[59] Separation of pteroyl-oligo-γ-l-glutamates by high-performance liquid chromatography

The effect of various anions on the mediated influx and efflux of [3H]methotrexate by L1210 cell plasma membrane vesicles in a HEPES buffer system was studied. Our results show that flux is stimulated to the same extent in either direction when SO4, Pi, or folate compounds (1,L5-CHO-folate-H4, methotrexate), but not Cl- was present in the opposite compartment. This implies the property of directional symmetry, a condition in which differential mobility of loaded and unloaded carriers occurs in both directions. We also observed a similarity in the specificity of the interaction between various anions and carrier in each orientation of the membrane, in the order, Cl- much less than Pi approximately equal to SO2-4 much less than methotrexate less than 1,L5-CHO-folate-H4. Also, the absolute differential in mobility of loaded and unloaded carrier (assumed from the extent of transstimulation obtained) varied substantially among the anions examined. No stimulation was obtained with Cl-, and stimulation was twofold with Pi, SO2-4 and methotrexate and fourfold with 1,L5-CHO-folate-H4. Transstimulation of flux from either external or internal compartment only occurred when a positive gradient of total anions was maintained in the opposite compartment. Also, no stimulation occurred when the same equivalence of two different anions are present in opposing compartments. The concentration of anions required to transstimulate [3H]methotrexate influx was increased four- to 10-fold when vesicles were equilibrated in 145 mM NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…[3H]Methotrexate was purified prior to use by high-performance liquid chromatography [1]. Radiochemical purity of the final preparation was >97%.…”

Section: Methodsmentioning

confidence: 99%

Interaction between anions and the reduced folate/methotrexate transport system in L1210 cell plasma membrane vesicles: Directional symmetry and anion specificity for differential mobility of loaded and unloaded carrier

1984

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“…Intracellular polyglutamylate accumubonucleotide (GAR), and aminoimidazole carboxamide lation was measured using a method previously described. 18 Briefly, (AICAR) transformylases are inhibited, as well. 8,9 The amount of long chain polyglutamates in cells represents a result of the balance between the activity of two intracellular levels of MTX, Tomudex, and 1843U89 and their determination of the mitochondrial, cytosolic, and lysosomal markers.…”

Section: Tion Of Intracellular Mtx Polyglutamates and As A Resultsmentioning

confidence: 99%

Disparate Affinities of Antifolates for Folylpolyglutamate Synthetase From Human Leukemia Cells

Longo

Görlick

Tong

et al. 1997

Blood

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Previous work showed that acute myelocytic leukemia that interfering substances in crude lysates were not responblasts accumulate less long chain polyglutamates of methosible for this difference. We postulate that there may be trexate (MTX) than acute lymphocytic leukemia blasts when differences in the properties of this enzyme related to tissue incubated with this radiolabeled antifolate. This difference specific expression. In contrast to MTX, both Tomudex (Zenlikely explains the increased sensitivity of lymphoid leukeeca Pharmaceuticals, Wilmington, DE) and 1843U89, potent mias to short-term exposure of MTX as compared with myinhibitors of thymidylate synthetase, have low K m s for folyleloid leukemias. In this study, we examined the basis for polyglutamate synthetase, and polyglutamate forms of differences between long chain MTX polyglutamate accuthese inhibitors are accumulated to the same degree in both mulation between different leukemia cell types using both myeloid and lymphoid acute leukemia cells, paralleling the leukemia cell lines and blasts freshly isolated from blood equivalent cytotoxicity found between myeloid and of leukemic patients. The major difference found between lymphoid leukemia cell lines. Based on these results, we leukemia cells that accumulate long chain polyglutamates believe a clinical trial of Tomudex in patients with acute and those that do not were differences in K m values for the myeloid leukemia is warranted. enzyme folylpolyglutamate synthetase. K m values did not ᭧ 1997 by The American Society of Hematology. change with partial purification of this enzyme, indicating humidified atmosphere in RPMI 1640 media containing 10% fetal

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“…Samples of unlabeled methotrexate and aminopterin were provided by the Drug Procurement and Synthesis Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD. Both radioactive and nonradioactive samples were found by rechromatography (22) to be Ͼ98% pure. All other chemicals were reagent grade.…”

Section: Isolation Of Plasma Membranes and Purification Of [ 3 H]amt-mentioning

confidence: 97%

Ligand-directed Immunoaffinity Purification and Properties of the One-carbon, Reduced Folate Transporter

Chiao

Yang

Roy

et al. 1995

Journal of Biological Chemistry

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Almost complete purification (> 95%) of the 46-kDa murine, one-carbon, reduced folate transporter (RFT) at a recovery of 20% was obtained by ligand-directed immunoaffinity fractionation from transporter overproducing L1210/R83 cells. These cells were labeled with the N-hydroxysuccinimide ester of [3H]aminopterin (AMT), the isolated plasma membrane alkaline washed to remove nonintegral membrane proteins, detergent-solubilized, and RFT-separated on an anti-AMT antibody-protein G-Sepharose column followed by preparative SDS-polyacrylamide gel electrophoresis. Anti-RFT antibody, subsequently derived, differentially blotted (L1210/R83 >> L1210/0) a 46-kDa protein during SDS-polyacrylamide gel electrophoresis of plasma membrane from L1210/R83 and L1210 cells and in L1210/R83 cells after trichloroacetic acid precipitation. In contrast to that reported for human tumor cells, glycosidase treatment of RFT revealed no common N- or O-linked core oligosaccharides associated with this protein. The same 46-kDa protein at different relative levels was revealed in a Western blot of plasma membrane from other murine tumors. Blotting of plasma membrane from methotrexate resistant, transport defective L1210 cell variants exhibited wild-type levels of a less electrophoretically mobile RFT or greater levels of the same 46-kDa RFT which could not be affinity labeled with N-hydroxysuccinimide-[3H]AMT. The same antibody differentially blotted a 83-kDa plasma membrane protein from human HL-60 and CCRF-CEM cells with different levels of reduced folate transport and affinity labeling of RFT, verifying the conserved nature of this protein consistent with earlier functional studies.

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[59] Separation of pteroyl-oligo-γ-l-glutamates by high-performance liquid chromatography

Cited by 48 publications

References 5 publications

Interaction between anions and the reduced folate/methotrexate transport system in L1210 cell plasma membrane vesicles: Directional symmetry and anion specificity for differential mobility of loaded and unloaded carrier

Interaction between anions and the reduced folate/methotrexate transport system in L1210 cell plasma membrane vesicles: Directional symmetry and anion specificity for differential mobility of loaded and unloaded carrier

Disparate Affinities of Antifolates for Folylpolyglutamate Synthetase From Human Leukemia Cells

Ligand-directed Immunoaffinity Purification and Properties of the One-carbon, Reduced Folate Transporter

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