Biomedical subjects

S P Rothenberg

Publications and source records attributed to S P Rothenberg.

At least 37 records · Page 2Linked to original sources

PubMed1992

Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium.

KB cells express a folate-binding protein that is anchored to the plasma membrane by a glycosylated phosphatidylinositol (GPI) tail and these cells can grow in medium containing a very low folate concentration (1 nM). In contrast, mouse 3T3 cells do not express a membrane-associated folate-binding protein and cannot grow under similar low folate conditions. In these studies, 3T3 cells were transfected with a vector containing the cDNA that codes for the KB cell folate-binding protein. In contrast to the wild-type 3T3 cells, the transfected 3T3 cells express a level of folate-binding protein similar to KB cells, 1 and 1.4 ng/micrograms protein, respectively. The capacity for binding [3H] folate to the surface of transfected 3T3 cells cultured in folate-deficient medium is 7.7 pmol/10(6) cells, and this is approximately 50% of the surface binding capacity of KG cells under similar culture conditions. Moreover, after treatment of the transfected 3T3 cells with phospholipase C specific for phosphatidylinositol, the binding of [3H] folate to the surface of these cells is reduced by 90%, indicating that, like the KB cells, the folate-binding protein is anchored to the plasma membrane by a GPI tail. Although the doubling time of wild-type 3T3 cells markedly increases after 13 d of culture in folate-deficient medium, the doubling time of both the transfected 3T3 cells and KB cells do not change. The results of these experiments indicate that the GPI-anchored folate-binding protein provides a mechanism to maintain a level of folate that permits the folate-dependent metabolic functions necessary for cell survival under low folate conditions.

3T3 Cells↗

PubMed1992

Identification of a novel reductase in folate metabolism.

A protein with a molecular weight of 27,500 was co-purified with the enzyme dihydrofolate reductase (molecular weight 22,000) from the liver of mice less than 8 weeks of age using a methotrexate-Sepharose affinity matrix. This 27.5 kDa protein crossreacts with dihydrofolate reductase against an antiserum raised to the purified 22 kDa enzyme. The protein could also reduce dihydrofolate to tetrahydrofolate, thus demonstrating the catalytic properties of dihydrofolate reductase. The expression of this 27.5 kDa protein also appears to be age-dependent because it could not be isolated from liver of mice older than four months.

Aging↗

PubMed1991

The cDNA sequence and the deduced amino acid sequence of human transcobalamin II show homology with rat intrinsic factor and human transcobalamin I.

The cellular uptake of cobalamin (Cbl, vitamin B12) is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TCII and, therefore, served as the source of the complementary DNA (cDNA) library from which the TCII cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5'-untranslated segment of 37 nucleotides, and a 3'-untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R-IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene.

Amino Acid Sequence↗

PubMed1991

Evidence for kinetic and immunologic heterogeneity of dihydrofolate reductase in L1210 leukemia cells.

Two species of DHFR were identified in wild-type L1210 murine leukemia cells by analysis of the kinetics of the binding of MTX and dissociation of the MTX-enzyme complex at pH 5.0 and pH 7.2. The two forms of DHFR were also distinguished by immunoinhibition of the binding of MTX and the catalytic reduction of FH2 to FH4 using an antiserum raised to the purified high affinity form of DHFR. The Ka for the binding of MTX by the low affinity form of the enzyme is 4.5 x 10(7) M-1, substantially lower than the reported Ka for the binding of this drug by the high affinity enzyme. The low affinity form of the enzyme catalyzed the reduction of FH2 to FH4 at a rate slower than the high affinity form of DHFR.

Animals↗

PubMed1989

Age-dependent expression of a novel protein in mouse liver immunologically and functionally homologous with dihydrofolate reductase.

Dihydrofolate reductase with a molecular weight of 22,000 has been purified by salt precipitation and methotrexate-Sepharose affinity chromatography from mouse livers having a mean weight of 2.4 g each. When the same purification procedure was followed using livers with a mean weight of 1.4 g or less, a protein with a molecular weight of 27,500 co-purified with dihydrofolate reductase. This 27.5 kDa species was recovered with dihydrofolate reductase following a second passage through the affinity column and it reacted by Western immunoblotting with a monospecific polyclonal antiserum raised to the purified 22 kDa enzyme. The two proteins could not be separated in a native state to compare their functional activity, but the 27.5 kDa protein appeared to have catalytic reductase activity when assayed directly on the polyacrylamide gel following non-denaturing electrophoresis. The catalytic activity of the mixture of the purified proteins was stoichiometrically inhibited by a molar equivalent of methotrexate.

