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Biomedical subjects

F M Huennekens

Publications and source records attributed to F M Huennekens.

At least 37 records · Page 2Linked to original sources
PubMed1988

Chemotherapeutic potential of methotrexate peptides.

MTX peptides in which the amino acid was linked to the alpha-carboxyl group have been prepared and examined for cytotoxicity before and after treatment with proteolytic enzymes. The alanine, aspartic acid and arginine derivatives (MTX-ala, MTX-asp and MTX-arg) were synthesized by a regio-specific route, following the general procedures of Rosowsky and Montgomery. Each compound was obtained in good yield, and purity was established by TLC, HPLC, absorbance spectra and elemental analyses. The MTX peptides were not hydrolyzed by a variety of proteolytic enzymes (e.g., trypsin, plasmin, urokinase, aminopeptidase). Pancreatic carboxypeptidase A, however, hydrolyzed MTX-ala readily, MTX-asp slowly and MTX-arg not at all. The MTX-ala and, to a lesser extent, MTX-arg were substrates for pancreatic carboxypeptidase B. MTX-arg was also hydrolyzed by the endogenous carboxypeptidase N in human serum. The cytotoxicity of these MTX peptides toward L1210 cells was measured in a microculture assay system using a tetrazolium dye. MTX-ala was weakly cytotoxic (ID50 = 2.0 x 10(-6)M) compared to MTX (ID50 = 2.4 x 10(-8)M). When MTX-ala was tested in the presence of carboxypeptidase A, the ID50 value improved to 8.5 x 10(-8)M. MTX-arg gave an ID50 of 5.0 x 10(-8)M, which was not unexpected in view of its susceptibility to hydrolysis by the carboxypeptidase activity present in the fetal calf serum of the culture medium. Inclusion of carboxypeptidase B lowered the ID50 value to 2.5 x 10(-8)M. Possible clinical uses of MTX peptides are discussed.

Animals↗
PubMed1987

L1210 dihydrofolate reductase. Kinetics and mechanism of activation by various agents.

Dihydrofolate reductase from a methotrexate-resistant subline (R6) of L1210 mouse leukemia cells is activated (i.e. has its catalytic activity increased severalfold) by treatment with (a) sulfhydryl-modifying agents (p-chloromercuribenzoate (pCMB) or 5,5'-dithiobis(2-nitrobenzoic acid], (b) salts (KCl or NaCl), or (c) chaotropes (urea or guanidinium hydrochloride). With b or c activation is rapid (less than 10 s), but with a the process is much slower; at 25 degrees C, pseudo first-order rate constants for activation by excess pCMB or 5,5'-dithiobis(2-nitrobenzoic acid) are 0.45 and 0.08 min-1, respectively. Activation can also be monitored by conformational changes in the protein as indicated by enhanced fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate or by increased intrinsic fluorescence of tryptophan residues in the enzyme. Pseudo first-order rate constants for the pCMB-induced conformational change, measured by these fluorimetric procedures (0.45 min-1 and about 0.4 min-1, respectively), are in good agreement with the value obtained from the increase in catalytic activity. The rate of modification of the single cysteine residue in the enzyme by excess 14C-labeled pCMB, however, is faster than the rate of activation, indicating that the conformational change follows derivatization and is the rate-limiting step in the overall process. Activated forms of the enzyme are more labile to thermal denaturation or proteolysis than the untreated enzyme; the former process, however, is retarded by the presence of bovine serum albumin. Activation by the various agents is considered to involve a common mechanism in which interaction of the enzyme with the agents is followed by conformational changes in the enzyme, producing a series of forms that differ in microstructure, catalytic activity, and lability.

Animals↗
PubMed1987

Antibody for detection and quantitation of membrane-associated folate-binding protein from Lactobacillus casei.

