Biomedical subjects

B Seetharam

Publications and source records attributed to B Seetharam.

At least 55 records · Page 3Linked to original sources

PubMed1992

Intrinsic factor receptor activity and cobalamin transport in bile duct-ligated rats.

The intrinsic factor (IF)-cobalamin (Cbl) receptor activity in the mucosal homogenates progressively decreased after bile duct ligation in the rat, and 80% of the receptor activity was decreased in 96 h after ligation. The activity was restored to normal values of 5.5-6 pmol of IF-[57Co]Cbl bound/g mucosa when the assays were performed with both conjugated and unconjugated bile acids. When [57Co]Cbl bound to intrinsic factor was orally administered, the tissue levels of [57Co]Cbl were decreased by 75-80% in bile duct-ligated rats. The apical membrane receptor activity was also decreased after bile duct ligation; however, the activity was stimulated twofold by the addition of ileal cytosol and threefold with the addition of both ileal cytosol and taurocholate (1 mM). Enhanced binding of IF-[57Co]Cbl to the apical ileal brush-border membrane occurred with the use of dialyzed ileal cytosol but not with cytosol isolated from duodenal or proximal jejunal mucosa. The enhanced binding obtained with ileal cytosol was abolished after its treatment with trypsin. These results suggest that luminal bile acids optimize the binding of IF-Cbl by the ileal membrane receptor via interactions with a cytosolic factor and thus influence the gastrointestinal absorption of cobalamin.

Animals↗

PubMed1991

Functional expression of transcobalamin II cDNA in Xenopus laevis oocytes.

The products of in vitro transcription of human transcobalamin II (TC II) cDNA when microinjected into Xenopus laevis oocytes yielded a single secretory protein of 43 kDa. The mobility of the 43 kDa band did not change following digestion with peptide N-glycosidase F. [57Co]Cbl bound to the medium was immunoprecipitated with anti-serum to human TC II, but not to other Cbl binders. In addition, the [57Co]Cbl complex also bound to placental microsomes. These results suggest that TC II mRNA transcribed encodes TC II which contains both the Cbl and receptor binding domains. Furthermore, Xenopus oocytes can be used as a screening system to define structural elements important in TC II's secretion and binding reactions.

Animals↗

PubMed1991

Synthesis and secretion of cobalamin binding proteins by opossum kidney cells.

Opossum kidney epithelial cells synthesize and secrete two Cobalamin (Cbl) binding proteins of Mr 66,000 and 43,000. When grown on culture inserts, the apical medium contained both these proteins while the basolateral medium contained only the 43 kDa Cbl binder. Colchicine, a microtubule disruptive drug, increased two fold the apical but not the basolateral secretion of the Cbl binding proteins. Although the opossum Cbl binders did not cross react with anti-serum raised to Cbl binders from other species, the identity based on Cbl binding and size suggest that the 66 kDa and 43 kDa proteins are haptocorrin and transcobalamin II.

Animals↗

PubMed1991

Functional expression of intrinsic factor-cobalamin receptor by renal proximal tubular epithelial cells.

Previous studies from our laboratory (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489-4494) have identified and isolated a 230-kDa receptor from rat and canine kidney which binds with high affinity [57Co]cyanocobalamin (Cbl) complexed to gastric intrinsic factor (IF). Although these studies have identified a renal receptor which binds intrinsic factor-cobalamin (IFCR), it is not known whether the binding is specific for IF-Cbl and whether renal cells internalize [57Co]Cbl bound to IF and transport [57Co]Cbl across the cell. Using a variety of renal cells, our results show that IF-[57Co]Cbl binding activity is detected in proximal tubular-derived epithelial cells from opossum (OK) and porcine kidney (LLC-PK1) but not in distal tubular-derived cells from canine kidney cells (MDCK). Metabolic labeling studies with Tran 35S-label confirmed the presence of a 230-kDa IFCR in OK and LLC-PK1 cells. Cell surface labeling and binding studies demonstrated that IFCR is targeted to the apical membrane. This apical expression of IFCR in OK cells is inhibited by the microtubule-disruptive drugs, colchicine and nocodazole. Opossum kidney cells when grown on culture inserts are polarized and transport [57Co]Cbl only when bound to IF and not to other Cbl binders. Furthermore, the transport of [57Co]Cbl occurred unidirectionally from the apical to the basolateral surface. Treatment of cells with colchicine or nocodazole inhibited the surface binding of IF-[57Co]Cbl as well as the transcytosis of [57Co]Cbl by 70-75%. IFCR retained intracellualarly by incubation of cells with colchicine or nocodazole is degraded by leupeptin-sensitive proteases. Based on these results, we suggest that proximal tubular-derived epithelial cells transport [57Co]Cbl bound to IF in a saturable way via receptor-mediated endocytosis.

