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Biomedical subjects

F M Huennekens

Publications and source records attributed to F M Huennekens.

At least 55 records · Page 3Linked to original sources
PubMed1980

Irreversible inactivation of the methotrexate transport system of L1210 cells by carbodiimide-activated substrates.

Methotrexate and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide react to form a relatively unstable product (activated methotrexate) that irreversibly inhibits methotrexate transport in L1210 mouse leukemia cells. The rate of methotrexate transport was reduced 50% by pretreatment of cells with activated methotrexate at a concentration of 1 microM and by 98% upon exposure to an excess (40 microM) of this agent. Specificity was demonstrated by the fact that activated methotrexate had no effect on other transport systems (i.e. leucine and phosphate) and that complete protection against inactivation was afforded by either the unlabeled substrate (methotrexate) or a competitive inhibitor (phosphate). The carbodiimide-generated derivatives of aminopterin, folate, and p-aminobenzoylglutamate were also irreversible inhibitors of methotrexate transport, and their relative effectiveness paralleled the ability of the parent compounds (aminopterin greater than methotrexate greater than folate greater than p-aminobenzoylglutamate) to act as reversible transport inhibitors. Measurements at 4 degrees C showed that activated methotrexate (Ki = 0.8 microM) was a competitive inhibitor for binding of methotrexate (KD = 0.4 microM) to the transport protein. Treatment of the cells with sufficient activated methotrexate to inhibit transport increased the KD for methotrexate to 0.9 microM but had no effect on total methotrexate-binding activity. Methotrexate efflux was also reduced but was not blocked by an excess of this reagent.

Animals↗
PubMed1980

Adherence of L1210 murine leukemia cells to sephacryl-aminopropylcobalamin beads treated with transcobalamin-II.

Sephacryl beads containing an immobilized aminopropylcobalamin-transcobalamin-II complex serve as foci for the adherence of L1210 murine leukemia cells. Bead-cell interaction does not occur when (A) nonderivatized beads are used; (B) transcobalamin-II is omitted or presaturated with cyanocobalamin in the preparation of the bead complex; (C) intrinsic factor replaces transcobalamin-II; and (D) the complex is removed from beads by photolysis. These observations suggest that adherence results from the ability of transcobalamin-II to form a bridge between immobilized cobalamin on the bead and receptors in the plasma membrane of the cell.

Acrylic Resins↗
PubMed1979

Photoinactivation of the methotrexate transport system of L1210 cells by 8-azidoadenosin 5'-monophosphate.

Methotrexate transport in L1210 cells is mediated by a carrier protein that can bind organic and inorganic phosphate compounds in addition to the various folate substrates. The photoaffinity labeling agent, 8-azidoadenosine 5'-monophosphate (8-azido-AMP), also interactis (Ki = 140 microM) with the receptor site for this transport system, and upon irradiation with ultraviolet light, irreversibly inhibits methotrexate uptake. Protection against this inactivation is afforded by either a substrate (methotrexate) or a competitive inhibitor (inorganic phosphate). The light-induced reaction proceeds rapidly (t1/2 = 2 min at 23 degrees C under the conditions described) and produces half-maximal reduction in the transport rate when the 8-azido-AMP concentration is 65 microM. complete photoinactivation of methotrexate transport could not be obtained from a single exposure to 8-azido-AMP (up to 1.0 mM), but it could be achieved by the repetitive illumination of cells in a fresh medium. The phosphate and folate/adenine transport systems of L1210 cells are not affected by irradiation in the presence of 8-azido-AMP.

Adenine Nucleotides↗
PubMed1979

Cryptofluorescent analogs of cobalamin coenzymes: synthesis and characterization.