Lactobacillus casei cells contain a 25 kDa, membrane-associated, folate-binding protein (fbp), which is a component of the folate transport system. Polyclonal antibody to fbp (anti-fbp) has been prepared, and conditions have been established for detection and quantitation of the protein. Anti-fbp did not block [3H]folate transport or binding in L. casei cells. As judged by Western blots, the antibody reacted only with fbp on sodium dodecyl sulfate electrophoretograms of Triton X-100 extracts of L. casei membranes. Anti-fbp showed no cross-reactivity with L. casei dihydrofolate reductase, L. casei 5,10-methenyltetrahydrofolate synthetase, L1210 dihydrofolate reductase, rat liver dihydrofolate reductase, or L1210 folate-binding protein. Enzyme-linked immunosorbent assay measurements indicated the presence of an fbp in membranes of Lactobacillus salivarius and two transport-defective sublines of L. casei. Anti-fbp was used to demonstrate selective extraction, with n-butanol, of fbp from a mixture of Triton-solubilized L. casei membrane proteins; repression of fbp in membranes of L. casei cells grown on high levels of folate; and localization of fbp by electron microscopy, using anti-fbp in conjunction with goat anti-rabbit IgG gold conjugate, in L. casei membranes.

Animals↗
PubMed1987

Platinum-folate compounds: synthesis, properties and biological activity.

Cis-diamminediaquaplatinum(II)-ion, the biologically active form of the anticancer agent Cisplatin, reacted readily with tetrahydrofolate at pH 7 and 37 degrees C to produce a stable complex. The reaction was monitored spectrophotometrically by the change in absorbance maximum from 298 nm (tetrahydrofolate) to 275 nm (complex); occurrence of isobestic points at 282 and 327 nm indicated that a single product was formed. Purity of platinum-tetrahydrofolate, after isolation in ca. 70% yield, was established by TLC and HPLC. Elemental analysis, absorbance spectra at various pH values and nmr spectra provided evidence that the diammine platinum moiety was bridged across the N-5 and N-10 positions of tetrahydrofolate. Complexation also occurred with 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, Methotrexate and aminopterin, but not with folate or 7,8-dihydrofolate. Biological implications of these observations have been investigated. Intracellular folates in L1210 cells have been identified and quantitated via reverse phase HPLC (C18 column; tetrabutylammonium phosphate as the pairing ion) and changes in the levels of these compounds, after exposure of cells to Cisplatin, have been measured. Platinum derivatives of tetrahydrofolate or other reduced folates were not found, but there was a decrease in the level of 5,10-methenyltetrahydrofolate, accompanied by an increase in 5-formyl and 10-formyltetrahydrofolate (and perhaps tetrahydrofolate). The chemical interaction of the diaqua form of Cisplatin with Methotrexate resulted in decreased uptake of the latter by L1210 cells. The platinum complex of tetrahydrofolate was a reasonably good inhibitor (Ki = 4 microM) of L1210 dihydrofolate reductase and of the folate transport system (50% inhibition at ca. 200 microM) of L1210 cells.

Animals↗
PubMed1987

Folic acid metabolism and its disruption by pharmacologic agents.

Folic acid metabolism in eukaryotic cells consists of a network of enzymatic reactions in which 1-carbon (C1) units at three different oxidation states are 1) interconverted while linked to the 5- and/or 10-positions of tetrahydrofolate, or 2) added to, or taken from, tetrahydrofolate. Particularly important in the latter category are reactions involving C1-tetrahydrofolate adducts in the synthesis of inosinate, thymidylate, serine, and methionine. Tetrahydrofolate, a central component of the network, can be generated from: 1) folate, via the NADPH-dependent dihydrofolate reductase; 2) 5-methyltetrahydrofolate via the methyl B12-dependent methionine synthetase; or 3) 5-formyltetrahydrofolate via a sequence of reactions beginning with the ATP-dependent isomerization to 5,10-methenyltetrahydrofolate or via transfer of the formyl group to glutamate. Because of the close relationship of folic acid metabolism to cell replication, folate-dependent enzymes provide excellent targets for cancer chemotherapy. This potential has not yet been realized, however, except for dihydrofolate reductase and thymidylate synthetase, which are strongly inhibited by the anti-cancer agents methotrexate (MTX) and FUra. The following enzymes are particularly attractive as targets for future exploitation in chemotherapy: 1) the two transformylases involved in purine nucleotide synthesis, 2) serine hydroxymethyltransferase, 3) methionine synthetase, and 4) methylenetetrahydrofolate dehydrogenase. Suggestions are also made for the development of new agents based upon a strategy of enzyme-targeted chemotherapy.