Animals↗

PubMed1991

In vitro translation and expression of renal intrinsic factor-cobalamin receptor.

The primary translation product of intrinsic factor (IF)-cobalamin receptor (IFCR) mRNA from rat kidney is a single polypeptide chain of Mr = 215,000. When expressed in Xenopus laevis oocytes the IFCR binding activity is expressed with mRNA of a size between 5 to 7 kb. These results suggest that IFCR mRNA transcripts are present in the renal tissue and encode a single chain, large molecular weight precursor. Furthermore, Xenopus oocytes can be used as a screening system in the expression cloning of the renal IFCR.

Animals↗

PubMed1991

Defective brush-border expression of intrinsic factor-cobalamin receptor in canine inherited intestinal cobalamin malabsorption.

Ligand binding activity of intrinsic factor-cobalamin receptor (IFCR) was determined in homogenates and isolated brush-border membranes (BBM) of ileum and kidney from dogs exhibiting simple autosomal recessive inheritance of selective cobalamin malabsorption (Fyfe, J. C., Giger, U., Hall, C. A., Jezyk, P. F., Klumpp, S. A., Levine, J. S., and Patterson, D. F. (1991) Pediatr. Res. 29, 24-31). IFCR activity of affected dog ileal homogenates was 3-4-fold higher than normal whereas IFCR activity in affected dog kidney homogenates was one-tenth of normal. The recovery of IFCR activity in the BBM of ileum and renal cortex of affected dogs was 30- and 20-fold less than normal, respectively. The dissociation constant (Kd) for intrinsic factor-cobalamin was similar in BBM of both tissues and was the same in affected and normal dogs. In the affected dog ileal BBM, activities of alkaline phosphatase and sucrase-isomaltase and vesicular transport of glucose and Na(+)-taurocholate were normal. Immunoblots showed no IFCR cross-reactive material in the ileal or renal BBM of affected dogs. IFCR purified by affinity chromatography from kidney of both normal and affected dogs had an Mr = 230,000. However, amino acid analysis revealed that the affected dog IFCR had more lysine than the normal, and protease cleavage of the purified IFCRs revealed different peptide maps. Asparagine-linked oligosaccharides of both proteins were sensitive to peptide N-glycosidase F cleavage, but only the affected dog IFCR was endoglycosidase H sensitive. These results suggest that cobalamin malabsorption in this canine family is caused by inefficient BBM expression of IFCR due to a mutation of IFCR and its retention in an early biosynthetic compartment.

Alkaline Phosphatase↗

PubMed1991

Expression of cobalamin transport proteins and cobalamin transcytosis by colon adenocarcinoma cells.