Upper-axial (beta-position) ligand analogs of the B12 coenzymes 5'-deoxy-5'-adenosylcob(III)alamin and methylcob(III)alamin have been synthesized by reaction of the 5'-chloro-5'-deoxy derivatives of fluorescent nucleosides (1,N6-ethenoadenosine, formycin, 2-amino-nebularine, and 2,6-diaminonebularine) and a fluorescent alkyl halide (dansylamidopropyl chloride) with cob(I)alamin. These analogs were nonfluorescent, but fluorescent products could be generated by photolysis or cyanolysis of the carbon-cobalt bonds. Under anaerobic and aerobic conditions, the major fluorescent photolysis products of 1,N6-ethenoadenosylcob(III)alamin were 1,N6-etheno-5',8-cyclic-5'-deoxyadenosine, and the 5'-aldehyde of 1,N6-ethenoadenosine, respectively. The cryptofluorescent property of these analogs was utilized to follow the kinetics of aerobic photolysis. First-order rate constants determined by this method were comparable to those obtained spectrophotometrically [via appearance of of aquacob(III)alamin]. Pseudo-first-order rate constants determined fluorometrically for the cyanolysis (at 25 degrees C) of 1,N6-ethenoadenosylcob(III)alamin, 2,6-diaminonebularinylcob(III)alamin, 2-aminonebularinylcob(III)alamin and formycinylcob(III)alamin were 5.8 X 10(-2), 2 X 10(-2), 1.8 X 10(-2), and 3 X 10(-5) min-1, respectively; values in good agreement were obtained spectrophotometrically (via appearance of dicyanocobalamin). Dansylamidopropylcob(III)alamin was stable in the presence of cyanide. The nucleoside alpha-ribazole is fluorescent in the free state but nonfluorescent when present as the lower axial (alpha-position) ligand in cobalamin coenzymes. Thus fluorescence of ligands in both the alpha- and beta-positions of cobalamins is quenched, probably as a result of intramolecular energy transfer between the ligands and the nonfluorescent corrinoid.

Aerobiosis↗
PubMed1979

Mechanism of folate transport in Lactobacillus casei: evidence for a component shared with the thiamine and biotin transport systems.

Lactobacillus casei cells have been shown previously to utilize two separate binding proteins for the transport of folate and thiamine. Folate transport, however, was found to be strongly inhibited by thiamine in spite of the fact that the folate-binding protein has no measurable affinity for thiamine. This inhibition, which did not fluctuate with intracellular adenosine triphosphate levels, occurred only in cells containing functional transport systems for both vitamins and was noncompetitive with folate but competitive with respect to the level of folate-binding protein. Folate uptake in cells containing optimally induced transport systems for both vitamins was inhibited by thiamine (1 to 10 muM) to a maximum of 45%; the latter value increased to 77% in cells that contained a progressively diminished folate transport system and a normal thiamine system. Cells preloaded with thiamine could transport folate at a normal rate, indicating that the inhibition resulted from the entry of thiamine rather than from its presence in the cell. In a similar fashion, folate (1 to 10 muM) did not interfere with the binding of thiamine to its transport protein, but inhibited thiamine transport (to a maximum of 25%). Competition also extended to biotin, whose transport was strongly inhibited (58% and 73%, respectively) by the simultaneous uptake of either folate or thiamine; biotin, however, had only a minimal effect on either folate or thiamine transport. The nicotinate transport system was unaffected by co-transport with folate, thiamine, or biotin. These results are consistent with the hypothesis that the folate, thiamine, and biotin transport systems of L. casei each function via a specific binding protein, and that they require, in addition, a common component present in limiting amounts per cell. The latter may be a protein required for the coupling of energy to these transport processes.

Biological Transport, Active↗
PubMed1979

Coupling of energy to folate transport in Lactobacillus casei.

Lactobacillus casei cells can accumulate folate to an intracellular concentration in excess of 500 muM and to concentration gradients (relative to the extracellular compartment) of several thousand-fold. Maximum rates of folate transport are achieved rapidly (t(1/2) < 1 min) after the addition of glucose to energy-depleted cells and occur at intracellular adenosine 5'-triphosphate concentrations above 625 muM. The rate of folate transport and the adenosine 5'-triphosphate content of cells are both extremely sensitive to arsenate and decrease in parallel with increasing concentrations of the inhibitor, indicating a requirement for phosphate-bond energy in the transport process. The energy source is not a membrane potential or a pH gradient generated via the membrane-bound adenosine triphosphatase, since dicyclohexylcarbodiimide (an adenosine triphosphatase inhibitor) and carbonyl cyanide m-chlorophenylhydrazone (a proton conductor) have little effect on the uptake process. The K(+)-ionophore, valinomycin, is an inhibitor of folate transport, but does not act via a mechanism involving dissipation of the membrane potential. This can be deduced from the facts that the inhibition by valinomycin is relatively insensitive to pH, is considerably greater in Na(+)- than in K(+)-containing buffers, and is not enhanced by the addition of proton conductors. Folate efflux is not affected by valinomycin, glucose, or various metabolic inhibitors, although a rapid release of the accumulated vitamin can be achieved by the addition of unlabeled folate together with an energy source (glucose). These results suggest that the active transport of folate into L. casei is energized by adenosine 5'-triphosphate or an equivalent energy-rich compound, and that coupling occurs not via the membrane-bound adenosine triphosphatase but by direct interaction of the energy source with a component of the transport system.