Aging↗

PubMed1989

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.

Amino Acid Sequence↗

PubMed1989

Endothelial cells from human umbilical vein secrete functional transcobalamin II.

Transcobalamin II (TCII) is a cobalamin (Cbl) binding protein in the plasma that mediates the cellular uptake of Cbl. Although the synthesis of TCII by a variety of cultured mammalian cells and by some isolated perfused organs has been reported, no single tissue has been identified as the source of TCII in vivo. In this study, we demonstrate that cultured human umbilical vein endothelial cells secrete a protein that binds CN[57Co]Cbl, elutes from a Sephacryl S-200 column in the same position as TCII, and precipitates with an antiserum to purified human TCII. The biosynthesis of TCII by these cells was confirmed by demonstrating the incorporation of [35S]methionine into a nascent protein that immunoprecipitated with anti-TCII and which, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) had an Mr of 43,000, the same as human TCII. This secreted protein also had the functional properties of TCII because it facilitated the uptake of CN[57Co]Cbl by the same endothelial cells that secreted it as well as other cell lines that express the membrane receptor for TCII. We also present evidence that the venous endothelium could be the source of TCII in vivo by showing that an intact umbilical vein in an isolated umbilical cord, when perfused with medium containing [35S]methionine, secretes a radiolabeled nascent protein with the same immunoreactive and electrophoretic properties as human TCII. These studies demonstrate that the endothelial cell, which has been shown to secrete a number of plasma proteins, also synthesizes and secretes TCII both in vitro and as an intact endothelium in situ, and therefore, could be the source of circulating TCII in vivo.

Cells, Cultured↗

PubMed1989

Isolation of the complementary DNA for human transcobalamin II.

A complementary DNA (cDNA) clone coding for transcobalamin II (TCII) has been isolated from a human umbilical vein endothelial cell cDNA library. The cDNA is 1.9 Kb and includes the nucleotide sequence which encodes the NH2-terminal 19 amino acids of human TCII. The size of the cDNA is sufficient to code for the entire protein and also contains the nucleotide sequence coding for a 24 amino acid leader peptide and a long untranslated 3' region. The availability of this cDNA will provide the opportunity to characterize genetic disorders of TCII.

Amino Acid Sequence↗

PubMed1988

Characterization of the folate-binding proteins associated with the plasma membrane of rat liver.

Unsaturated folate-binding proteins (i.e., apo forms) have been identified with the plasma membranes of rat liver by the binding of [3H]pteroylglutamic acid. Normal rat liver contains very little of the folate-binding apoproteins, but the folate-binding capacity increases substantially when the rats are made folate-deficient. This increase appears to be due to unsaturation of the folate-binding holoproteins rather than to synthesis of additional protein, because the binding capacity of the plasma membranes from normal rat liver following dissociation of the bound folate is equivalent to the binding capacity of the preparation from folate-deficient liver. Two molecular forms of folate-binding protein were identified by gel filtration of the solubilized plasma membrane fraction, a high-molecular-weight form (Mr less than 100,000), representing 25% of the binding capacity, and a smaller protein (Mr approximately equal to 55,000), representing 75% of the binding capacity. Whereas the larger species can be solubilized only with a detergent, the smaller form appears to be hydrophilic and dissociates spontaneously from the membrane preparation. The binding of [3H]pteroylglutamic acid by the membrane preparation was specific, saturable, and pH- and temperature-dependent. Scatchard analysis of the binding could be fitted to a curvo-linear plot, indicating at least two orders of binding sites which probably correspond to the two molecular forms identified by gel filtration. Competitive inhibition by folate analogues demonstrated that the apoproteins have higher affinity for oxidized folate than for N5-methyltetrahydrofolate and virtually no affinity for N5-formyltetrahydrofolate or methotrexate.

Animals↗

PubMed1988

A specific radioimmunoassay for 5'-deoxyadenosyl cobalamin in serum.