Human colon adenocarcinoma (Caco-2) cells express both intrinsic factor-cobalamin receptor and transcobalamin II (TC II). The expression of these activities began to rise by day 6 and reached peak levels between 10 and 15 days in culture. The postconfluent Caco-2 cell membranes bound approximately 30-35 fmol of intrinsic factor (IF) [57Co]Cbl/mg protein. The size of the mature receptor expressed in the apical brush border had a relative molecular mass of 230 kDa. The intracellular form of TC II had a Mr of 43, 5 higher than the secreted form of TC II. TC II was secreted unidirectionally via the basolateral direction when Caco-2 cells were grown on culture inserts. When grown on culture inserts, the Caco-2 cells were polarized (electrical resistance greater than 200 omega/cm2) and transcytosed [57Co]Cbl bound to IF from apical-to-basal but not from basal-to-apical direction. Under these conditions, [57Co]Cbl complexed to haptocorrin was not transported. These cells also transcytosed free [57Co]Cbl, although less efficiently. The [57Co]Cbl transcytosed using either IF[57Co]Cbl or free [57Co]Cbl as ligands was bound exclusively to TC II. Intracellular [57Co]Cbl decreased during transcytosis with a slow (t1/2 = 4 h) transfer of [57Co]Cbl from IF to TC II. These results show that the transport of Cbl in Caco-2 cells is very similar to the human enterocyte system.

Adenocarcinoma↗

PubMed1990

Renal brush border membrane bound intrinsic factor.

A highly active receptor for intrinsic factor (IF)-cobalamin (Cbl) complex has been detected and reported in mammalian kidney earlier (Seetharam, B., et al. (1988) J. Biol. Chem. 263, 4443-4449). The physiological role of this receptor in normal Cbl homeostasis is not known. In addition to binding of exogenously added IF-[57Co]Cbl, the renal apical membranes contain endogenous IF or IF-Cbl. Washing with pH 5/EDTA buffer enhanced the binding of exogenously added IF-[57Co]Cbl to renal apical but not basolateral membranes. The pH 5/EDTA extract from renal apical membranes bound [57Co]Cbl. The complex also bound to rat ileal brush border membrane and promoted ileal transport of [57Co]Cbl. On immunoblots using monospecific antiserum to IF a 62 kDa protein was identified in renal and intestinal apical membranes, serum and in tissue extracts of unperfused rat liver, kidney and heart. The 62 kDa band was eliminated from the renal apical membranes following pH 5/EDTA wash. Rat urine demonstrated unsaturated [57Co]Cbl binding (0.2 to 0.4 pmol/day) of which only 30-40% was immunoprecipitated with anti IF and could be identified on immunoblots. The identification of IF in rat renal apical membranes (160-200 ng/mg protein) and secretion of only traces of IF in urine suggest that the renal IF-Cbl receptor may play a role in sequestering IF/IF-Cbl and prevent urinary loss of Cbl.

Animals↗

PubMed1990

Isolation and characterization of a cDNA encoding porcine gastric haptocorrin.

A cDNA encoding the gastric haptocorrin was isolated from a porcine gastric mucosal lambda gt11 cDNA library using oligonucleotide probes. The 1.4-kb cDNA contains a 1.25-kb open reading frame and 178 nucleotides of 3' noncoding region. Although no initiator methionine is present, primer extension analysis indicated that the transcription initiation site is only 100 bp upstream of the 5' end of this clone. Northern blot analysis showed that a single mRNA species of 1.6 kb exists in hog gastric mucosa. There was no cross-hybridization between this cDNA and the mRNA for haptocorrin in rat submaxillary gland or gastric RNA by Northern blot analysis. This lack of cross-reactivity was also seen on Southern blots, where cow, sheep and dog but neither rat nor mouse genomic DNAs showed cross-hybridizing bands. Comparison of the deduced amino acid sequence of this cDNA with that previously reported for rat intrinsic factor [Dieckgraefe, B.K. et al. (1988) Proc. Natl Acad. Sci. USA 85, 46-50] showed considerable similarity, suggesting that these cobalamin-binding proteins may have a common evolutionary origin.

Amino Acid Sequence↗

PubMed1990

Effect of lectins on the cobalamin-protein binding reactions: implications for the tissue uptake of cobalamin.

