Maria da Costa

Protein-methotrexate-IgG complexes in the serum of patients receiving high-dose antifolate therapy.

Three patients who were treated with repetitive courses of high‐dose methotrexate and leucovorin rescue experienced hypersensitivity symptoms during infusion of the drug. Serum from all three patients contained one complex of methotrexate bound to a protein similar in size to albumin and a second complex of the protein‐bound methotrexate coupled to IgG. This immune complex was not found in 4 patients similarly treated who did not experience hypersensitivity symptoms. Cancer 46:471–474, 1980.

Properties of purified folate-binding proteins from chronic myelogenous leukemia cells

Folate-binding protein(s) from chronic myelogenous leukemia cells have been purified using acid dialysis, ammonium sulfate fractionation and affinity chromatography. The purified preparation which migrates as a single band on disc electrophoresis could be separated by DEAE agarose chromatography into two folate-binding proteins (binders I and II) which bind molar equivalents of folic acid. One binder (I) eluted from DEAE at 1 mM sodium phosphate, pH 6.0, and the other (II) at 100 mM sodium phosphate, pH 7.4. Analysis of the purified mixture, which contained more than 90% binder II, by sedimentation equilibrium centrifugation indicated a homogeneous protein with a calculated molecular weight of 44000. Antiserum raised against the purified mixture gave a single precipitin line by immunodiffusion against a preparation of partially purified cell lysate. Hydrolysis of the more acidic binder (II) with neuraminidase converted it to a weakly acidic protein similar to binder I, suggesting that these binders are glycoproteins which differ in sialic acid content. With isoelectric focusing, the binding of folic acid could be demonstrated at pH 6.7, 7.3, 7.8 and 8.2 for binder I, and at pH 5.1, 5.8, and 6.5 for binder II. Binders I and II had equally high affinity for folic acid and dihydrofolate, lower affinity for N5-methyl-tetrahydrofolate, and no apparent affinity for N5-formyltetrahydrofolate or methotrexate.

A simplified radioenzymatic assay for dihydrofolate reductase using [3H]dihydrofolate

Abstract This report describes a simple method to measure the activity of dihydrofolate reductase using the substrate [ 3 H]dihydrofolate, which is generated by preincubation of [ 3 H]folic acid for 10 min with dithionite before the enzymatic reaction. The procedure then measures the direct reduction of [ 3 H]dihydrofolate to [ 3 H]tetrahydrofolate by coprecipitating the unreduced substrate with excess unlabeled folic acid and acidified zinc sulfate. The advantage of this method is that [ 3 H]dihydrofolate, which is not commercially available, can be generated from high specific activity [ 3 H]folic acid, which is commercially available, immediately before initiating the enzymatic reaction. By this modification, the two important advantages of radioenzymatic assays for dihydrofolate reductase can be more easily exploited; namely, increased sensitivity because much less substrate need be used, and the ability to measure enzyme activity in crude tissue preparations without interference by precipitating proteins or nucleotide oxidases.

Preparation of a stable folate-sepharose complex for affinity chromatography.

Abstract A stable folic acid affinity gel has been developed for the purification of nanograms of protein that bind folic acid or its derivatives. The affinity gel was prepared by first coupling folic acid covalently to bovine serum albumin, followed by covalent coupling of the albumin to p-benzoquinone-activated Sepharose. After the albumin-folic acid complex was formed, it was treated with charcoal to remove ionically bound folate which would otherwise elute from the gel and decrease the recovery of the binding protein. The p-benzoquinone activation resulted in a more stable binding of the albumin to the Sepharose.

The Synthesis of Folate‐binding Protein in Lymphocytes during Transformation

The blast transformation of human lymphocytes exposed to phytohaemagglutinin was associated with an increase in the intracellular concentration of a folate‐binding protein. The protein was measured by a radioinimunoassay and by immunofluorescent microscopy using an antiserum raised against purified folatebinding protein prepared from chronic myelogenous leukaemia cells. Because it has been previously shown that protein‐bound folate cannot function as a cofactor or substrate, the synthesis of this binder during some phase of the replicative cycle may serve to modulate folate‐dependent RNA and DNA synthesis by controlling the concentration of free folate.

