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S P Rothenberg

Publications and source records attributed to S P Rothenberg.

At least 19 recordsLinked to original sources
PubMed2000

Radioenzymatic assay for reductive catalysis of N(5)N(10)-methylenetetrahydrofolate by methylenetetrahydrofolate reductase.

Methylenetetrahydrofolate reductase catalyzes the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate. Because this substrate is unstable and dissociates spontaneously into formaldehyde and tetrahydrofolate, the customary method to assay the catalytic activity of this enzyme has been to measure the oxidation of [14C]N(5)-methyltetrahydrofolate to N(5), N(10)-methylenetetrahydrofolate and quantify the [14C]formaldehyde that dissociates from this product. This report describes a very sensitive radioenzymatic assay that measures directly the reductive catalysis of N(5),N(10)-methylenetetrahydrofolate. The radio-labeled substrate, [14C]N(5),N(10)-methylenetetrahydrofolate, is prepared by condensation of [C(14)]formaldehyde with tetrahydrofolate and the stability of this substrate is maintained for several months by storage at -80 degrees C in a pH 9.5 buffer. Partially purified methylenetetrahydrofolate reductase from rat liver, incubated with the radio-labeled substrate and the cofactors, NADPH and FAD at pH 7. 5, generates [14C]N(5)-methyltetrahydrofolate, which is stable and partitions into the aqueous phase after the assay is terminated with dimedone and toluene. A K(m) value of 8.2 microM was obtained under conditions of increasing substrate concentration to ensure saturation kinetics. This method is simple, very sensitive and measures directly the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate, which is the physiologic catalytic pathway for methylenetetrahydrofolate reductase.

Animals↗
PubMed2000

Coding sequence, genomic organization and expression of a folate binding protein gene in the rat.

The complementary DNA (cDNA) and the gene encoding the folate binding protein alpha isoform (FBPalpha) have been reported for the human and mouse protein. However, there is no information about this gene in the rat, an animal that could be a model to study expression of this protein in vivo when folate metabolism is modified. Accordingly, the cloning and characterization of this gene in the rat have been the subject of this research. The gene has seven exons and six introns and is approximately 10kb in size. The organization and nucleotide sequence of the coding exons are similar to those of the corresponding human and mouse genes, which are the only other mammalian FBP genes cloned. However, the amino acid sequence of the rat FBPalpha is less homologous, having 48% identity with the published sequences for all the mammalian FBP isoforms. A finding not previously reported is the expression of two FBPalpha transcripts in the kidney that differ in the length of the 5' untranslated sequences, as determined by rapid amplification of cDNA end-polymerase chain reaction amplification (RACE-PCR). The brain expresses a single transcript intermediate in size between the two transcripts expressed in the kidney. The kidney transcripts are encoded by the same gene and appear to be regulated either from two independent promoters or from a single promoter in association with alternative RNA splicing.

Amino Acid Sequence↗
PubMed2000

Folate deficiency reduces the GPI-anchored folate-binding protein in rat renal tubules.

A folate-binding protein (FBP) anchored to cell membranes by a glycosyl phosphatidylinositol (GPI) adduct is constitutively expressed in some transformed and cultured cell lines. Its expression is upregulated when these cells are grown in medium containing low folate, but whether this occurs in vivo with nutritional folate deficiency is unknown. To address this question, the GPI-FBP in the liver, kidney, and brain of rats on control and folate-deficient (FD) diets was measured. The GPI-FBP in the kidney of FD rats decreased significantly in contrast to the upregulation of this protein in cultured cells. Northern blot analysis and nuclear run-on assays indicated that transcription of the GPI-FBP gene in the kidney was not reduced by folate deficiency. This decrease of the GPI-FBP appears to result from its proteolysis, similar to the enzymatic degradation of the apoprotein that occurs in vitro. Because the GPI-FBP is on the brush borders of the proximal renal tubules and provides for the reabsorption of folate, this function diminishes when the protein decreases in folate deficiency.

Animals↗
PubMed1999

Transcobalamin II synthesized in the intestinal villi facilitates transfer of cobalamin to the portal blood.

This study was designed to identify the cellular component of the intestinal villus where transcobalamin II (TCII) is synthesized, because this protein provides an essential function in the intestinal absorption of vitamin B(12) (cobalamin, Cbl). When a segment of proximal or distal small intestine of the guinea pig is cultured in medium containing [(57)Co]Cbl, TCII-[(57)Co]Cbl appears within 15 min. Northern blot analysis of RNA from both proximal and distal small intestine identified the TCII transcript. In situ hybridization of the distal ileum with (35)S-labeled TCII antisense transcript localized grains predominantly in crypts and in the lower third and central core of the villi. Grains were also evident at the base of the enterocytes in close apposition with the vascular network, whereas few grains appeared in the apical region of the columnar cells. This study provides evidence that TCII is constitutively expressed in the intestinal villi where vascular endothelium is abundant. In the distal ileum, where the intrinsic factor (IF) receptor is expressed, after uptake of IF-Cbl and the subsequent binding of free Cbl to TCII synthesized in the villi, the TCII-Cbl complex enters the microcirculation and passes into the portal blood.

