Phosphorylation modulates the activity of glycine N-methyltransferase, a folate binding protein. In vitro phosphorylation is inhibited by the natural folate ligand

Glycine N-methyltransferase (EC 2.1.1.20) was recently identified as a major folate binding protein of rat liver cytosol (Wagner, C., and Cook, R. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3631-3634). Activity of the enzyme is inhibited when the natural folate ligand, 5-methyltetrahydropteroylpentaglutamate (5-CH3-H4PteGlu5), is bound. It has been suggested that glycine N-methyltransferase plays a role in regulating the availability of methyl groups in the liver. Purified transferase was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase. If 5-CH3-H4PteGlu5 was first bound to the transferase, phosphorylation was inhibited. Phosphorylation of glycine N-methyltransferase in vitro increased its activity approximately 2-fold. 5-CH3-H4PteGlu5 inhibited the activity of newly phosphorylated enzyme as well as native enzyme. Freshly isolated rat hepatocytes incorporated 32P-labeled inorganic phosphate into this folate binding protein. Chemical analysis of purified enzyme showed about 0.55 mol of phosphate present per mol of glycine N-methyltransferase subunit. These results indicate that phosphorylation of glycine N-methyltransferase may provide a mechanism for modulating the activity of this enzyme and support its role in regulating the availability of methyl groups.     

The conversion of the human membrane-associated folate binding protein (folate receptor) to the soluble folate binding protein by a membrane-associated metalloprotease.

The membrane-associated (M-FBP) and soluble (S-FBP) forms of human folate binding proteins (FBP) have been well characterized. Although related in a precursor-product manner, the mechanism of conversion and the basis for differences between M-FBP and S-FBP are not known. The conversion of M-FBP to S-FBP in crude human nasopharyngeal carcinoma (KB) cell preparations is demonstrated based on characteristic gel filtration elution profiles of M-FBP and S-FBP (Ve/V0 = 1.3 and 1.7, respectively) in Triton X-100. M-FBP is stoichiometrically converted to S-FBP in a time- and temperature-dependent reaction by a metalloprotease which is: heat-labile; particulate; contained in human KB cell and placental membranes, and rat kidney homogenates; inhibited by EDTA, 1,10-phenanthroline, and parahydroxymercuribenzoate; requires divalent cations; is maximally active at neutral pH; and is active in the presence or absence of detergent. The purified soluble FBP product appears to be identical to S-FBP. Conversion of purified endogenously [3H]leucine-labeled M-FBP yields a soluble FBP characterized by a 45% decrease in specific activity (moles of 3H/mol folate bound) relative to M-FBP and a non-folate binding fragment which contains 45% of the [3H]leucine from M-FBP, requires detergent and/or urea to remain soluble, and migrates aberrantly on gel filtration in 1% (v/v) Triton X-100 and 8 M urea. Based on changes in the specific activity and the gel filtration elution profiles of purified labeled M-FBP associated with conversion to S-FBP, the endoproteolytic cleavage site is predicted between residues 226 and 229 of the cDNA predicted human FBP amino acid sequence. These results suggest that the cDNA predicted hydrophobic carboxyl terminus (residues 227-257) remains intact on the fully processed, membrane-anchored M-FBP, contains the Triton binding domain, and is involved in the formation of the membrane anchor of M-FBP.     

The effect of autoblood in a hyposmotic state on the colony-forming activity of hematopoietic stem cells]

It has been shown that hypoosmotic autoblood injected sub- or intracutaneously stimulates the colony-forming activity of haemopoietic stem cells in mice. Autoblood injected to animals immediately after their irradiation stimulates haemopoiesis even after a single dose. When mice are injected with autoblood prior to irradiation, the time between the first injection and the day of irradiation is critical for manifestation of the immunomodulating effect. Autoblood infusions immediately before, the day before, or two days before irradiation markedly deteriorate the clinical status of experimental animals and cause death in some of them. It is suggested that stimulation of haemopoiesis is associated with the appearance in the blood stream of a population of radiosensitive cells, apparently T-cell precursors.     