Folic Acid↗
PubMed1986

Transport of folate compounds in L1210 cells: kinetic evidence that folate influx proceeds via the high-affinity transport system for 5-methyltetrahydrofolate and methotrexate.

Folate is transported into L1210 mouse leukemia cells by the same system that mediates the uptake of methotrexate and reduced folate compounds. This conclusion is supported by the following observations: (a) methotrexate competitively inhibits the influx of folate and the Ki is comparable to the Kt for methotrexate influx; (b) the profile for inhibition of folate influx by methotrexate is monophasic and complete inhibition is achieved at high concentrations of the competitor; (c) folate inhibits the influx of methotrexate and the Ki is comparable to the Kt for folate influx; (d) the N-hydroxysuccinimide ester of methotrexate, a potent and specific irreversible inhibitor of the reduced folate system, also blocks the influx of folate; (e) folate and methotrexate influx are both inhibited by low concentrations of p-chloromercuriphenylsulfonate; and (f) folate influx fluctuates with the anionic composition of the medium in the same fashion as the influx of methotrexate. Measurements of folate influx can be complicated by the fact that the 3H-labeled substrate is susceptible to decomposition and that labeled breakdown products at concentrations as low as 1% contribute appreciably to the observed uptake. Of these products, 6-hydroxymethylpterin appears to account for most of the extraneous uptake. Impurities can be eliminated by subjecting the [3H]folate to preparative thin-layer chromatography immediately prior to use.

Animals↗
PubMed1985

Polymorphism of dihydrofolate reductase from a methotrexate-resistant subline of L1210 cells.

Dihydrofolate reductase, purified to homogeneity (as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), from a subline of L1210 murine leukemia cells resistant to 10(-6) M methotrexate, was resolved into two principal forms (1 and 2) by polyacrylamide gel electrophoresis at pH 8.3 or isoelectric focusing. In the latter procedure, these forms had pI values of 7.4 and 8.2, respectively; both stained for protein and catalytic activity. Form 1 appears to be a single component, comprising ca. 10% of the total protein and at least 20% of the total catalytic activity. It is also more sensitive to inhibition by MTX, more heat-stable, and less susceptible to activation than form 2. Multiple components of 2 were observed by narrowing the pH range in isoelectric focusing, and further resolution was achieved by urea denaturation. Substrate and inhibitor complexes of 1 and 2, differentiated by polyacrylamide gel electrophoresis or isoelectric focusing, provided information about the ability of the enzyme to undergo conformational changes. Interconversion of 1 with one of the components of 2 may also involve conformational isomerism. These conclusions are consistent with the well-known ability of eukaryotic dihydrofolate reductases to exhibit increased catalytic activity (attributed to transformations to more open conformations) when treated with salts, chaotropes, or cysteine-modifying agents. Treatment of the L1210/R6 enzyme preparation with one of these activating agents, 5,5'-dithiobis(2-nitrobenzoic acid), derivatized both 1 and 2 (changing their pI values to 7.3 and 6.9, respectively) and altered the enzyme such that stoichiometric inhibition for MTX was observed.

Animals↗
PubMed1985

L1210 dihydrofolate reductase: activation and enhancement of methotrexate sensitivity.

Dihydrofolate reductase, purified to homogeneity from a subline of L1210 murine leukemia cells resistant to 10(-6) M Methotrexate, was resolved into two principal forms (1 and 2) by isoelectric focusing. These forms had pI values of 7.4 and 8.2, respectively; both stained for protein and catalytic activity. Form 1 was a single component, comprising ca. 10% of the total protein, but multiple components of 2 were observed by narrowing the pH range in the electrophoretic procedure. Urea-denatured enzyme exhibited two bands of approximately equal intensity upon isoelectric focusing. These results were interpreted to mean that the enzyme consists of a set of conformationally different forms, arising from two primary structures. Inhibition of the native enzyme by Methotrexate was polyphasic, and appreciable activity remained when the drug was present at an equimolar concentration. Various agents (KCl, H+, urea, and SH-modifiers), known to "activate" dihydrofolate reductases, produced a monophasic, stoichiometric inhibition. Activating agents appear to induce a more open (and labile) conformation of the enzyme. This leads to increased affinity for MTX accompanied, in some instances, by increased catalytic activity.