Animals↗
PubMed1999

Increasing the dietary intake of folate: pros and cons.

Studies providing unambiguous evidence that the occurrence and recurrence of pregnancies complicated by neural tube malformations were reduced by folic acid supplementation at the time of conception have prompted the Food and Drug Administration (FDA) to approve the fortification of cereal-grain products with this vitamin. Additional enthusiasm for this decision has emanated from studies that show an association of hyperhomocysteinemia with vascular disease and neural tube defects. Despite the apparent logic for the folic acid food fortification program, there are some concerns about the danger of such a policy to segments of the public who have unrecognized vitamin B12 deficiency because folate can mask the hematologic abnormalities and allow the neurological complications to progress or even accelerate. Thus, the apparent benefits of the folic acid fortification and the potential dangers of such a program have polarized opinions in favor of (pro) and in opposition to (con) this FDA policy. The purpose of this review is to present the evidence on which each of these two groups base their opinions.

Diet↗
PubMed1997

Antibodies to transcobalamin II block in vitro proliferation of leukemic cells.

The plasma protein transcobalamin II (TCII) binds and delivers cobalamin (Cbl; vitamin B12) to all cells, which internalize the TCII/Cbl complex by receptor-mediated endocytosis. Congenital deficiency of TCII results in intracellular Cbl deficiency, one effect of which is to disrupt DNA synthesis, leading to megaloblastic anemia. We report here an in vitro culture system in which cell growth is dependent on delivery of Cbl to cells by TCII. Recombinant human holo-TCII was shown to support in dose-dependent manner the growth of the human erythroleukemic cell line K562 and the murine lymphoma cell line BW5147. Free Cbl also supported cell growth; however, at 100- to 1,000-fold higher concentrations than those effective in the presence of apo-TCII. To determine if cellular depletion of Cbl could be achieved by interfering with interactions between TCII/Cbl and its cell-surface receptor, several monoclonal antibodies raised against human TCII were studied. Three antibodies, found to compete for the same binding site on TCII, proved to be effective inhibitors of TCII/Cbl-dependent cell growth. Our results suggest that monoclonal anti-TCII antibodies that block the function of this protein may prove useful in antitumor therapies.

Antibodies, Monoclonal↗
PubMed1996

Characterization of monoclonal antibodies to epitopes of human transcobalamin II.

Cellular uptake of cobalamin (Cbl) is mediated by transcobalamin II (TCII), a Cbl binding protein in the plasma. The TCII-Cbl complex binds to a cell surface receptor and is internalized by endocytosis. We have generated monoclonal antibodies (mAbs) to human TCII that can be distinguished into three functional types on the basis of interaction with three different regions of the protein. Type 1: Receptor blocking. This mAb binds holo-TCII and inhibits the cellular uptake of Cbl. Type 2: Cbl blocking. This mAb binds apo-TCII at or near the Cbl binding domain and inhibits the formation of holo-TCII. Type 3: Precipitating. This mAb binds both holo-TCII and apo-TCII but does not interfere with Cbl binding. Whereas type 1 and type 2 mAb, following incubation with TCII-[57Co]Cbl or apo-TCII, respectively, inhibit the uptake of radio-labeled Cbl by K562 cells, type 3 mAb has no such activity with either form of TCII. These properties of type 1 and type 2 mAb that inhibit the cellular uptake of Cbl, may serve to induce rapid Cbl deficiency and provide a model to study the effect of selective Cbl depletion on cell division and differentiation as well as on the pathways dependent on the two Cbl cofactors, methyl-Cbl and 5'-deoxyadenosyl-Cbl.

Antibodies, Monoclonal↗
PubMed1996

Purification and characterization of folate binding proteins from rat placenta.

Rat placenta contains virtually no unsaturated (i.e., apo-form) folate binding protein. However, by lowering the pH of a solubilized membrane preparation of this tissue to 3.5, the endogenous bound folate was dissociated from the protein and adsorbed to charcoal. The apo-form of the folate binding protein thus obtained was purified by affinity chromatography using pteroylglutamic acid covalently coupled to Sepharose 4B. A single protein band with an apparent M(r) of 36,000 was observed by SDS-polyacrylamide gel electrophoresis of the eluate from the affinity matrix. Western blot of this preparation using a rabbit antiserum raised with the affinity eluate also identified a single 36 kDa protein band. However, peptide sequencing of the N-terminal region of the proteins in the affinity eluate established that it contained two homologous proteins. Computer alignment of the first 22 N-terminal amino acids of each rat placental protein with human, bovine milk and mouse folate binding proteins showed 50-64% identical homology and 27% homology when the eight proteins were aligned together. The affinity of both rat proteins is highest for pteroylglutamic acid (Ka = 1.6.10(9) l/mol) lower for N5-methyltetrahydrofolate and substantially lower for N5-formyltetrahydrofolate. In the dose-response range studied there was no apparent affinity for methotrexate. The folate binding proteins could be released from a preparation of placental membranes using phospholipase C indicating that these proteins belong to the class of proteins anchored to the plasma membrane by a glycosyl phosphatidylinositol adduct.

