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R J Cisneros

Publications and source records attributed to R J Cisneros.

At least 19 recordsLinked to original sources
PubMed1993

Studies of 5-fluorodeoxyuridine 5'-monophosphate binding to carboxypeptidase A-inactivated thymidylate synthase from Lactobacillus casei.

The binding of 5-fluorodeoxyuridylate (FdUMP) to carboxypeptidase-inactivated thymidylate synthase obtained from methotrexate-resistant Lactobacillus casei was investigated using [3H]FdUMP in a trichloroacetic acid precipitation assay and by 19F nuclear magnetic resonance spectroscopy. The cleavage of 1 valine residue from the carboxyl terminus of one of the identical subunits of the enzyme dimer correlates with complete loss of thymidylate synthesis (Aull, J. L., Loeble, R. B., and Dunlap, R. B. (1974) J. Biol. Chem. 249, 1167-1172). We have further investigated the phenomenon of carboxypeptidase A-dependent inactivation of thymidylate synthase by employing immobilized carboxypeptidase A in order to facilitate the isolation and characterization of the inactivated enzyme. The time course of carboxypeptidase treatment of thymidylate synthase has been profiled by the spectrophotometric assay, tritium release assay, trichloroacetic acid precipitation assay (covalent adduct analysis), 19F nuclear magnetic resonance spectroscopy, and amino acid analysis. The techniques utilized in this study yielded results which showed that the completely inactivated enzyme (failure to catalyze thymidylate formation) continued to catalyze both covalent FdUMP-enzyme interactions and the formation of the covalent inhibitory ternary complex with the cofactor, 5,1O-methylenetetrahydrofolate, although to a reduced extent, thus effectively uncoupling these processes from thymidylate synthesis activity.

Binding Sites↗
PubMed1991

Purification and characterization of selenomethionyl thymidylate synthase from Escherichia coli: comparison with the wild-type enzyme.

Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques [Yang et al. (1990) Science (Washington, D.C.) 249, 1398-1405]. The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet. We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E. coli Rue10. An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E. coli in 90% yield in no more than 8 h. The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values. Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h). The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen. Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes. In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of recombinant mouse thymidylate synthase.

Recombinant mouse thymidylate synthase (TS) expressed at high levels in Escherichia coli was purified to homogeneity in greater than 70% yield by a rapid three-step procedure. Both 0.1% Triton X-100 and 10% glycerol were required to stabilize the enzyme whose activity remained unchanged after 1 month when stored at -20 degrees C. Thermal inactivation of the enzyme was a first-order process at 37 degrees C, with t1/2 values of 6.9, 15.6 and 3.0 min at pH 5.5, 7.0 and 8.5, respectively. The presence of saturating levels of dUMP at pH 8.5 increased the t1/2 of inactivation of 38 min. The pH profile for enzyme activity showed a narrow optimum region centered at pH 7.0, which was mirrored by the shape of the Km, dUMP/Vmax plot. The pH dependence of Kd for the covalent inhibitory ternary complex of enzyme, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate exhibited a broad minimum between pH 5.5 and 8.5, and ranged between 3.1, 0.8 and 1.1 nM at pH 5.5, 7.0 and 8.5, respectively. The UV/VIS spectrum of the native enzyme exhibited a maximum at 280 nm (epsilon = 98,200 M-1 cm-1), while that of the inhibitory ternary complex showed an additional maximum at 320 nm. The 19F-NMR spectrum of the mouse enzyme:FdUMP binary complex revealed two new resonances at -2.8 and -34.8 ppm. The most deshielded resonance represented the noncovalent binary complex while the other resonance was assigned to the nucleotide covalently bound to the enzyme. The alteration of nucleotide binding equilibria produced by addition of H4 folate was exemplified by both an increase in intensity and a 5 ppm deshielding of the resonance attributed to the covalent FdUMP-enzyme complex. Addition of formaldehyde to the latter mixture produced the covalent ternary complex which resulted in the collapse of the resonances at -2.8 and -39.5 ppm and the appearance of a new resonance at -12.4 ppm.

Animals↗
PubMed1990

Characterization of the parameters affecting covalent binding stoichiometry in binary and ternary complexes of thymidylate synthase.

Covalent binding stoichiometries for both the enzyme:5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) binary complex and the enzyme:FdUMP:5,10-methylenetetrahydrofolate (inhibitory ternary) complex at equilibrium were measured by the trichloroacetic acid precipitation assay and shown to be a function of temperature, time, pH, salt concentration, buffer composition and thiol concentration. Incubation at 37 degrees C yielded the maximum covalent binding ratio (mol FdUMP/mol enzyme) for the latter binary (0.7) and ternary (1.7) complexes. In most buffers studied, the maximum covalent binding ratio (1.5-1.7) for the inhibitory ternary complex occurred over a broad pH range (4.5-8.0), while the optimum covalent binding ratio for binary complex was observed at a much narrower region centered between pH 5.5-6.5. In the presence of increasing concentrations of phosphate buffer, the maximum binding ratio for the covalent binary complex decreased from 0.63 in the absence of phosphate to 0.1 in the presence of 225 mM phosphate, while that for the inhibitory ternary complex was unchanged. When a ternary complex was formed with enzyme, FdUMP and (+/-)-tetrahydrofolate in the absence of phosphate, the FdUMP:enzyme covalent binding ratio was 1.8, while in the presence of 75 mM phosphate, the binding ratio was only 1.0. When exogenous thiol was removed by centrifugal column chromatography, the maximum binding stoichiometry of the resulting inhibitory ternary complex was 1.7 and was independent of added thiol over a 2 h incubation period at 37 degrees C. When extensive dialysis at 5 degrees C was used to remove the thiol, the maximum binding stoichiometry of the resulting inhibitory ternary complex was found to be dependent on both the concentration of added thiol and the time of incubation at 37 degrees C and did not exceed a value of 1.0.