Animals↗
PubMed1984

Purification and properties of 5,10-methenyltetrahydrofolate synthetase from Lactobacillus casei.

5,10-Methenyltetrahydrofolate synthetase (EC 6.3.3.2), which catalyzes the ATP- and Mg2+ -dependent isomerization of 5-formyl- to 5,10-methenyltetrahydrofolate, has been purified 10,000-fold from Lactobacillus casei using sequential affinity chromatography on immobilized 5-formyltetrahydrofolate and ATP. The enzyme is homogeneous when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is monomeric with a molecular mass of 23,000 Da, and contains a high proportion of hydrophobic amino acids and a single cysteine residue. At 30 degrees C, the turnover number is 88 min-1, and the Km values at pH 6 for 5-formyltetrahydrofolate and Mg-ATP are 0.6 and 1.0 microM, respectively. The enzyme is specific for (6S)-5-formyltetrahydrofolate, but ATP can be replaced by other nucleoside 5'-triphosphates with varying efficiency. The purified enzyme is markedly stabilized by the non-ionic detergent, Tween 20.

Amino Acids↗
PubMed1984

Multiple forms of L1210 dihydrofolate reductase differing in affinity for methotrexate.

Dihydrofolate reductase, purified from a Methotrexate-resistant subline (R6) of L1210 mouse leukemia cells, consists of two forms (designated 1 and 2) differing in affinity for the drug. Form 1 is more sensitive to inhibition by Methotrexate. Form 2 is more heat-labile, but it can be stabilized by bovine serum albumin or NADPH. Isoelectric focusing resolves 1 and 2; pI values are 7.4 and 8.2. Forms 1 and 2 comprise about 10 and 90% of the total protein, but 1 has at least a 2-fold higher specific activity. Binding measurements with [3H]Methotrexate provide KD values of ca. 1 and 27 nM for 1 and 2.

Animals↗
PubMed1984

Enzymatic activation of 5-formyltetrahydrofolate via conversion to 5, 10-methenyltetrahydrofolate.

The ATP-dependent conversion of 5-formyltetrahydrofolate (folinate) to the 5,10-methenyl derivative, catalyzed by 5,10-methenyltetrahydrofolate synthetase (EC 6.3.3.2), is of considerable importance in cancer chemotherapy, since it provides the basis for the administration of folinate to counteract the deleterious effects of high-dose Methotrexate regimens. Methenyltetrahydrofote synthetase has been purified 10,000-fold from L. casei using sequential affinity chromatography on immobilized folinate and ATP. The monomeric enzyme is homogeneous upon SDS-polyacrylamide gel electrophoresis, has a molecular weight of 23,000 (confirmed by gel filtration), and contains a single cysteine residue. The turnover number is ca. 250 min-1, and the Km values at pH 6 for 5-formyltetrahydrofolate and Mg-ATP are 0.6 and 1.0 microM, respectively; the equilibrium constant is 0.7-1.0 mM. Methotrexate, 5-methyltetrahydrofolate, and folate are not inhibitory. The mechanism for the reaction is proposed to involve phosphorylation of the formyl group to create an enol phosphate; subsequent attack on the methenyl carbon by N-10 would generate a tetrahedral intermediate, with release of the phosphate providing the driving force for ring closure.

Animals↗
PubMed1982

Accumulation of 5-methyltetrahydrofolate in cobalamin-deficient L1210 mouse leukemia cells.

Cobalamin-deficient L1210 mouse leukemia cells were propagated on [3H]folate under conditions designed to completely label the intracellular folate compounds. The latter, present almost entirely as polyglutamate forms, were converted to the corresponding monoglutamates by treatment with gamma-glutamylcarboxypeptidase; the monoglutamates were subsequently separated and identified by chromatography on Sephadex G-25. 5-Methyltetrahydrofolate accounted for approximately 70% of the total folate pool (17.8 nmol/10(9) cells), while the remainder was comprised largely of 5-formyltetrahydrofolate (13.0%), and tetrahydrofolate (9.5%). The identity of 5-methyltetrahydrofolate were further confirmed by chromatography on DEAE-cellulose and by its quantitative conversion to tetrahydrofolate after treatment with a highly purified preparation of methionine synthetase (5-methyltetrahydrofolate:homocysteine methyltransferase, EC 2.1.1.13). In contrast, cobalamin-replete cells contained only 5.3% 5-methyltetrahydrofolate, along with 30% each of the other three reduced folate compounds. These results provide direct experimental proof of the "methyl trap" hypothesis, which predicted that 5-methyltetrahydrofolate should accumulate in cobalamin-deficient cells.