Effect of human milk folate binding protein on folate intestinal transport

The present study examined the effect of human milk folate binding protein (FBP) on the intestinal transport of 5-methyltetrahydrofolate (5-CH3H4PteGlu). This was performed by examining the transport of radiolabeled 5-CH3H4PteGlu bound to FBP using everted sacs of rat intestine. In the jejunum at pH 6, transport of 27 nM bound 5-CH3H4PteGlu was linear with time for 30 min of incubation. Transport of 13 nM bound 5-CH3H4PteGlu was higher in the jejunum than in the ileum at both pH 6 (2.1 +/- 0.3 and 0.36 +/- 0.03 pmol/g wet wt/25 min, respectively) and pH 8 (1.9 +/- 0.3 and 0.32 +/- 0.02 pmol/g wet wt/25 min, respectively). In the jejunum, transport of 13 nM bound 5-CH3H4PteGlu at pH 6 was less than transport of an equimolar concentration of free 5-CH3H4PteGlu (2.1 +/- 0.3 and 5.1 +/- 0.5 pmol/g wet wt/25 min, respectively) but was similar at pH 8 (1.9 +/- 0.3 and 2.47 +/- 0.3 pmol/g wet wt/25 min, respectively). In the ileum transport of bound and free 5-CH3H4PteGlu was similar at pH 6 (0.36 +/- 0.03) and 0.41 +/- 0.06 pmol/g wet wt/25 min, respectively) and pH 8 (0.32 +/- 0.02 and 0.43 +/- 0.1 pmol/g wet wt/25 min, respectively). The transport process of bound 5-CH3H4PteGlu in the jejunum was energy, temperature, and Na+ dependent, but not pH dependent, and was competitively inhibited by sulfasalazine. Ninety-two percent of the transport substrate that appeared in the serosal compartment following incubation with bound 5-CH3H4PteGlu was found to be free (unbound) 5-CH3H4PteGlu. These results show that human milk FBP decreases the rate of transport of 5-CH3H4PteGlu in the jejunum and suggest that FBP-bound 5-CH3H4PteGlu may utilize the same transport system as free 5-CH3H4PteGlu. The results also suggest a role for human milk FBP in regulating the nutritional bioavailability of folate.     

Role of the membrane-associated folate binding protein (folate receptor) in methotrexate transport by human KB cells

The uptake of methotrexate by KB cells was observed to be dependent on time, temperature, and concentration of extracellular methotrexate. The Kd for methotrexate surface binding to KB cells was approximately 200 nM. Following exposure of KB cells to trace quantities of [3H]methotrexate for periods ranging from 6 min to 24 h, the cellular methotrexate was progressively formed into methotrexate polyglutamates and was bound to dihydrofolate reductase as well as to a particulate folate binding protein. To further study the mechanism of methotrexate uptake in KB cells, the N-hydroxysuccinimide ester of methotrexate was used to covalently label the surface of KB cells and to inhibit transport of methotrexate. The N-hydroxysuccinimide ester of methotrexate was bound to a species of protein with an apparent molecular weight of 160,000 in 1% (v/v) Triton X-100 that bound folic acid and was specifically precipitated by antiserum raised against the previously purified high-affinity folate binding protein (the folate receptor) from human KB cells. In addition, trypsin was utilized to remove surface-accessible covalently bound methotrexate. The amount of covalently bound methotrexate that could be released by trypsin initially decreased on incubation at 37 degrees C, suggesting that the methotrexate and binding protein were internalized. However, with time, trypsin could again release the covalently bound methotrexate, suggesting that the binding protein cycles from the external cell surface to the inside of the cell and out again.     