Plant lectins have been thought to impair nutrient absorption, both by specific and nonspecific interference in the absorptive process. The main objective of this investigation was to study the effect of lectins on the various binding reactions involving cobalamin (cbl)-protein complexes and their receptors, and to identify the rate-limiting step important in maintaining tissue levels of cobalamin. Among the lectins tested in vivo, only concanavalin A (ConA) was able to inhibit the transport of cobalamin to the tissues and caused a 70% to 75% inhibition of [(57)Co] cobalamin transported to the liver and kidney. The inhibition of transport to the tissues was independent of route of administration of cobalamin, whether intragastric or systemic, and was not due to decreased gastrointestinal uptake. When tested in vitro, concanavalin A inhibited the binding of transcobalamin II-cbl to its receptor, but not the binding of cobalamin to intrinsic factor or intrinsic factor-cobalamin complex to the ileal receptor. These results suggest that late events during transcellular transport of cobalamin through the enterocytes is the rate-limiting step determining tissue levels of cobalamin and that ConA inhibits these latter events.

Journal Article↗

PubMed1989

Effects of dietary fish oil supplementation on membrane fluidity and enzyme activity in rat small intestine.

Rats were fed either a fat-free diet supplemented with 10% menhaden oil or a control diet for four months. Intestinal brush border membranes were isolated; phospholipid fatty acid analysis revealed that the membranes from the fish-oil fed animals had higher levels of palmitoleic (C16:1) and eicosapentaenoic (C20:5) acids and lesser levels of stearic (C18:0) linoleic (C18:2) acids compared with controls. The membranes from the fish-oil fed animals had increased levels of alkaline phosphatase activity compared with controls but disaccharidase levels were equivalent in the two groups. Rocket immunoelectrophoresis studies revealed that the increase in alkaline phosphatase activity was due to an increase in the specific activity of the enzyme rather than an increase in the amount of enzyme. Membrane fluidity was assessed by fluorescence anisotropy using diphenylhexatriene and 12-anthroyl stearate as fluorescent probes. The anisotropy of both probes was similar in the two membranes. These studies indicate that fish-oil supplementation alters the fatty acid composition of the intestinal brush border membrane and increases alkaline phosphatase activity without affecting membrane fluidity. Thus the effects of changes in membrane lipid composition on alkaline phosphatase activity appear to result from changes in the local lipid environment of the enzyme rather than from changes in the biophysical characteristics of the membrane.

Alkaline Phosphatase↗

PubMed1989

Immunohistochemical survey of cobalamin-binding proteins.

Dietary cobalamin is bound to intrinsic factor (IF) and nonintrinsic factor (R protein) in the gastrointestinal tract. Distribution of IF has previously been described only in the stomach (parietal, chief cells), duodenum (mucous cells), and pancreatic juice. Cells containing R protein have been reported to be more widespread, including mucin-secreting cells of the intestinal tract. To further clarify the cells producing these cobalamin binders in the various organs, we surveyed a number of tissues in four species, with monospecific antisera raised against rat IF and hog R protein, using indirect immunoperoxidase histochemistry. A positive staining pattern was confirmed after blocking with the appropriate protein purified to homogeneity. Strong IF staining was noted over chief cells in the rat stomach, with weaker but substantial staining in the parietal cells as well. Intrinsic factor was found only in parietal cells in all other species examined. R protein was found in parietal cells of all species tested, except in the hog, where mucous cells of the basal crypt stained. The area of the brush border and apical cytoplasm in small intestinal epithelial cells was also positive for both IF and R protein, probably reflecting uptake. Accessory digestive organs, such as salivary gland and pancreas were also positive for both IF and R protein in the secretory ducts. Accessory digestive organs contribute to the secretion of IF as well as R protein, but the physiologic importance of this IF secretion is not clear.

Animals↗

PubMed1989

Cobalamin release from intrinsic factor and transfer to transcobalamin II within the rat enterocyte.

