Sheldon P. Rothenberg

Further observations on the folate-binding factor in some leukemic cells

The lysates of peripheral cells as well as the serum from some patients with chronic myelogenous leukemia, contained a macromolecular factor which bound tritiated folic acid. Bound tracer folate filtered through Sephadex G-75 and G-100 columns with the early effluent and appeared with the inner volume through a Sephadex G-200 column. Bound tracer could not be extracted from solution by coated charcoal or the anion exchange resin Dowex 2-X8 and could not be reduced to tetrahydrofolate by folate reductase. The velocity of the binding reaction was very rapid and dissociation of bound tracer extremely slow. Binding decreased sharply below pH 5.0 and the binding factor as well as the folate-binder complex, resisted 56 degrees C for 30 min. The binding factor in the leukemic lysate could be separated from endogenous folate reductase by filtration through a G-75 Sephadex column. Competitive inhibition studies demonstrated little or no inhibition of binding of tritiated folic acid by formyltetrahydrofolate and methyltetrahydrofolate. Diopterin (pteroyldiglutamate), pteropterin (pteroyltriglutamate), methotrexate, and dihydrofolate inhibited binding of tracer folate but not as effectively as unlabeled folic acid. The function of this folate binder is unknown. However, that it reacts with dihydrofolate suggests some relationship (physiologic or pathologic) to DNA synthesis since this folate cofactor is essential for the de novo synthesis of thymidylate from deoxyuridylate. In addition, these findings also suggest that the binding of methotrexate may, like folate, inhibit its reaction with folate reductase, and thus be a mechanism by which leukemic cells become resistant to this drug.

Correlation of low serum IgM levels with absence of functional splenic tissue in sickle cell disease syndromes

Abstract Serum immunoglobulin concentrations were determined in 21 patients with sickle cell disease syndromes and correlated with the size and visualization of the spleen. Whereas serum immunoglobulin A (IgA) levels were elevated in all patients studied, immunoglobulins G (IgG) and M (IgM) were elevated in those patients with demonstrable increase in splenic mass. When even minimal splenic visualization could be demonstrated, serum IgM was normal. However, when there was complete absence of splenic vi-sualization, or following splenectomy, serum IgM concentration was significantly below normal. This finding of low serum IgM concentration may explain the increased susceptibility to infection observed in some groups of patients with sickle cell disease.

Formation of Transcobalamin II-Vitamin B12 Complex by Guinea-Pig Ileal Mucosa in Organ Culture After in Vivo Incubation with Intrinsic Factor- Vitamin B12

Summary. The in vivo incubation of intrinsic factor‐[57Co]vitamin B12 in an ileal loop of a guinea‐pig followed of in vitro culturing of segments of the ileum for 180 min has been used to study the transepithelial transport vitamin B12. Analysis of the solubilized supernate of mucosa following the in vivo phase demonstrated that 44% of the [57Co]vitamin B12 was bound to intrinsic factor (IF), 26% was free, and 16% was bound to transcobalamin II (TCII). Following culture, similar analysis demonstrated that 18% of the vitamin was now bound to IF, 49% was free, and 35% was bound to TCII. In the culture medium, 54% of the [57Co]vitamin B12 was free and 37% was bound ot TCII. The formation of TCII‐[57]vitamin B12 did not occur if homogenized mucosa was incubated with free [57Co]vitamin B12 but it did form in cultures of ileal segments from animals given an excess of unlabelled vitamin to saturate all circulating TCII, and in the medium containing puromycin. Indirect immunoflourescence using chicken anti‐TCII demonstrated that TCII was associated with the mucosal cells of both the ileum and jejunum.

In vitro and in vivo effect of hypotonic saline on the sickling phenomenon

The number of erythrocytes in the sickle form decreased in parallel with the decrease in salt concentration when deoxygenated blood from homozygous sickle cell (SS) anemia patients was mixed with hypotonic solutions of NaCl. This occurred without evidence of red cell hemolysis. There was no change in the proportion of sickle cells when oxygenated erythrocytes were similarly mixed with hypotonic saline solutions, indicating that it was the reversibly rather than the irreversibly sickled cell which unsickled as osmolality decreased. The rapid administration of 0.45 per cent saline solution to patients with SS and SC disease in painful crisis was followed in seven of nine trials by a lowering of serum osmolality; a decrease in the proportion of circulating sickle cells increased 10 to 24 hours after starting therapy, even with lowered osmolality, suggesting release of trapped cells from areas poorly perfused before therapy. When osmolality did not decrease, there was no significant decrease in the proportion of circulating sickle cells. This study indicates (1) lower osmolality reverses the sickling of reversibly sickled cells, and (2) the administration of sufficient hypotonic saline to lower serum osmolality decreases the proportion of circulating sickle cells and may be beneficial for the emergency treatment of painful sickle cell crises.

