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Biomedical subjects

C M Baugh

Publications and source records attributed to C M Baugh.

At least 19 recordsLinked to original sources
PubMed1986

Interaction of polyglutamyl derivatives of methotrexate, 10-deazaaminopterin, and dihydrofolate with dihydrofolate reductase.

Polyglutamyl derivatives of methotrexate (MTX) and 10-deazaaminopterin (10-DAM) containing a total of one through six glutamate residues (Glu residues) were tested as inhibitors of dihydrofolate reductase (DHFR) derived from sheep, chicken, and beef liver. The ability of dihydropteroylpentaglutamate to antagonize the inhibitory activity of these analogues was also studied. The most striking effects were seen with sheep liver DHFR, where polyglutamylation of MTX causes stepwise decreases in the concentration required for 50% inhibition (IC50) with each additional Glu residue until MTX with a total of six Glu residues has an IC50 value 1/3 that of MTX. With 10-DAM the pattern is more complex. The IC50 values increase with addition of Glu residues until a maximum is reached with 10-DAM having a total of three Glu residues which has a value twice that of 10-DAM. 10-DAM with a total of four Glu residues and 10-DAM with a total of five Glu residues have progressively lower IC50 values, the latter being equipotent with 10-DAM. With dihydropteroylpentaglutamate as substrate instead of dihydrofolate, the IC50 values are increased 2- to 5-fold for both MTX and 10-DAM derivatives. The results obtained with chicken liver and beef liver DHFR are generally similar to those described for the sheep liver enzyme, but the effects of polyglutamylation are less pronounced. The addition of 0.2 M KCl to the assay system reduces the differences in inhibitory potency of the polyglutamyl derivatives with all three enzymes tested. We conclude that polyglutamylation can alter the interaction of folate analogues and dihydrofolate with DHFR.

Aminopterin↗
PubMed1985

Channeling between the active sites of formiminotransferase-cyclodeaminase. Binding and kinetic studies.

Formiminotransferase-cyclodeaminase, a circular tetramer of dimers, binds four tetrahydropteroylpolyglutamates/octamer, which indicates that these polyglutamate sites are formed by one type of subunit interface. The transferase and deaminase are separate catalytic sites as determined by inhibition studies with (6R)-tetrahydropteroylglutamate and by the observation that the activities can operate simultaneously. Under conditions where the transferase is saturated with tetrahydropteroyl(glutamate)n substrate, exogenously added formimino intermediate is utilized by the deaminase only if at least one of the substrate/intermediate pair is a monoglutamate. These properties indicate the existence of only one polyglutamate site/pair of catalytic sites. Kinetic specificity for each activity as measured by Vm/Km increases for longer polyglutamates, but does not differentiate among 4, 5, 6, and 7 glutamates. The enzyme shows distinct preference for hexaglutamate based on Kd as well as on Km values. With all substrates, Vm of the deaminase is greater than that of the transferase, allowing for potential channeling of the intermediate between active sites. Efficiency of channeling, optimal with pentaglutamate, does not correspond with affinity for binding. This demonstrates that a steric requirement predominates over simple sequestering of intermediates on the enzyme surface as the fundamental mechanism for channeling.

Ammonia-Lyases↗
PubMed1985

The binding of folyl- and antifolylpolyglutamates to hemoglobin.

A binding method that detects only the strongest binding site for a ligand on a protein has been used to show that folates and folate analogs, conjugated with poly-gamma-glutamates, are bound to hemoglobin. When the concentration of hemoglobin is much larger than that of the polyglutamate, as is the case in the red cell, the fraction bound is a direct function of the hemoglobin concentration and is independent of the total polyglutamate concentration. Binding to deoxyhemoglobin tetramers is competitive with 2,3-diphosphoglycerate. In oxyhemoglobin the folyl and methotrexate polyglutamates are bound preferentially by free alpha beta dimers, but removal of the pteridine moiety leads to tetramer binding even in oxyhemoglobin. Changes in the length of the polyglutamate side chain and alterations of the pteridine structure such as reduction and/or methylation have a much larger effect on the constant for binding to deoxyhemoglobin tetramers than on that for oxyhemoglobin dimers. The implications of these results for the storage of pteroylpolyglutamates in the erythrocyte and their release from the red cell under the influence of the degree of oxygenation and variations in the 2,3-diphosphoglycerate level are discussed.

Folic Acid↗
PubMed1985

Preparation of tritium-labeled tetrahydropteroylpolyglutamates of high specific radioactivity.

Tritium-labeled [6S]-tetrahydropteroylpolyglutamates of high radiospecific activity were prepared from the corresponding pteroylpolyglutamates. Malic enzyme and D,L-[2-3H]malate were used as a generating system to produce [4A-3H]NADPH which was coupled to the dihydrofolate reductase-catalyzed reduction of chemically prepared dihydropteroylpolyglutamate derivatives. Passage of the reaction mixtures through a column of immobilized boronate effectively removed NADPH, and the tetrahydropteroylpolyglutamates were subsequently purified by chromatography on DEAE-cellulose. Overall yields of the [6S]-tetrahydro derivatives were 18-48% and the radiospecific activities were 3-4.5 mCi X mumol-1.