To ascertain the mechanism of release of cobalamin (Cbl) from intrinsic factor (IF) and subsequent formation of transcobalamin II (TC-II)-Cbl complex, we studied the intracellular distribution of 57Co-labeled Cbl after its uptake in suckling and adult rats. The amount of Cbl bound to IF, to the IF-Cbl receptor via IF, and to TC-II was determined by immunoprecipitation with monospecific antisera raised to these proteins. IF-Cbl receptor activity was found to be very low in suckling rats up to 12 days after birth. Oral administration of leupeptin in amounts known to alter protein turnover had no effect on the release of Cbl from IF nor did it inhibit the formation of the TC-II-Cbl complex in either adult or suckling animals. However, oral administration of chloroquine resulted in a transient increase in the intestinal concentration of Cbl in both adult and suckling rats and in total inhibition of Cbl released from IF in adults rats. Chloroquine prevented completely the transfer of Cbl to TC-II in adult rats and inhibited the transfer by 50% in suckling rats. These data demonstrate that in adult mucosa utilizing receptor-mediated endocytosis, Cbl is transferred from IF to TC-II. This transfer does not require the IF-Cbl receptor, as it occurs in suckling rats. Finally, transfer of Cbl to TC-II is decreased by a drug that alters vesicular pH. Because Cbl can be released at acid pH from IF, it is proposed that release of Cbl from IF and its transfer to TC-II occurs in an acidic vesicle.

Administration, Oral↗

PubMed1988

Purification, properties, and immunochemical localization of a receptor for intrinsic factor-cobalamin complex in the rat kidney.

High levels of receptor for intrinsic factor-cobalamin (vitamin B12) were detected in human, canine, and rat kidneys. The ratio of specific activity (picomoles/mg of protein) for kidney relative to intestine was 116, 20, and 797, respectively, in these species. The receptor was purified about 3000-fold from 200 g of rat kidney with a recovery of 16% and exhibited a single band on nondenaturing gel electrophoresis. Quantitative amino acid analysis of the receptor gave a value of 457,310 g of amino acid/mol of intrinsic factor-cobalamin binding activity. The pure receptor revealed an Mr of 430,000, as assessed by filtration with Bio-Gel A-5m. Treatment with papain resulted in the production of active monomers of Mr to about 205,000-210,000. Electrophoresis in the presence of sodium dodecyl sulfate confirmed the monomer Mr to be 230,000. The monomer receptor did not reveal the presence of any further subunits upon reductive alkylation. Following cyanogen bromide cleavage the kidney receptor revealed three peptides of Mr 115,000, 60,000, and 54,000. The pI of these peptides was 5.17, 6.17, and 6.17, respectively. Western blot analysis using antiserum raised to the receptor demonstrated a protein with an Mr of 175,000 and 230,000 for intestinal and kidney membrane receptors, respectively. Immunologically, the rat kidney receptor was identical to the rat ileal receptor but was distinct from the canine ileal receptor. Ultrastructural localization revealed the presence of the receptor in the apical surface membrane of proximal tubular cells of the kidney and absorptive cells of the ileum. The kidney is the best source for obtaining this receptor in reasonable quantities.

Amino Acids↗

PubMed1988

Isolation and structural characterization of a cDNA clone encoding rat gastric intrinsic factor.

Rat intrinsic factor (IF) has been purified and proteolytic fragments were sequenced. A cDNA library was constructed from size-enriched gastric poly(A)+ RNA and screened for IF-positive clones by antibody and synthetic oligodeoxynucleotide probe hybridization. An IF clone was isolated and sequenced, revealing a predicted primary amino acid sequence in the coding region of 421 amino acids and a putative signal sequence of 22 amino acids. The primary translation product of IF produced in a cell-free translation system displayed cobalamin (Cbl)-binding activity without proteolytic processing or glycosylation. The amino-terminal region of IF showed significant secondary structural and hydropathic homologies with the nucleotide-binding domain in NAD-dependent oxidoreductases. Alignment of the first 80 residues of IF, following the signal peptide, demonstrated homology with the nucleotide-binding domain of cytoplasmic malate dehydrogenase. Based on these data, we propose a model of IF tertiary structure in which the Cbl-binding domain resides in the NH2-terminal half of the protein.

Amino Acid Sequence↗

PubMed1988

Developmental regulation of rat intrinsic factor mRNA.