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.

Purification of the intestinal receptor for intrinsic factor by affinity chromatography

The intestinal receptor for the intrinsic factor vitamin B-12 complex has been solubilized and then purified from the guinea pig ileum using a double structured affinity resin comprised of intrinsic factor coupled to vitamin B-12 which, in turn, was covalently linked to Sepharose 4B. The receptor purified approximately 57 000-fold from the crude homogenate, appears to be a homogenous protein which may be composed of two subunits which separated when the preparation was subjected to polyacrylamide disc gel electrophoresis. Ethylenediaminetetraacetic acid induced dissociation of the complex between the purified receptor and intrinsic factor-B-12 could not be reversed by the addition of excess Ca2+, unlike the effect of EDTA with semipurified receptor or crude ileal homogenates. Calcium reversed the EDTA effect only after the mixture was subjected to extensive dialysis suggesting that the chelating agent interacts directly with the receptor protein. Intrinsic factor-vitamin B-12 competively inhibited the binding of intrinsic factor-[57Co] vitamin B-12 to the purified receptor whereas vitamin B-12 free intrinsic factor did not, even at a 100-fold greater concentration.

Identification of an Immunoreactive Folate in Serum Extracts by Radioimmunoassay

Summary. A radioimmunoassay for folic acid using antibodies with binding determinants specific for this vitamin has been used to identify an immunoreactive folate in serum and serum extracts. Because reduced folates such as methyltetra‐hydrofolate, formyltetrahydrofolate and dihydrofolate did not inhibit the antibody binding of [3H]folic acid while unreduced folates did, the immunoreactive folate in serum and serum extracts was most likely an unreduced folate. Serum extracted by boiling in the absence of ascorbate had a significantly higher concentration of immunoreactive folate than did whole serum. The extraction procedure apparently released or converted endogenous folate to an immunoreactive form because serum extracted with ascorbate contained very low concentrations of the immunoreactive form. The range of recovery of crystalline folic acid added to serum and assayed in extracts prepared without ascorbate was 82‐132%, with a mean of 104%.

A Macromolecular Factor in Some Leukemic Cells Which Binds Folic Acid

Summary The reduction of 0.57 μμmoles of 3H-folic acid to tetrahydrofolic acid by folate reductase in the cell lysates from two patients with chronic myelogenous leukemia was not apparent until the total substrate concentration of the reaction mixture was raised by the addition of stable folic acid. In addition, these leukemic cell lysates inhibited the reduction of tracer 3H-folic acid by a leukemic cell lysate containing “normal” folate reductase activity. Gel-filtration experiments demonstrated that this phenomenon was due to binding of the tracer folate to a large molecule because 3H-folic acid preincu-bated with these peculiar leukemic cell lysates and filtered through Sephadex G-75 appeared in the early or excluded volume indicating that the substrate was bound to a factor with a molecular weight of at least 50,000. This binding of 3H-folic acid could be competitively inhibited by stable folate. I thank Carol Schreiber for her technical assistance for part of these studies, and Drs. Robert Goldstein and Solomon Berson for their helpful criticism in the preparation of this manuscript.

A radioassay for folic acid reductase.

This report describes a radioassay for folic reductase using tritiated folic acid as the substrate. The assay measures directly the disappearance of the labeled substrate, thus avoiding some of the difficulties other indirect assays encounter. By employing high specific activity tritiated folic acid, the reduction of substrate levels in the range of 10−12 mole can be monitored, permitting in turn the assay of correspondingly small amounts of enzyme. A special method of paper chromatography has been used whereby the tritiated folate substrate is separated from the tritiated H4-folate product, and the disappearance of the substrate can thus be quantitated. The enzymic reaction is TPNH dependent and can be inhibited by the folic acid antagonist, amethopterin.