Buffers↗
PubMed1990

Development of a trichloroacetic acid precipitation assay for covalent adducts of thymidylate synthase.

The use of trichloroacetic acid as a protein precipitant and denaturant in the quantitative measurement of covalent complexes of thymidylate synthase is described. Enzyme inactivated with N[3H]ethylmaleimide and inhibitory ternary complex (formed with native enzyme, 5-[6-3H]fluoro-2'-deoxyuridylate, and methylenetetrahydrofolate) served as reagents which were used to establish the conditions under which trichloroacetic acid precipitation, washing, and solubilization steps provided quantitative results. The ternary complex formed by dihydrofolate reductase with [3H]methotrexate and NADPH was used as a control to assess whether tight, but noncovalent, enzyme:ligand complexes survived trichloroacetic acid precipitation. The fact that no counts above background were detected in the pellet of precipitated protein demonstrated that the noncovalent complexes were completely dissociated by this treatment. The dynamic range of linear response for the inhibitory ternary complex of thymidylate synthase spanned five orders of magnitude, and the assay detected levels of enzyme as low as 10 fmol, a value which was essentially limited by the specific radioactivity of 5-[6-3H]fluoro-2'-deoxyuridylate. The ability of the enzyme to bind 5-[6-3H]fluoro-2'-deoxyuridylate specifically, as measured by the trichloroacetic acid assay, generated a specific binding value of 13.4 nmol of enzyme/mg protein (assuming a binding ratio of 1.5 for the inhibitory ternary complex). Specific binding values were compared to specific activity values (obtained from the spectrophotometric assay) at each stage of purification of the enzyme from Lactobacillus casei and were found to give parallel results. The characteristics of the trichloracetic acid assay procedure, which exclusively detects covalent enzyme-ligand adducts, are compared to those for other ligand binding assays for thymidylate synthase.

Bacterial Proteins↗
PubMed1990

Site-directed mutagenesis of mouse thymidylate synthase: alteration of Arg44 to Val44 in a conserved loop guarding the active site has striking effects on catalysis and nucleotide binding.

The arginine located at position 44 of mouse thymidylate synthase is in a highly conserved loop that is in close proximity to the active site cleft of the enzyme. Structural analyses have suggested that this arginine forms hydrogen bonds with the alpha-carboxylate of the C-terminal amino acid and the phosphate of the substrate analog, FdUMP (D. A. Matthews, K. Appelt and S. J. Oatley, (1989) Adv. Enz. Reg., 29, 47-60). We have used protein engineering techniques to change this amino acid residue to valine. This alteration leads to large reductions in the ability of the enzyme to form covalent complexes with substrate (dUMP) or inhibitor (FdUMP) and at least a 100-fold reduction in catalytic activity. These observations show that this arginine plays an important role in maintaining catalytic activity of the enzyme.

Amino Acid Sequence↗
PubMed1989

Efficient synthesis of mouse thymidylate synthase in Escherichia coli.

The coding region of the mouse thymidylate synthase (TS)-encoding cDNA (ts) was inserted downstream from the phage T7 promoter and translation initiation signals of the expression vector, pET-3a, and transformed into Escherichia coli BL21(DE3)[pLysS]. When the wild-type (wt) cDNA sequence was used, mouse TS was synthesized in the bacterial cells in response to induction, but the level of expression was low. When the second codon (Leu) was changed from CUG, found in the normal mRNA, to CUU, the level of expression increased 17-fold and TS represented 5-10% of total cell protein. The recombinant enzyme was purified to homogeneity by affinity chromatography. The recombinant TS had the same Mr as the enzyme from cultured mouse fibroblasts. Kinetic studies with the recombinant enzyme showed that the apparent Km values for deoxyuridylate and 5,10-methylenetetrahydrofolate were 10.5 and 22 microM, respectively, which were similar to the values for TS from mouse cell extracts. The mouse ts expression vector will be useful for the large-scale production of the wt enzyme and for the creation and analysis of mutant enzymes by protein engineering techniques.

Animals↗
PubMed1988

Is the risk of failure increasing in the hospital industry?

Is the risk of failure increasing? According to a survey of U.S. hospitals conducted by Touche Ross & Co., 46 percent of the hospitals responding to the survey say their institutions are at risk of failure within the next five years, an increase of 3 percent from two years ago. Hospitals in all bed size categories express a greater risk of failure in 1988 than in 1986, and the smaller hospitals, which represented the largest portion at risk of failure in 1986, feel even more at risk now. Clearly, the health-care industry is moving into a period of transition that will affect the very structure of health-care delivery.

Attitude of Health Personnel↗
PubMed1982

Evaluation of family planning communications in El Salvador.

During the 1970s, El Salvador had one of the most active communication programmes for family planning (FP) of any Latin American country. The current study, carried out nationwide among women of reproductive age in El Salvador, indicates that over 90% of women have been reached FP messages via mass or interpersonal channels. Levels of exposure were found to be relatively lower among women who live in rural areas, who work at home or not at all, who have little education, who are not married or live in union, and who are under 19 or over 40. The study was completed at a time of political stability in the country, and these data were to be used as a guideline for designing future communication programmes with respect to content, target population, and channels. With regard to communication research, this study yielded findings which usefully supplement those already recorded through a number of investigations of the subject.

Adult↗