Amino Acid Sequence↗
PubMed1995

The cloning and characterization of the human transcobalamin II gene.

Transcobalamin II (TCII) is a plasma protein that binds vitamin B12 (cobalamin; Cbl) and facilitates the cellular uptake of the vitamin by receptor-mediated endocytosis. In genetic disorders that are characterized by congenital deficiency of TCII, intracellular Cbl deficiency occurs, resulting in an early onset of megaloblastic anemia that is sometimes accompanied by a neurologic disorder. To define the genetic basis for TCII deficiency, we have cloned and characterized the human gene that encodes this protein. The gene spans a minimum of 18 kbp and contains nine exons and eight introns, with a polyadenylation signal sequence located 509 bp downstream from the termination codon and a transcription initiation site beginning 158 bp upstream from the ATG translation start site. The 5' flanking DNA does not have a TATA or CCAAT regulatory element, but a 34-nucleotide stretch beginning just upstream of the CAP site contains four tandemly organized 5'-CCCC-3' tetramers. This sequence is a motif for a trans-active transcription factor (ETF) that regulates expression of the epidermal growth factor receptor gene (EGFR), which also lacks TATA and CCAAT regulatory elements. A GC-rich sequence that binds the SP1 protein is located 356 nucleotides upstream from the first of the series of CCCC tetramers. Although this GC sequence is at an unusual location with respect to the CAP site, a 507-bp fragment containing this GC box drives the chloramphenicol acetyltransferase (CAT) reporter gene after transient transfection into NIH 3T3 cells. No CAT activity was observed when a 420-bp fragment lacking this GC box but containing the ETF-binding domains was similarly transfected into this cell line. One consensus and two atypical motifs for the c-myc ligand are located downstream and upstream, respectively, of the GC box, and this could explain the elevated plasma TCII observed in some patients with multiple myeloma, as the c-myc product is overexpressed in some myeloma cells. Restriction endonuclease digestion of genomic DNA from eight normal subjects with Taq I, Hinfl, Msp I, and Bgl I identified three patterns of restriction fragment length polymorphism (RFLP). A number of the exon/intron splice junctions of human TCII, TCI, and IF genes are located in homologous regions of these proteins, providing evidence that these genes have evolved by duplication of an ancestral gene. This characterization of the TCII gene and the RFLP should facilitate the identification of the mutation(s) responsible for the genetic abnormalities of TCII expression.

Amino Acid Sequence↗
PubMed1995

Transcobalamin II and the membrane receptor for the transcobalamin II-cobalamin complex.

Transcobalamin II is a plasma protein that binds vitamin B12 (cobalamin) as it is absorbed in the terminal ileum and distributes it to tissues. The circulating transcobalamin II-cobalamin complex binds to receptors on the plasma membrane of tissue cells and is then internalized by receptor-mediated endocytosis. A number of genetic abnormalities are characterized either by a failure to express transcobalamin II or by synthesis of an abnormal protein. These disorders result in cellular cobalamin deficiency and megaloblastic anaemia. In this chapter we review the structural and functional properties of transcobalamin II, the receptor for the transcobalamin-cobalamin complex and the clinical disorders that are associated with perturbation of circulating transcobalamin II. In addition, we provide emerging data about the molecular genetics of transcobalamin II which has emanated from our own and other laboratories.

Animals↗
PubMed1994

Characterization of the gene encoding a folate-binding protein expressed in human placenta. Identification of promoter activity in a G-rich SP1 site linked with the tandemly repeated GGAAG motif for the ets encoded GA-binding protein.