The isolation and properties of multiple forms of folate binding protein in cultured KB cells

The folate binding proteins (FBPs) of KB cells which were cultured in normal (N) and folate-deficient (D) medium have been characterized. The 200,000 g supernate of lysed cells contained two FBPs which could be separated by DEAE-Bio-Gel A chromatography, indicating that they differ in ionic charge although they could not be separated by gel filtration through Sephadex G-100 (apparent Mr approximately 40,000). Two species of FBP, a major form of apparent Mr approximately 160,000 and a minor form of apparent Mr approximately 40,000, were identified by gel filtration through Sephadex G-150 in the membrane component of the cells after solubilization with Triton X-100. An additional FBP was isolated and purified by affinity chromatography from the medium in which these cells were cultured. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent Mr of this FBP was approximately 44,000. The association constants for pteroylglutamic acid of the FBPs in the 200,000 g cell lysate supernate, culture medium, and Triton-solubilized membrane were similar and the relative affinity of folate analogs for the FBP, vis-à-vis pteroylglutamic acid, was similar for all species. An antiserum raised to the purified FBP from the culture medium precipitated the FBPs in the 200,000 g cell lysate supernate, Triton-solubilized membrane, and culture medium, indicating antigenic homology among these FBPs. There was no unsaturated FBP in the 200,000 g cell lysate supernate or medium when KB cells were cultured in N medium. However, when cells were cultured in D medium, the unsaturated FBP of the 200,000 g cell supernate and culture medium was substantial (9.2 and 14.1 pmol/mg protein, respectively). Unsaturated FBP was detected in the membrane of normal cells but this also increased when these cells were cultured in D medium (4.5 to 756 pmol/mg protein), indicating that the FBPs of these cellular compartments are normally saturated by folate. After 16 weeks of culture in D medium, the total folate binding capacity of the membrane-associated FBP was twofold greater than that of normal KB cells, indicating the induction of FBP.     

The effect of erythropoietin on the growth and development of spleen colony-forming cells

The progressive growth and development of spleen colonies was studied in heavily irradiated host mice in which erythropoiesis was modified by various procedures. Erythropoietic activity in non-polycythemic hosts bearing spleen colonies was not increased by injections of exogenous erythropoietin. Detectable levels of erythropoietin were found in the heavily irradiated host mice suggesting that the failure of exogenous erythropoietin to modify erythropoiesis was because the host mice were already maximally stimulated by the high endogenous erythropoietin levels. Spleen colonies do not become erythroid in polycythemic mice. The injection of exogenous erythropoietin into heavily irradiated polycythemic hosts did not decrease the total number of spleen colonies produced by a given bone marrow transplant, as would be expected if erythropoietin acted directly on the colony-forming cells. Comparison of growth curves for colony-forming cells in the spleens of polycythemic hosts either receiving or not receiving erythropoietin indicated that the overall doubling time of colony-forming cells during the first ten days after transplantation was not changed by the daily injection of erythropoietin. These experiments are consistent with the concept that erythropoietin is necessary for the development of erythroid colonies. Erythropoietin acts upon some progeny of the colony-forming cell rather than the colony-forming cell itself.     

A high-affinity folate binding protein in human semen

The presence of a folate binding protein of high-affinity type (affinity constant 3.10¹⁰M^–1, maximum folate binding 1.4 nM) in human semen was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Radioligand dissociation from the binding protein was slow at pH 7.4, but rapid at pH 3.5. By use of rabbit antibodies against 25 kDa human milk folate binding protein we determined the concentration of folate binding protein in 16 speciments of human semen in an enzyme-linked immunosorbent assay. The concentration of immunoreactive folate binding protein was independent of the number of spermatozoa in individual specimens. Gel filtration showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one of 100 kDa and a minor one of 25 kDa.     

Interleukin-2 stimulates the colony-forming activity of in vitro irradiated bone marrow

The data are presented on the stimulatory effect of interleukin-2 on the formation of spleen exocolonies from bone marrow irradiated in vitro with doses of 0.5 to 2.5 Gy. It is suggested that the effect observed is associated with the increased proliferation of CFUs survived after irradiation.     

