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Biomedical subjects

R L Kisliuk

Publications and source records attributed to R L Kisliuk.

At least 91 records · Page 5Linked to original sources
PubMed1975

Interaction of deoxyuridylate with thymidylate synthetase.

The reaction of deoxyuridylate with Lactobacillus casei thymidylate synthetase (methylenetetrahydrofolate: deoxyuridylate C-methyltransferase) in the absence of folate or thiols can be followed by changes in the circular dichroic spectra at 267 nm. These changes at the maximum absorption wavelength of the pyrimidine suggest that on binding the interaction of the pyrimidine ring and the deoxyribose moiety is altered. The binding curve follows the form expected for single site binding. There was no change in the ultraviolet absorption spectra. The dissociation constant of the deoxyuridylate enzyme complex was calculated to be 4 x 10-minus 7 M. Iodoacetamide also reacts stoichiometrically with one of the two subunits of thymidylate synthetase and inactivates the enzyme. This reaction is blocked by the presence of deoxyuridylate or chloromercuribenzoate. Complement fixation and immunodiffusion studies gave no indication that iodoacetamide treatment caused conformational changes or breakdown of the enzyme into subunits. Treatment with 0.1 M mercaptoethanol restored activity to one-half the value obtained with native enzyme, suggesting that mercaptoethanol enabled the unreacted subunit to become functional.

Amino Acids↗
PubMed1975

Study of thymidylate synthetase-function by laser Raman spectroscopy.

The Laser-Raman spectra of thymidylate synthetase have been obtained with 488 nm excitation from an argon ion laser. Raman bands observed in the range 600-800 cm-minus-1 have been assigned to functional groups of constituent amino acids. The band positions and intensities in the Amide I (1600-1700 cm-minus-1) and Amide III (1200-1300 cm-minus-1) regions, suggest that the enzyme is a mixture of alpha-helical and unordered conformations. Low levels of beta-structure cannot be excluded. The spectra of the ternary complex formed by reacting thymidylate synthetase with (+)-L-methylenetetrahydrofolate and fluorodeoxyuridylate reveals a new band at 1618 cm-minus-1 assigned to the C=N stretching vibration. This band may be due to formation of dihydrofolate or an iminium ion. The overall secondary structure of thymidylate synthetase does not change on formation of the ternary complex. However, the spectrum of the complex indicates local changes in groups such as ionized carboxyl (1400 cm-minus-1), tryptophan (1003 cm-minus-1) and CH-3, CH-2 deformation modes (1440-1470 cm-minus-1).

Binding Sites↗
PubMed1975

Synthesis and biological activity of 10-thia-10-deaza analogs of folic acid, pteroic acid, and related compounds.

The 10-thia analogs of pteroic acid, folic acid, their esters, and their 4-amino analogs were synthesized through a reaction sequence involving, as a key step, the condensation of 2-amino-3-cyano-5-chloromethylpyrazine with appropriately substituted thiols. The abilities of the products to inhibit the growth of methotrexate (MTX)-sensitive and MTX-resistant microorganisms were investigated as were their abilities to inhibit dihydrofolic acid reductase and thymidylic acid synthetase. Several compounds had high activity.

Enterococcus faecalis↗
PubMed1974

Conformational changes induced in dihydrofolate reductase by folates, pyridine nucleotide coenzymes, and methotrexate.

Dihydrofolate reductase (EC 1.5.1.3; 5,6,7,8-tetrahydrofolate:NADP(+) oxidoreductase) from antifolate-resistant Lactobacillus casei has been isolated in pure form and examined in solution by high resolution proton magnetic resonance spectroscopy. The 220 MHz proton magnetic resonance spectrum of this small enzyme (about 15,000 daltons) consists of several distinct resonance peaks that provide a sensitive nonperturbing probe of its conformational state. Comparison of catalytically active enzyme with preparations denatured in 6 M urea demonstrates dramatically the overall contribution of secondary and tertiary structure to its proton magnetic resonance spectra. More subtle differences existing among several catalytically active enzyme forms may also be readily differentiated by proton magnetic resonance spectroscopy, e.g., the spectra of the ligand-free enzyme and forms containing stoichiometric amounts of tightly bound folate and dihydrofolate, each obtained separately by affinity chromatography, are easily identified. Addition of ligands to these spectroscopically distinct forms may induce changes in their proton magnetic resonance spectra. For example, addition of equimolar dihydrofolate to the apoenzyme converts its relatively featureless aromatic proton magnetic resonance spectrum to one indistinguishable from that of the original enzyme-dihydrofolate binary complex obtained chromatographically. Interaction of the pyridine nucleotide coenzymes NADP(+) or NADPH or of the antifolate Methotrexate with apoenzyme induces additional distinct spectral changes. Enzyme-NADPH and enzyme-Methotrexate binary complexes, which have different aromatic region proton magnetic resonance spectra, are converted to ternary complexes having quite similar spectra by addition of Methotrexate and NADPH, respectively, thus suggesting that an ordered addition of ligands is not required.

Folic Acid↗