The gene encoding a folate-binding protein (FBP) expressed in human placenta has been cloned by screening a genomic library with the KB cell FBP complementary DNA. This gene, contained in a 10-kilobase EcoRI fragment of this genomic clone, has 5 exons, 4 introns, the AATAA polyadenylation signal in the 3'-untranslated region, and a 5'-flanking sequence which contains the promoter elements, all of which span approximately 5 kilobases. Transcription initiation was mapped by RNase protection to a site 73 base pairs downstream from a G-rich sequence linked to a tandemly repeated GGAAG sequence which is a motif that the ets oncogene encoded GA-binding protein (GABP) transcription factor binds. Gel-shift and supershift mobility assays indicate that the G-rich sequence and the ets motif bind specifically to SP1 and GABP, respectively. These cis regulatory elements in tandem drive expression of the chloramphenicol acetyltransferase reporter gene in transiently transfected mouse 3T3 cells. The location of these elements upstream of transcription initiation in this gene, which lacks an appropriately located TATA box promoter, indicates that this SP1-GA binding region most probably regulates expression of this placental FBP. The gene encoding this placental FBP has been assigned the FBP/PL-1 gene because it is a member of a multigene family that includes a gene encoding a FBP expressed in both KB cells and placenta and its unprocessed pseudogene.

3T3 Cells↗
PubMed1994

Characterization of the human placental membrane receptor for transcobalamin II-cobalamin.

A specific receptor on the plasma membrane of mammalian cells facilitates the uptake of vitamin B12 (cobalamin, Cbl) by receptor-mediated endocytosis of transcobalamin II-bound Cbl (TCII-Cbl). Purification of this receptor has proven to be difficult because of the lability of the protein during solubilization. Using human placental membranes as the source of the receptor, we have investigated alternative methods for solubilization of this protein and characterized a number of functional and structural properties. Homogenized and washed placental membranes show specific, saturable binding of TCII-Cbl with a Ka of 0.26 nM-1. Following solubilization of the membranes in 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), the most efficient nonionic detergent tested, 21% of the receptor activity remained with the residual insoluble membrane fraction, a property of membrane proteins that are bound tightly to the cell cytoskeleton. Whereas 10 mM Chapso removed 79% of the receptor activity from the membrane preparation, only 3.7% of the TCII-Cbl binding activity was recovered in the solubilized fraction. The unstable TCII-Cbl binding in the soluble fraction was protected by the addition of 15% glycerol to the preparation and storage at -20 degrees C. The apparent M(r) of the receptor estimated by SDS-PAGE of the crosslinked receptor 125I-TCII-Cbl is approximately 58,000. The decrease in M(r) following digestion with several glycosidases and neuraminidase indicates that approximately 29% of the protein is carbohydrate which accounts for a core polypeptide of 41 kDa. Selective binding to a battery of lectins has established that the carbohydrate moiety of the receptor contains a large proportion of N-acetylglucosamine and terminal X-linked mannose.

Cell Membrane↗
PubMed1993

Functional human transcobalamin II isoproteins are secreted by insect cells using the baculovirus expression system.

Transcobalamin II (TCII) is a cobalamin (Cbl, vitamin B12)-binding protein in mammalian plasma that facilitates the cellular uptake of the vitamin. To obtain human TCII in sufficient quantity for analytical studies, the complementary DNA (cDNA) encoding TCII was inserted into the plasmid PVL 1393, and the baculovirus expressing TCII was obtained by homologous recombination in Spodoptera frugiperda (SF9) insect cells by cotransfection with the wildtype virus. Under optimized conditions, SF9 cells infected with the recombinant virus secreted 2 to 4 micrograms of TCII per milliliter of culture medium. TCII did not accumulate in the SF9 cells and seemed to be constitutively secreted as observed previously in cultured human endothelial cells. The purified recombinant TCII has the same molecular weight by SDS-PAGE as purified human TCII. The recombinant TCII cross-reacts with an antiserum to native human TCII, binds Cbl and facilitates the uptake of Cbl in eukaryotic cells by binding to the receptor for TCII-Cbl on the plasma membrane of K562 cells. Amino acid sequence analysis of the purified recombinant TCII identified two polypeptides, one identical to the amino acid sequence deduced from the cDNA and a second lacking the first and second N-terminal residues. These sequences are identical to two TCII polypeptides purified from Cohn fraction III of pooled human plasma. The two forms of recombinant TCII have the same isoelectric points as the two predominant isoprotein forms of TCII in human serum. Since the baculovirus construct contains a single cDNA that can encode only one amino acid sequence, the two isoproteins in recombinant TCII must be generated by a mechanism other than allele specific expression. A plausible mechanism for generating isoproteins of nonglycosylated peptides, such as TCII, may be by splicing of the leader peptide at alternative sites.

Amino Acid Sequence↗
PubMed1992

Genomic organization of the gene and a related pseudogene for a human folate binding protein.

An unprocessed pseudogene which is 90% homologous with the cDNA encoding a folate binding protein in KB cells has been cloned from a human genomic library. This pseudogene contains TGA stop codons, base deletions and substitutions and lacks a 5' region. The size of the exons and the intron-exon sites are almost identical to the organization of the gene encoding this protein which has now been characterized from genomic DNA using the polymerase chain reaction with selected primers to the cDNA.

Amino Acid Sequence↗
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↗