A specific radioimmunoassay for 5'-deoxyadenosyl cobalamin (ado-Cbl) has been developed using an antiserum raised to this cobalamin (Cbl). At a 1:100 or greater dilution the antiserum did not bind radiolabelled methyl-, hydroxo-, sulphito- or cyano-Cbl and these Cbl analogues did not compete in the radioimmunoassay even at 100-fold higher concentration. The serum concentration (range and mean +/- SD) of ado-Cbl in 30 normal subjects was 47-134 and 81 +/- 16 pg/ml. The corresponding values for total Cbl in these sera were 189-610 and 355 +/- 144 pg/ml, and the computed values for methyl-Cbl were 142-476 and 274 +/- 127 pg/ml. The coefficient of variation was substantially greater for methyl-Cbl than for ado-Cbl (46% v. 21%, respectively). The ado-Cbl concentration was in the normal range (75-95 pg/ml) in five healthy subjects with a low normal concentration (189-217 pg/ml) of total Cbl. In two subjects with low total Cbl (118 and 170 pg/ml) and without any clinical evidence of Cbl deficiency, ado-Cbl was normal (63 and 95 pg/ml, respectively). Thus, in this group, low methyl-Cbl accounted for the lower total Cbl. The concentration (mean +/- SD) of serum ado-Cbl and methyl-Cbl in six patients with low total Cbl (57 +/- 25 pg/ml) and clinical evidence for Cbl deficiency was 35 +/- 12 pg/ml and 22 +/- 22 pg/ml, respectively. This difference from the normal mean for each cofactor was highly significant (P less than 0.001). The decrease in the concentration of methyl-Cbl in Cbl deficiency was relatively greater than the decrease in ado-Cbl (92% v. 57%, respectively) which raised the relative concentration of ado-Cbl in Cbl deficiency to 61% of the total Cbl. Although a low serum methyl-Cbl is a sensitive indicator of Cbl deficiency, it may not be as specific as a decrease in serum ado-Cbl. Factor(s) other than tissue stores of Cbl may lower serum methyl-Cbl below the 95% confidence limit in the absence of clinical Cbl deficiency.

Cobamides↗

PubMed1988

Molecular cloning of the complementary DNA for a human folate binding protein.

The complementary DNA for a human folate binding protein has been cloned from a lambda gt11-cDNA library prepared from cultured KB cells. A number of clones were selected by immunoscreening with a monospecific antiserum and by oligonucleotide probes corresponding to the NH2-terminal sequence of the folate binding protein. A partial nucleotide sequence of the cDNA was determined directly from the lambda gt11 phage and after subcloning into M13. The 18 amino acids deduced from the initial 19 codons were exactly the same as the amino acid sequence obtained by peptide analysis of the purified protein providing proof that this clone is the folate binding protein cDNA.

Amino Acid Sequence↗

PubMed1987

Purification, properties, and immunological characterization of folate-binding proteins from human leukemia cells.

A new matrix for affinity chromatography using pteroylglutamic acid coupled to an epoxy-activated matrix via hexanediamine resulted in negligible ligand leakage and permitted the purification of soluble and membrane-associated folate-binding proteins from human leukemia cells contained in a human spleen. Two species of membrane-associated folate-binding proteins were purified from the solubilized membrane fraction of the tissue using 2 M guanidine-HCl to elute the proteins from the affinity matrix. The higher molecular weight binding protein had an Mr of approximately 310,000 and the smaller species had an Mr of approximately 28,000 by gel filtration. By SDS-polyacrylamide gel electrophoresis the smaller species of membrane-associated protein had a molecular weight of 35,500, but the molecular weight of the larger membrane-associated species could not be determined by this method because of the high concentration of residual Triton X-100 in the sample which interfered with the silver staining of the gel. Two folate-binding proteins, which by SDS-polyacrylamide gel electrophoresis had molecular weights of 34,500 and 32,000, were purified from the 44,000 X g supernatant fraction of the tissue homogenate by acid elution from the affinity matrix. Despite the different cell components from which the soluble and membrane-associated folate-binding proteins were purified, the amino acid compositions were similar, especially with respect to the apolar amino acids. All these forms of folate-binding proteins had higher affinity for oxidized than for reduced folates, and very low affinity for 5-formyltetrahydrofolate and methotrexate. Although these proteins cross-react with one antiserum raised previously to a folate-binding protein from other human leukemia cells, they do not cross-react with the folate-binding proteins purified from two other sources of human leukemia cells, from human placenta, or from the human KB cell line.

Antibodies, Neoplasm↗

PubMed1987

Purified membrane and soluble folate binding proteins from cultured KB cells have similar amino acid compositions and molecular weights but differ in fatty acid acylation.