Chromatography↗
PubMed1984

Studies on the polyglutamate specificity of methylenetetrahydrofolate dehydrogenase from pig liver.

Methylenetetrahydrofolate dehydrogenase, which is one of the activities of a trifunctional folate-dependent enzyme isolated from pig liver, displays an ordered bi-bi kinetic mechanism when methylenetetrahydropteroylmonoglutamate is used as the folate substrate [Cohen, L., & MacKenzie, R. E. (1978) Biochim. Biophys. Acta 522, 311-317]. We have studied the inhibition of this activity by a series of pteroylglutamates containing one to seven glutamyl residues. Inhibitors with one to four glutamyl residues exhibit a kinetically determined KD of about 56 microM for binding at the folate site of the enzyme, while inhibitors with five to seven glutamyl residues exhibit a KD of about 16 microM. These results suggest that folylpolyglutamates are bound to the trifunctional enzyme relatively weakly, with the major interaction involving the fifth glutamyl residue of the polyglutamate "tail". A free energy decrease of about 0.74 kcal (3.1 kJ) is associated with this interaction. The possibility of a swinging arm mechanism for the trifunctional enzyme is discussed. We have also measured the kinetic parameters Vmax and the Km values for NADP+ and the folate substrate associated with catalysis using a series of methylenetetrahydropteroylpolyglutamate substrates. The variation in these parameters with the length of the polyglutamate tail is small.

Animals↗
PubMed1983

Molecular basis for the interaction of polyglutamates of folic acid and its analogs with dihydrofolate reductase.

Fluorimetric titration has been used to measure the dissociation constants for the complexes of folate, pteroyltriglutamate and pteroylheptaglutamate with dihydrofolate reductase purified from Lactobacillus casei, Streptococcus faecium (isoenzyme 2) and bovine liver. Effects of pH, temperature, salt concentration and second ligands have been examined. The method is shown to be unsuitable for methotrexate complexes. The polyglutamates do not bind more tightly than folate to the S. faecium reductase under any conditions examined, but bind somewhat more tightly than folate to the L. casei reductase at low pH (less than 7) and to the bovine liver enzyme at pH 7-9. Increasing concentrations of KC1 decrease the binding of all three ligands to the L. casei and bovine liver enzymes. Increasing pH markedly raises the dissociation constants for all complexes of the L. casei reductase, but has only slight effects on the complexes of the S. faecium reductase. Complexes of the bovine enzyme are affected to an intermediate degree by pH, but the folate complex is affected much more than those of the polyglutamates. Model building studies have been performed with a three-dimensional model of the complex of L. casei reductase with NADPH and methotrexate. Additional glutamyl groups were added in gamma-linkage to the glutamate moiety of the complexed methotrexate. A proposed mode of binding of the pteroyl polyglutamates is discussed and sequence comparisons are used to predict residues that might be involved in polyglutamate binding by reductase from other sources.

Animals↗
PubMed1983

Interaction of tetrahydropteroylpolyglutamates with two folate-dependent multifunctional enzymes.

The naturally occurring pteroylpolyglutamate derivatives are substrates for the folate-mediated reactions in cells, including the reactions catalyzed by two multifunctional folate dependent enzymes in eucaryotes. The appropriate derivatives of tetrahydropteroyl (glutamate)n where n = 1, 3, 5, or 7 were used to determine the specificity for, and kinetic advantages of the extra glutamyl residues with two multifunctional proteins from pig liver: methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase, and formiminotransferase-formininotetrahydrofolate cyclodeaminase. Specificity for the polyglutamate derivatives ranged from 10- to 70-fold as indicated from Km values or from the ability to inhibit the five different enzyme activities. With the sequential activities of the transferase-deaminase enzyme, it was demonstrated that when the tetrahydropteroyl pentaglutamate is used as a substrate, the intermediate formimino-compound does not accumulate in the medium. That this kinetic observation is due to preferential transfer of the pentaglutamate- but not monoglutamate intermediate from transferase to deaminase sites without its release from the enzyme molecule was supported by three types of experiments. Chemical modification to yield monofunctional derivatives of the transferase-deaminase affected the kinetics of the recombined activities only with the pentaglutamate substrate, causing a lag in the appearance of final product. Inhibition studies demonstrated that the deaminase activity could preferentially be inhibited only with the monoglutamate substrate. The deaminase activity with the monoglutamate substrate was increased by providing elevated formiminotetrahydrofolate in the assay mixture; no effect was observed when the reaction was carried out with pentaglutamate. Preliminary binding studies indicate a single folate site per subunit of the octameric enzyme, suggesting a type of combined transferase-deaminase site.