Using a full-length rat intrinsic factor (IF) cDNA clone, we have examined gene expression of IF in selected tissues from the adult rat and during postnatal development in the rat stomach. IF mRNA was expressed only in the rat stomach, and in situ hybridization revealed that the expression was limited to the chief cells at the base of the oxyntic glands. IF mRNA concentration in the stomach increased with postnatal age and reached a peak at the 30th day. Postnatally the amount of IF mRNA hybridized increased in both individual cells and in total number of cells. Administration of cortisol to, or adrenalectomy of, rats between 5 and 12 days of age resulted in increase or decrease of IF mRNA levels, respectively. Other hormones tested (pentagastrin, thyroxine, and triiodothyronine) had no significant effect. During the suckling period, IF bound well to the ileal receptor, despite lower than adult levels of activity and different glycosylation, as assessed by binding to concanavalin A-Sepharose beads. These results show that developmental expression of IF activity in rat is closely related to the expression of its mRNA and that corticosteroids play an important role in such an expression.

Adrenalectomy↗

PubMed1987

The role of choline on the activity-temperature relationship of brush-border alkaline phosphatase.

We have studied the effect of choline on the activity and temperature dependency of the brush-border alkaline phosphatase isoenzymes from rat intestine (tissue-specific type), and from kidney and placenta (tissue-nonspecific type). The removal of choline with phospholipase D resulted in the loss of enzyme activity in all the membranes, whereas in situ loss in the discontinuity of Arrhenius plots occurred in the kidney and the placental membranes, but not in the intestinal membranes. The lost activity was restored either by addition of free choline or phosphatidylcholine or by the removal of the enzyme from the membrane surface. Intestinal enzyme was removed by papain, while the tissue-nonspecific enzyme was released by subtilisin and by phosphatidylinositol-specific phospholipase C. The enzyme from kidney and placental membranes aggregated (rho = 1.13) upon removal of choline, and addition of choline resulted in disaggregation (rho = 1.03). Conversion of discontinuous to continuous linear plots of alkaline phosphatase in the kidney and placental membranes paralleled the increase in membrane phosphatidic acid content, and the decrease in total phosphatidylcholines. The intestinal enzyme produced plots with break points at all phosphatidic acid/phosphatidylcholine ratios. The change brought about by treatment with phospholipidase D was not due to changes in the half-saturation kinetics (Km) for the substrate. Based on these studies we conclude that the active site of the tissue-nonspecific phosphatase is approximated to exterior membrane cholines, as in the case of the intestinal isoenzyme; that despite similar effects on the membrane content of phospholipids, phospholipase D treatment caused much greater effects on the tissue-nonspecific enzyme, as assessed by Arrhenius plots and density centrifugation; that these effects are due to different protein structures rather than to a lipid milieu unique to each brush-border membrane.

Alkaline Phosphatase↗

PubMed1987

Hydrophobic interactions of brush border alkaline phosphatases: the role of phosphatidyl inositol.

Tissue-specific (intestinal) and tissue-nonspecific (kidney) rat alkaline phosphatases are released from their respective brush border membranes by different enzymes. To elucidate the mechanism underlying their membrane attachment, we tested the ability of these enzymes to partition into lipid or aqueous phases both before and after treatment with phospholipases and proteases. Interaction with Triton X-114 micelles was eliminated or decreased by treatment of intestinal enzyme with phospholipase A2 or papain, while only phosphatidylinositol (PI)-specific phospholipase C (PIPLC) and subtilisin were effective with the kidney enzyme. Binding to octyl Sepharose for the intestinal enzyme was decreased by phospholipase A2 more than by PIPLC, whereas the reverse was true for the kidney enzyme. Treatment with phospholipases decreased the apparent mass of the phosphatases by 50-80 kDa, presumably due to loss of bound lipid and detergent. PIPLC treatment of the kidney, but not the intestinal enzyme, prevented binding of the phosphatase to phospholipid vesicles. These results show that both enzymes are bound to respective membranes by hydrophobic anchor peptides to which phospholipids are bound. However, their sensitivity to phospholipases is different. The data are consistent with the hypothesis that, in the kidney enzyme, the PI is bound covalently, while with the intestinal enzyme, binding of PI appears to be tight but not covalent.

Alkaline Phosphatase↗