A membrane-associated folate binding protein (FBP) and a soluble FBP, which is released into the culture medium, have been purified from human KB cells using affinity chromatography. By NaDodSO4/PAGE, both proteins have an apparent Mr of approximately 42,000. However, in the presence of Triton X-100, the soluble FBP eluted from a Sephadex G-150 column with an apparent Mr of approximately 40,000 (similar to NaDodSO4/PAGE) but the membrane-associated FBP eluted with an apparent Mr of approximately 160,000, indicating that this species contains a hydrophobic domain that interacts with the detergent micelles. The amino acid compositions of both forms of FBP were similar, especially with respect to the apolar amino acids. In addition, the 18 amino acids at the amino termini of both proteins were identical. The membrane FBP, following delipidation with chloroform/methanol, contained 7.1 mol of fatty acid per mol of protein, of which 4.7 mol was amide-linked and 2.4 mol was ester-linked. The soluble FBP contained only 0.05 mol of fatty acid per mol of protein. These studies indicate that the membrane FBP of KB cells contains covalently bound fatty acids that may serve to anchor the protein in the cell membrane.

Acylation↗

PubMed1987

Dietary folate and biogenic amines in the CNS.

Abnormal biogenic amine biosynthesis has been observed in humans and animals with endogenous and exogenous disturbances in folate metabolism. In an attempt to study this interaction biochemically, rats were depleted or repleted with folate for 10 weeks. Folate levels in depleted animals in serum and CSF correlated with stores in liver and brain, respectively. In depleted or repleted animals, there was no significant effect on biogenic amine metabolism in the CNS, as determined by quantitation of biogenic amines in brain and their respective metabolites in brain and CSF. These results are contrary to studies by other investigators. We suspect, however, that specific genetic defects in folate metabolism do result in impaired biogenic amine metabolism and probably at the level of disturbed biopterin cofactor functions.

Animals↗

PubMed1986

Mouse brain contains a high molecular weight nonfunctional protein with antigenic homology to dihydrofolate reductase.

The cytosol of brain tissue from mature BDF1 mice contains very little dihydrofolate reductase activity but it does contain a high molecular weight nonfunctional protein which cross-reacts in a radioimmunoassay for the active enzyme. Liver cytosol contains less of this high molecular weight cross-reacting protein and more of the functional dihydrofolate reductase. The cytosol from kidney contains very little of the high molecular weight cross-reacting protein, 95% of the immunoreactive proteins being the functional form of dihydrofolate reductase. The modification of dihydrofolate reductase into a nonfunctional form may be an intrinsic property of some cells and this finding could explain the variability in measuring the activity of this enzyme in brain tissue of mature animals.

Animals↗

PubMed1986

Purification and molecular characterization of human transcobalamin II.

Transcobalamin II (TCII) has been purified from Cohn fraction III of human plasma by batchwise binding to and then elution from carboxymethyl-Sephadex, affinity chromatography using photo-labile aminopropyl cobalamin coupled to activated Sephacryl S-200, and finally chromatography through carboxymethyl cellulose. The yield was approximately 80%. The addition of protease inhibitors in all steps of the purification procedure and extensive washing of the carboxymethyl-Sephadex prior to eluting the TCII minimized degradation of the protein and the final preparation of holo-TCII contained 1 mol of cobalamin/mol of protein. A single polypeptide of 43,000 daltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 19 amino acids have been determined for human TCII. 12 of the amino acids are homologous with rabbit TCII and six are homologous with human R-binder, but there is no homology with human intrinsic factor.

Amino Acid Sequence↗

PubMed1986

Characterization of multiple forms of folate-binding protein from human leukemia cells.

Folate-binding proteins were isolated from the particulate fraction (44,000 X g pellet) and the soluble fraction (44,000 X g supernate) of the homogenate of a spleen obtained from a patient who had an acute leukemic (blast) transformation of chronic myelogenous leukemia. The folate-binding activity which was obtained from the particulate fraction by solubilization with 1% Triton X-100 could be resolved into two binding proteins (Mr 310,000 and 28,000) by gel filtration through Sephadex G-200 after incubation with excess [3H]pteroylglutamic acid (PteGlu). The folate-binding protein in the solubilized particulate fraction and the soluble folate-binding protein in the 44,000 X g supernatant cytoplasm were purified by affinity chromatography. Only a 32 kDa protein was identified by SDS-polyacrylamide gel electrophoresis in the final preparation of the purified folate-binding protein from the particulate, whereas two protein bands (Mr 42,000 and 32,000) were identified by SDS-polyacrylamide gel electrophoresis in the purified preparation of the soluble folate-binding protein. Both of these species were immunologically crossreacting. Both the purified folate-binding protein from the particulate fraction and the purified soluble form had higher affinity for oxidized folate than for the reduced folate cofactors, and both proteins had very low affinity for the antifolate compound, methotrexate. The amino-acid composition of the soluble folate-binding protein was similar with regard to the content of apolar amino acids to that reported for the membrane-derived folate-binding protein purified from milk and human placenta.

Amino Acids↗

PubMed1986

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.

Carrier Proteins↗