Aminohydrolases↗
PubMed1983

The interaction of pteroylpolyglutamates with calf thymus thymidylate synthase.

Calf thymus thymidylate synthase was purified to homogeneity using a pteroyltetraglutamyllysine Sepharose affinity column. Inhibition of the purified enzyme by folate or methotrexate was enhanced by addition of gamma-Glu residues. Tetrahydrofolate, tetrahydropteroyltriglutamate and tetrahydropteroylheptaglutamate, all having the natural configuration at C-6, all showed similar Km values near 15 microM. Thus, although calf thymus thymidylate synthase showed a higher affinity for pteroylpolyglutamates than pteroylmonoglutamate, this was not reflected in lowered Km values with the corresponding tetrahydropteroylpolyglutamate substrates.

Animals↗
PubMed1983

A pteroylpolyglutamate binds to tetramers in deoxyhemoglobin but to dimers in oxyhemoglobin.

The binding of a physiological concentration of pteroylhepta(glutamate) to oxy- and deoxyhemoglobin in large excess was measured by ultrafiltration. The variation of free to bound folate with hemoglobin concentration showed that a single molecule of the pteroylpolyglutamate is bound by deoxyhemoglobin tetramers and by alpha beta dimers in oxyhemoglobin. Although the binding sites are different, the affinity constants are the same and very similar to the 2,3-bisphosphoglycerate binding energy. Nevertheless, in view of the small proportion of dimers in oxyhemoglobin much more pteroylhepta(glutamate) is bound by deoxyhemoglobin over a wide range of hemoglobin concentrations. Because even 2% deoxyhemoglobin is enough to bind all of the erythrocyte folate as polyglutamate, the bulk of it will be bound at physiological oxygen pressures. Free folate could only be expected in fully oxygenated erythrocytes. Therefore, the reaction of pteroylpolyglutamates with hemoglobin represents an oxygenation-dependent storage mechanism that can account for the 40-fold excess of the vitamin in the erythrocyte over the amounts in the serum. Because methotrexate is also converted to polyglutamate derivatives in the erythrocyte, this drug is likely to be concentrated and stored there by the same mechanism.

Folic Acid↗
PubMed1981

Polyglutamyl derivatives of tetrahydrofolate as substrates for Lactobacillus casei thymidylate synthase.

Tetrahydropteroylpolyglutamates containing up to seven Glu residues were tested as substrates for Lactobacillus casei thymidylate synthase. The Km values decreased from 24 microM for the monoglutamate to 1.8 microM for the triglutamate. Addition of residues 4, 5, 6, and 7 did not decrease the Km further. When monoglutamate and polyglutamate substrates were simultaneously incubated with the enzyme, the rate observed was characteristic of the polyglutamate even when the monoglutamate concentration was 44 times that of the polyglutamate. Iodoacetamide treatment inhibited the enzyme to the same extent with monoglutamate and polyglutamate substrates. Addition of 0.3 M NaCl doubled the rate obtained with the polyglutamate substrate whereas the rate with the monoglutamate was inhibited 25%. MgCl2 stimulated the reaction only 10% with the polyglutamate substrate compared with 80% stimulation obtained with the monoglutamate. Inhibition by fluorodeoxyuridylate was similar with both mono- and polyglutamate substrates; however, with the phosphonate derivative of fluorodeoxyuridine, the polyglutamate substrate enhanced inhibition 5- to 8-fold.

Deoxyuridine↗
PubMed1980

Interactions of pig liver methylenetetrahydrofolate reductase with methylenetetrahydropteroylpolyglutamate substrates and with dihydropteroylpolyglutamate inhibitors.

Dihydrofolate and dihydropteroylpolyglutamates inhibit pig liver methylenetetrahydrofolate reductase. In all cases the inhibition is linearly competitive with respect to methylenetetrahydrofolate. The Ki values decrease with each additional glutamyl residue from one to six, from a value of 6.5 microM for dihydrofolate to 0.013 microM for dihydropteroylhexaglutamate. Dihydropteroylheptaglutamate has a Ki of 0.065 microM. These data indicate a free energy of binding of approximately 0.75 kcal/mol for each of the five terminal glutamyl residues in dihydropteroylhexaglutamate. Methylenetetrahydropteroylpolyglutamates are substrates for the enzyme, and the increased free energy of binding is reflected in increased values for Vmax/Km with polyglutamate substrates. Vmax is increased 1.76-fold on going from the mono- to the diglutamate substrate; additional glutamyl residues lead to decreases in Km values for methylenetetrahydropteroylpolyglutamates. Our results suggest that the in vivo activity of methylenetetrahydrofolate reductase may also be sensitive to fluctuations in the ratio of methylenetetrahydropteroylpolyglutamates to dihydropteroylpolyglutamates and that this ratio may be important in determining the relative fluxes of methylenetetrahydropteroylpolyglutamates into the pathways leading to thymidylate biosynthesis and methionine regeneration.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