kcal/mol. In the absence of Mg2+ ions a decrease of this value was observed, whereas the addition of a synthetic primer/template (18/36mer) results in an increase of dimer stability. Analyzing the effect of Mg2+ on the establishment of the binding equilibrium, a dramatic effect with a 100-fold acceleration of the association by the divalent ion was observed.

Acetonitriles↗
PubMed1993

4'-Thio-oligo-beta-D-ribonucleotides: synthesis of beta-4'-thio-oligouridylates, nuclease resistance, base pairing properties, and interaction with HIV-1 reverse transcriptase.

We present the synthesis and the study of properties of a new series of modified oligonucleotides, namely 4'-thio-oligo-beta-D-ribonucleotides (4'-S-RNA). Homo-oligonucleotides of this class (4'-SU6 and 4'-SU12) were prepared from the previously known thionucleosides using the phosphoramidite methodology. The comparison of the substrate properties of 4'-SU6 and its natural analog U6 with respect to four nucleases indicates that the former is much more resistant than the latter. Such resistance to nucleases in addition to relatively high Tm values for 4'-SU12 hybridized with Poly(A) show that these new 4'-S-RNA are good candidates for potential antisense effects. The oligonucleotides 4'-SU6 and 4'-SU12 have been also evaluated as non sequence specific inhibitors of HIV-1 reverse transcriptase. All available evidences, based primarily on fluorescence measurements, are consistent with the binding of 4'-SU6 and 4'-SU12 to RT at a site which is different from the polymerase site of the enzyme.

Animals↗
PubMed1993

Intrinsic tryptophan fluorescence of rat liver elongation factor eEF-2 to monitor the interaction with guanylic and adenylic nucleotides and related conformational changes.

Elongation factor 2 (eEF-2), which contains seven Trp residues, exhibited a tryptophan-characteristic intrinsic fluorescence with maximum excitation at 280 nm and an emission peak centered at 333 nm that suggested a hydrophobic environment of these tryptophans. Upon denaturation with 6 M guanidine hydrochloride, the maximum emission was shifted to 348 nm. Fluorescence quenching studies using acrylamide and iodide confirmed that the Trp residues were mainly buried in the native molecule and indicated an important heterogeneity, the fractional accessible fluorescence (fa) values being 0.50 and 0.25, respectively. Partial quenching of eEF-2 fluorescence by nucleotides proved the existence of an interaction of the factor in the absence of ribosomes, not only with GDP but also with GTP, nonhydrolyzable analogs, GMP, and adenylic, but not cytidylic, nucleotides. Saturating binding plots showed different maximal changes of fluorescence depending upon the nucleotides, from 6.4% with ADP to 24.5% with GDP, and suggested the existence of more than one binding site for each nucleotide. Among all the nucleotides tested, only GTP at saturating concentration modified the fa value obtained with acrylamide (-36%). The possibility that this modification is related to a conformational change of eEF-2 induced by GTP binding is discussed.

Adenine Nucleotides↗
PubMed1993

Chemical modification of alpha-subunit tryptophan residues in Schizosaccharomyces pombe mitochondrial F1 adenosine 5'-triphosphatase: differential reactivity and role in activity.

Chemical modification of mitochondrial F1-ATPase from Schizosaccharomyces pombe by the tryptophan-specific reagent N-bromosuccinimide (NBS) at pH 5.0 in the presence of 20% glycerol produced a characteristic lowering in both enzyme absorbance at 280 nm and intrinsic fluorescence at 332 nm that varied with NBS/F1 molar ratio up to a value of 130. Fluorometric titration of tryptophans and correlation to residual ATPase activity showed that modification of three reactive residues among the seven present on alpha- and epsilon-subunits did not markedly modify the enzyme activity but efficiently released endogenous ATP and abolished the fluorescence quenching related to GDP or ATP binding to the catalytic site. Additional modification of one, less reactive, tryptophan altered both negative cooperativity of ATP hydrolysis and sensitivity to azide inhibition and produced a nearly complete inactivation at high NBS/F1 molar ratio. NBS-induced inactivation of F1 was favored by catalytic-site saturation with GDP or low ATP concentration and on the contrary was prevented by noncatalytic-site saturation with ADP or high ATP concentration. When reactive tryptophans were selectively modified by NBS in the presence of ADP, and subunits were isolated after guanidine hydrochloride dissociation by one-step purification on reversed-phase HPLC, the absorbance of alpha-subunit at 280 nm was decreased, whereas that of epsilon-subunit was unchanged. Cyanogen bromide cleavage of alpha-subunit and fragments separation by reversed-phase HPLC showed that one peptide of 3 kDa apparent molecular mass had decreased absorbance. N-Terminal sequencing allowed its identification to fragment 255-282 that contains tryptophan257.

Amino Acid Sequence↗
PubMed1992

Differential nucleotide binding to catalytic and noncatalytic sites and related conformational changes involving alpha/beta-subunit interactions as monitored by sensitive intrinsic fluorescence in Schizosaccharomyces pombe mitochondrial F1.