Binding of N5,N10-methylene tetrahydrofolate and the inhibition of thymidylate synthesis by a folate-binding protein

A mixture of two ionic forms of a folate-binding protein purified from chronic myelogenous leukemia cells reversibly binds N5,N10-methylene tetrahydrofolate and prevents the coupling of this cofactor to thymidylate synthetase in a terniary complex with fluorodeoxyuridylate. The binding protein also inhibits the enzymic synthesis of thymidine monophosphate by preventing the methylation of deoxyuridylate. These findings suggest that one function of the folate-binding protein may be to regulate the intracellular concentration of free folate cofactors and, thereby, modulate their functional activity.

Studies with the Folate Binding Protein in Chronic Granulocytic Leukaemia Cells: I. SYNTHESIS AND RELEASE OF BINDER BY CELLS IN SHORT‐TERM CULTURE

Summary. Chronic granulocytic leukaemia (CGL) cells which contained a high concentration of unsaturated folate binding protein were incubated in suspension culture for a period of 5 h. Cell samples were periodically assayed for binder and these demonstrated active synthesis which was inhibited by puromycin, cyclo‐heximide, N‐ethylmaleimide, and by incubation at 4°, but not by actinomycin D.

Autoantibodies against folate receptors in women with a pregnancy complicated by a neural tube defect

BACKGROUND In the absence of clinical folate deficiency, periconceptional supplementation with folic acid reduces a womans risk of having an infant with a neural-tube defect. Since antiserum to folate receptors induces embryo resorption and malformations in rats, we hypothesized that autoantibodies against folate receptors in women may be associated with pregnancy complicated by a neural-tube defect. METHODS Serum from 12 women who were or had been pregnant with a fetus with a neural-tube defect and from 24 control women (20 with current or prior normal pregnancies and 4 who were nulligravid) was analyzed for autoantibodies by incubation with human placental folate receptors radiolabeled with [3H]folic acid. The properties of these autoantibodies were characterized by incubating serum and the autoantibodies isolated from serum with placental membranes, ED27 cells, and KB cells, which express the folate receptors. RESULTS Serum from 9 of 12 women with a current or previous affected pregnancy (index subjects) and 2 of 20 control subjects contained autoantibodies against folate receptors (P<0.001). The autoantibodies blocked the binding of [3H]folic acid to folate receptors on placental membranes and on ED27 and KB cells incubated at 4 degrees C and blocked the uptake of [3H]folic acid by KB cells when incubated at 37 degrees C. CONCLUSIONS Serum from women with a pregnancy complicated by a neural-tube defect contains autoantibodies that bind to folate receptors and can block the cellular uptake of folate. Further study is warranted to assess whether the observed association between maternal autoantibodies against folate receptors and neural-tube defects reflects a causal relation.

A radiochemical assay for N5-formyltetrahydrofolic acid (citrovorum factor) in serum and urine.

Abstract N 5 -Methyltetrahydrofolate, but not N 5 -formyltetrahydrofolate, can be measured in biological fluids by ligand-binding radioassay. Therefore, in order to measure N 5 -formyltetrahydrofolate by radioassay, it is chemically converted to N 5 -methyltetrahydrofolate by acidification followed by reduction with borohydride. By this method, 70–113% of N 5 -formyltetrahydrofolate added to serum and urine was recovered. The plasma clearance of the mixture of diastereoisomer of N 5 -formyltetrahydrofolate (Leucovorin) following intravenous administration to two normal subjects was rapid for the first 30 min, but then plateaued and cleared very slowly over the next 90 min, most probably because of the accumulation of the inactive isomer which was slowly excreted in the urine during this time period.

Impaired antibody response in folate deficient mice persisting after folate repletion.

Abstract Normal BDFl mice immunized against p-aminobenzoylglutamate (PABG) diazotized to bovine serum albumin produced antibodies which can be measured using [3H]-pteroylglutamate ([3H]-PGA) because the molecular structure of this compound has the PABG moiety. The antibody response to immunization with the same hapten-protein complex in mice made deficient in folic acid was markedly depressed and remained impaired for approximately eight months after folate repletion, even with repetitive immunizing injections. This depressed response was specific for the PABG-protein complex because the folate repleted mice responded normally to immunization with sheep red cells.