Animals↗
PubMed1982

Transport of folate compounds, pterins and adenine in L1210 mouse leukemia cells.

L1210 mouse leukemia cells provide a convenient model for examining the mechanisms and components involved in the active transport of various metabolites and drugs. One of these transport systems exhibits a broad specificity for folate compounds, including 4-amino antagonists such as methotrexate. The primary substrate for this system is 5-methyltetra-hydrofolate (Kt = 1 microM), the principal circulating form of the vitamin in mammals. 5-Formyltetrahydrofolate (Kt = 5 microM) and Methotrexate (Kt = 5 microM) are also taken up efficiently, but folate (Kt = 100 microM) is a relatively poor substrate. Vmax for this system is ca. 15 pmoles/min/mg protein. Energy for substrate internalization is provided by an anion-exchange mechanism, and regulation appears to be mediated by cyclic AMP. The system can be inhibited irreversibly by treatment of the cells with photo-activated azido AMP or carbodiimide-activated folate compounds. The latter method allows the membrane-associated binding protein to be labeled in situ, thereby providing a means for identifying it during subsequent solubilization and purification. Guidance for this latter project is provided by previous experience in the purification to homogeneity of a similar folate-binding protein from Lactobacillus casei. L1210 cells also contain an efficient system for the transport of adenine (Kt = 20 microM; Vmax = 200 pmoles/min/mg protein). Uptake of adenine is linked with its conversion to AMP via PRPP-dependent adenine phosphori-bosyltransferase. Pterins, which have a close structural similarity to adenine (as well as to a portion of the folate molecule), are also transported into L1210 cells. Transport of [3H] 6-hydroxymethylpterin (Kt = 20 microM) was inhibited by 6-formylpterin, 6-methylpterin and 6-carboxypterin with Ki values of 42, 100 and 350 microM, respectively. Adenine (Ki = 20 microM) and various other purines were also good inhibitors of pterin transport. Present evidence indicates that adenine and pterins use separate transport systems, but isolation of the components of these systems may further delineate their interrelationships.

Adenine↗
PubMed1981

Vitamin B12-dependent replication of L1210 mouse leukemia cells. A model system for cobalamin-folate inter-relationships.

L1210 mouse leukemia cells were made cobalamin-deficient by propagation in a medium from which cyanocobalamin was omitted and fetal bovine serum (containing protein-bound cobalamins) was replaced by bovine serum albumin. These cobalamin-deficient cells exhibited a normal replication time of 12 h, provided that the medium contained excess folate or 5-formyltetrahydrofolate. The cells responded poorly, however, to 5-methyltetrahydrofolate unless exogenous cobalamin was added. A cobalamin dependency was also observed when low levels of folate or 5-formyltetrahydrofolate were used. With 5-methyltetrahydrofolate, optimal stimulation of growth was observed with free and transcobalamin-II-bound cobalamin at 4,000 pM and 2 pM, respectively. Under cobalamin-replete conditions, cells contained 2,000 to 4,000 molecules of cobalamin/cell, and in the deficient state, this value declined to less than 10 molecules/cell; optimal replication on 5-methyltetrahydrofolate required approximately 180 molecules/cell. Cobalamin-deficient cells cultured in the absence of folate reached an arrested state from which limited replication could be induced by the addition of aquacobalamin; normal replication was induced by aquacobalamin plus 5-methyltetrahydrofolate. Results of this investigation are interpreted in terms of the requirement for tetrahydrofolate in cell replication and the production of this compound from folate and 5-formyltetrahydrofolate (via cobalamin-independent pathways) and from 5-methyltetrahydrofolate (via the cobalamin-dependent methionine synthetase).

Animals↗