Mitochondrial F1 from the yeast Schizosaccharomyces pombe exhibits an intrinsic tryptophan fluorescence sensitive to adenine nucleotides and inorganic phosphate [Divita, G., Di Pietro, A., Deléage, G., Roux, B., & Gautheron, D.C. (1991) Biochemistry 30, 3256-3262]. The present results indicate that the intrinsic fluorescence is differentially modified by nucleotide binding to either catalytic or noncatalytic sites. Guanine or hypoxanthine nucleotides, which selectively bind to the catalytic site, produce a hyperbolic saturation monitored by fluorescence quenching at 332 nm, the maximal emission wavelength. On the contrary, adenine nucleotides, which bind to both catalytic and noncatalytic sites, exhibit a biphasic saturation. High-affinity ATP binding produces a marked quenching as opposed to the lower-affinity one. In contrast, ADP exhibits a sigmoidal saturation, with high-affinity binding producing no quenching but responsible for positive cooperativity of binding to the lower-affinity site. The catalytic-site affinity for GDP is almost 20-fold higher at pH 5.0 as compared to pH 9.0, and the high sensitivity of the method allows detection of the 10-fold lower-affinity GMP binding. In contrast, high-affinity binding of ADP, or AMP, is not pH-dependent. The selective catalytic-site saturation induces a F1 conformational change decreasing the Stern-Volmer constant for acrylamide and the tryptophan fraction accessible to iodide. ATP saturation of both catalytic and noncatalytic sites produces an additional reduction of the accessible fraction to acrylamide.

Adenine Nucleotides↗
PubMed1991

Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions.

Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.8 M-1, respectively, in the presence of either acrylamide or iodide, indicate that tryptophans are mainly buried inside the native enzyme. Upon subunit dissociation and unfolding by 6 M guanidine hydrochloride (Gdn.HCl), the maximal emission is shifted to 354 nm, a value very similar to that obtained with N-acetyltryptophanamide, a solute-tryptophan model compound. The tryptophan content of each isolated subunit has been estimated by fluorescence titration in the presence of Gdn.HCl with free tryptophan as a standard. Two tryptophans and one tryptophan are found respectively in the alpha and epsilon subunits, whereas none is detected in the beta, gamma, and delta subunits. These subunit contents are consistent with the total of seven tryptophans estimated for native F1 with alpha 3 beta 3 gamma 1 delta 1 epsilon 1 stoichiometry. The maximal emission of the isolated epsilon subunit is markedly blue-shifted to 310-312 nm by interaction with the isolated delta subunit, which suggests that the epsilon subunit tryptophan might be a very minor contributor to the native F1 fluorescence measured at 332 nm. This fluorescence is very sensitive to phosphate, which produces a marked blue shift indicative of tryptophans in a more hydrophobic environment. On the other hand, ADP and ATP quench the maximal emission at 332 nm, lower tryptophan accessibility to acrylamide, and reveal tryptophan heterogeneity.

Acrylamide↗
PubMed1989

Structure-function relationships of mitochondrial ATPase-ATPsynthase using Schizosaccharomyces pombe yeast mutants with altered F1 subunits.

Phenotypic revertants have been selected from mutants of the yeast Schizosaccharomyces pombe devoid of either alpha or beta subunits of mitochondrial ATPase-ATPsynthase. In contrast to parental mutants, phenotypic revertants are able to grow on glycerol respiratory medium and show immunodetectable alpha and beta subunits. However, growth and cellular respiration are only partially restored as compared to the wild strain, indicating that the recovered subunits are mutated. ATPase activity of revertant submitochondrial particles shows markedly different parameters: more acidic optimal pH, absence of bicarbonate activation and decreased sensitivity to azide inhibition in the alpha subunit-modified R3.51. Opposite differences are observed in the beta subunit-modified R4.3: more alkaline optimal pH, much higher bicarbonate activation, and increased sensitivity to azide. The ITPase activity of R4.3 submitochondrial particles is also more sensitive to azide as compared to the wild strain. ATPase activity of purified F1 also exhibits marked differences: loss of bicarbonate-sensitive negative cooperativity, decreased sensitivity to both ADP and azide inhibitions in the R3.51 revertant. On the contrary, increased negative cooperativity and increased sensitivity to both ADP and azide inhibitions are observed for the R4.3 revertant enzyme which in addition exhibits a much lower maximal rate. The beta subunit-mutation of R4.3 also increases the sensitivity of ITPase activity to tripolyphosphate inhibition, whereas the alpha subunit-mutation of R3.51 is without any effect. Soluble F1 with beta subunit-mutation is very sensitive to high ammonium sulfate concentrations required for enzyme precipitation and concentration and known to partially deplete the enzyme from its endogenous nucleotides. On the contrary, poly(ethylene)glycol is very efficient for preparing from any strain a pure and very stable enzyme retain-ing high amounts of endogenous nucleotides. The R4.3 revertant F1 retains even more nucleotides than the wild-strain F1 and is much less sensitive to high iodide concentrations which favor enzyme dissociation and precipitation. The tryptophan intrinsic fluorescence of F1 is modified by both mutations that increase the maximal emission intensity. The most important effect is produced by beta subunit-mutation which decreases the quenchable fraction, one-third to one-half tryptophans being no longer accessible to iodide. The overall results suggest that both mutations modify enzyme-nucleotide interactions: the alpha subunit-mutation of R3.51 would favor ADP release by lowering interactions with the adenine moiety, whereas the beta subunit-mutation of R4.3 would lower ADP release by strengthening interactions with the phosphate chain moiety.

Adenosine Triphosphatases↗
PubMed1988

A case of pure dislocation of the ankle joint.

Pure dislocation of the ankle joint is extremely rare. This is due to the fact that the particular mechanism of injury required to produce it occurs equally rarely. The authors report a case in which a good clinical result was obtained with nonsurgical treatment.

Adolescent↗