The complete amino acid sequence of a human folate binding protein from KB cells determined from the cDNA

Two species of folate binding protein (FBP), an integral membrane-associated form and a soluble secreted form, have been previously purified from cultured human KB cells. The complete nucleotide sequence of the complementary DNA (cDNA) clone for the coding region of the mature membrane-associated FBP has now been determined, and the deduced amino acid sequence has been computer-analyzed for a prediction of the secondary structure of the protein. The clone has 857 nucleotides of which 678 comprise the coding region for 226 amino acids. The deduced amino sequence contains the identical sequence of the published 18 NH2-terminal amino acids of the purified FBP from KB cells and the published partial amino acid sequence of the human milk FBP except for 1 residue. There was also over 90% homology with the published amino acid sequence of the bovine milk FBP. A total of 16 cysteine residues has been conserved in the three proteins indicating that this amino acid may provide a tertiary structure which is required for its ligand binding function. Northern blot analysis using the cDNA probe identified a single band of 1.28-kilobase pair mRNA in KB cells which was 4.7-fold more intense in folate-depleted cells than in normal cells. These results indicate that the membrane FBP and the soluble FBP in the medium are translation products of the same gene. Computer analysis of the deduced amino acid sequence indicates that there is only one stretch of amino acids of sufficient hydrophobicity and length to span the lipid bilayer of the plasma membrane, but it lacked a predictable helical structure. Those regions of the sequence which did have a predictable helical structure lacked sufficient hydrophobicity required for a membrane anchor. Thus, it is likely that the fatty acids previously reported to be present in the membrane-associated FBP from these cells rather than a peptide sequence provide an important membrane anchoring function.     

The effect of riboflavin binding protein (RPB) on flavokinase catalytic activity

1. Flavokinase isolated from 18-day chick embryo liver shows optimal activity at pH 8.6, in the presence of the divalent cations Zn2+, Ca2+ and Mn2+. 2. The activity and stability of flavokinase depends on the RBP concentration. 3. Interaction between flavokinase and riboflavin binding protein was demonstrated by means of crossed immunoelectrophoresis and affinity chromatography.     

Charge heterogeneity of the folate binding protein in normal human leukocytes

The DEAE-Sepharose CL-6B chromatographic profile of supernatant from homogenized normal human leukocytes containing large amounts of folate binder revealed two peaks of binding activity. A minor binder (I) eluted with the equilibrating buffer (1 mM sodium phosphate of pH 6.0), while the major binder (II) first eluted after the initiation of a linear phosphate gradient with 200 mM sodium phosphate of pH 7.6 as the limiting buffer. Binder II was thus a more acidic protein since it required elution by a salt-pH gradient. Binding of [3H] folate to binder II was of a high-affinity type (K = 10(10) M-1) and displayed positive cooperativity.     

The distribution of colony-forming cells in the kidney

Abstract. A method is described for producing outgrowths of small nephron segments (average 24 cells) in culture. The method was used to estimate an overall colony-forming efficiency of 4.6% for cells constituting the segments. Efficiency was found to be lower for thick segments (1%) than for thin segments (6%) from Henle's loop. The latter higher level indicates that precursor cells are concentrated near the middle of the nephron. For comparison, a two-dose irradiation technique was used to calculate a mean number of 5 ± 2 (SE) clonogens per segment producing outgrowths. This tended to be higher than the value of about 1 calculated from the 65% of segments producing outgrowths, as expected if the remaining segments contained no clonogens.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

An L1210 cell line (JT-1), which can grow in medium supplemented with 1 nM folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37 degrees C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23 +/- 0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [3H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37 degrees C remained as unmetabolized folic acid. Binding was also rapid at 0 degrees C but uptake at the plateau was only one-half the value obtained at 37 degrees C. Half-maximal saturation of the binding component (KD) occurred at a folate concentration of 0.065 nM at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (KD = 2.0 nM). 5-Methyltetrahydrofolate was also bound by this component (Ki = 13 nM at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (Ki = 45 nM) and methotrexate (Ki = 325 nM). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500 nM caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)