Adenosine Triphosphate↗
PubMed1978

Cyclic adenosine 3':5'-monophosphate and methotrexate transport in L1210 cells.

Treatment of L1210 cells with glucose, 1-methyl-3-isobutylxanthine, theophylline, and prostaglandins E1 and E2 was used to increase to varying degrees the endogenous level of cyclic adenosine 3':5'-monophosphate (cAMP). In an inverse manner these agents decreased the rate of methotrexate (MTX) transport into the cells. Azide, conversely, lowered the cAMP level and enhanced MTX transport. Quantitative information about this relationship was obtained by plotting the reciprocal rates of MTX transport as a function of cAMP levels. These data suggest that MTX transport is regulated by the level of cAMP.

Animals↗
PubMed1977

Purification and properties of a membrane-associated, folate-binding protein from Lactobacillus casei.

A folate-binding protein has been solubilized from Lactobacillus casei by treatment of membrane preparations with Triton X-100 in the presence of [3H]folate. The protein-folate complex was purified 100-fold and recovered in a 22% yield by adsorption and elution from microgranular silica (Quso G-32), followed by passage through Sephadex G-150. When subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the purified preparations showed only a single, protein-staining band whose molecular weight was 25,000. Bound folate (34 nmol/mg of protein) corresponded to 0.85 mol/mol of protein. Analyses of the protein revealed relatively few charged or polar amino acids, an unusually high content of hydrophobic residues and methionine, and the absence of cysteine. The purified protein-folate complex was contained within a Triton micelle (molecular weight, 220,000; about 340 mol of detergent per mol of protein). Bound folate was retained when the micelle was exposed at 4 degrees to solutions whose pH values ranged between 3 and 12; at 23 degrees, however, stability was decreased, especially above pH 8. Folate could be released by treatment of the micelle with ethanol or with chaotropic agents such as guanidinium chloride, perchlorate, or thiocyanate.

Amino Acids↗
PubMed1976

Interaction of 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides with dihydrofolate reductase from amethopterin-resistant L1210 cells.

The 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides (TPN and TPNH) epsilon-TPN and epsilon-TPNH) have been synthesized and used as fluorescent probes to examine the pyridine nucleotide binding site of L1210 dihydrofolate reductase. Epsilon-TPNH (Km = 16.7 muM) was able to replace TPNH (Km = 3.8 muM) in the enzyme-catalyzed reduction of dihyrdofolate, and both epsilon-TPN and epsilon-TPNH formed binary complexes with the enzyme that were stable to polyacrylamide gel electrophoresis. The fluorescence of epsilon-TPN was enhanced and the emission maximum shifted from 415 to 405 nm when the nucleotide was bound to the enzyme. The ethenoadenine moiety in epsilon-TPNH behaved similarily, but the fluorescence changes were complicated by concurrent effects of binding upon the dihydronicotinamide fluorophore. Fluorescence enhancement titrations yielded values of 1.8 and 0.59 muM, respectively, for the dissociation constants of the enzyme-epsilon-TPN and enzyme-epsilon-TPNH complexes. Titration experiments based upon quenching of enzyme fluorescence gave similar values, viz., 2.1 and 0.53 muM for the dissociation constants of these complexes. Fluorimetric titration of the enzyme-TPNH complex with epsilon-TPN (or of the enzyme-TPN complex with epsilon-TPNH) failed to reveal the presence of a second pyridine nucleotide binding site. The fluorescence enhancement of enzyme-bound epsilon-TPN or dihydrofolate was quenched when amethopterin or epsilon-TPN, respectively, was added to form a ternary complex. These results provide information concerning the nature of the pyridine nucleotide binding site and its spatial relationship to the dihydrofolate/amethopterin binding site.

Animals↗