Release of vitamin B-12 in vitro from intrinsic factor-vitamin B-12 complex Purification and characterization of a releasing factor from rat ileal brush border

Cubilin is a 460-kDa multipurpose, multidomain receptor that contains an NH2-terminal 110-residue segment followed by 8 epidermal growth factor (EGF)-like repeats and a contiguous stretch (representing nearly 88% of its mass) of 27 CUB (initially found in complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domains. Cubilin binds to intrinsic factor (IF)-cobalamin (cbl, vitamin B12) complex and promotes the ileal transport of cbl. The 460-kDa form of cubilin is the predominant form present in the apical brush-border membranes of rat intestine, kidney, and yolk sac, but a 230-kDa form of cubilin is also noted in the intestinal membranes. In thyroidectomized (TDX) rats, levels of intestinal brush-border IF-[57Co]-labeled cbl binding, 460-kDa cubilin protein levels and tissue (kidney) accumulation of cbl were reduced by ∼70%. Immunoblot analysis using cubilin antiserum of intestinal total membranes from TDX rats revealed cubilin fragments with molecular masses of 200 and 300 kDa. Both of these bands, along with the 230-kDa band detected in the total membranes of control rats and unlike the 460-kDa form, failed to react with antiserum to EGF. Mucosal membrane cubilin associated with megalin was reduced from ∼12% in control to ∼4% in TDX rats, and this decreased association was not due to altered megalin levels. Thyroxine treatment of TDX rats resulted in reversal of all of these effects, including an increase to nearly 24% of cubilin associated with megalin. In vitro, megalin binding to cubilin occurred with the NH2-terminal region that contained the EGF-like repeats and CUB domains 1 and 2 but not with a downstream region that contained CUB domains 2–10. These studies indicate that thyroxine deficiency in rats results in decreased uptake and tissue accumulation of cbl caused mainly by destabilization and deficit of cubilin in the intestinal brush border.

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Some species differences in the intrinsic factor stimulation of B12 uptake by small intestine in vitro

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1959.197.4.926 ◽

1959 ◽

Vol 197 (4) ◽

pp. 926-928 ◽

Cited By ~ 21

Author(s):

T. Hastings Wilson ◽

Elliott W. Strauss

Keyword(s):

Small Intestine ◽

Guinea Pig ◽

Species Differences ◽

Intrinsic Factor ◽

Vitamin B ◽

Guinea Pig Ileum ◽

Rat Intestine ◽

Intrinsic Factors ◽

Stimulation Of

Sacs of everted small intestine from a variety of animals were incubated in bicarbonate-saline containing vitamin B12 with and without intrinsic factor (IF). B12 uptake by rat intestine was stimulated only by its own intrinsic factor. Guinea pig ileum responded to all intrinsic factors tested (guinea pig, rat, hog, hamster, human being and rabbit). The intestines of hamster and rabbit were intermediate in specificity, responding to some, but not all, of the IF preparations. Species differences occur in both the intestine and intrinsic factor preparations. The guinea pig ileum was suggested as a possible assay for both hog and human IF.

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Protamine messenger RNA: partial purification and characterization of a heterogeneous family of polyadenylated messenger components

Canadian Journal of Biochemistry ◽

10.1139/o79-115 ◽

1979 ◽

Vol 57 (6) ◽

pp. 945-958 ◽

Cited By ~ 8

Author(s):

Kostas Iatrou ◽

Lashitew Gedamu ◽

Gordon H. Dixon

Keyword(s):

Nucleotide Sequence ◽

Messenger Rna ◽

Rnase H ◽

Partial Purification ◽

Polyacrylamide Gels ◽

Purification And Characterization ◽

Sequence Variations ◽

Duplex Formation

Poly(A)+ protamine mRNA (pmRNA) components were isolated after separation on denaturing preparative polyacrylamide gels. The four size classes of protamine mRNA described previously were found to contain poly(A) tracts of different lengths. The pmRNA1 was found to be associated with (A)110, pmRNA2 with (A)90, pmRNA3 with (A)85, and pmRNA4 with (A)69. Following deadenylation with RNase H after duplex formation with oligo-dT, the isolated mRNAs were found to be still heterogeneous, although highly enriched in certain of the deadenylated components. DNA complementary to the isolated mRNAs (cDNA) was synthesized in vitro. Following depurination, the oligopyrimidine maps indicated that C7T4, corresponding to an Arg-Arg-Gly-Gly sequence in protamine and originally thought to be characteristic of all mRNA components, is present in only one or possibly two of the components. Cross-hybridizations between the cDNAs and the four poly(A)+ pmRNAs indicated that a basic polynucleotide unit of substantial length is common to all four mRNAs and that the existing nucleotide sequence variations probably originate from one or both of the non-coding portions of the mRNA molecules.

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Factors controlling B12 uptake by intestinal sacs in vitro

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1960.198.1.103 ◽

1960 ◽

Vol 198 (1) ◽

pp. 103-107 ◽

Cited By ~ 16

Author(s):

Elliott W. Strauss ◽

T. Hastings Wilson

Keyword(s):

Small Intestine ◽

Low Temperature ◽

Guinea Pig ◽

Calcium Ion ◽

Intrinsic Factor ◽

Intestinal Wall ◽

Vitamin B ◽

Anaerobic Conditions ◽

Intestinal Uptake

Sacs of everted small intestine were incubated in bicarbonate-saline containing radioactive vitamin B12 with or without a source of gastric intrinsic factor (IF). In both the hamster and guinea pig the lowest ileum was most active in B12 uptake in the presence of intrinsic factor, the upper jejunum showing little or no uptake. Low temperature and anaerobic conditions completely abolished the stimulatory effect of IF on B12 uptake. Intrinsic factor did not bind to the intestinal wall in the absence of B12 (even in the presence of calcium ion) as the IF activity could be completely removed by gentle washing of the tissue. The vitamin and intrinsic factor must be present together to cause intestinal uptake of B12.

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Vitamin B-12 and liver activity and expression of methionine synthase are decreased in fetuses with neural tube defects

American Journal of Clinical Nutrition ◽

10.1093/ajcn/nqy340 ◽

2019 ◽

Vol 109 (3) ◽

pp. 674-683

Author(s):

Ma'atem B Fofou-Caillierez ◽

Rosa-Maria Guéant-Rodriguez ◽

Jean-Marc Alberto ◽

Céline Chéry ◽

Thomas Josse ◽

...

Keyword(s):

Cord Blood ◽

Neural Tube ◽

Neural Tube Defects ◽

Intrinsic Factor ◽

Methionine Synthase ◽

Vitamin B ◽

Transcriptional Level ◽

Gene Variant ◽

Methionine Synthase Reductase ◽

Vitamin B 12

ABSTRACT Background The risk of neural tube defects (NTDs) is influenced by nutritional factors and genetic determinants of one-carbon metabolism. A key pathway of this metabolism is the vitamin B-12– and folate-dependent remethylation of homocysteine, which depends on methionine synthase (MS, encoded by MTR), methionine synthase reductase, and methylenetetrahydrofolate reductase. Methionine, the product of this pathway, is the direct precursor of S-adenosylmethionine (SAM), the universal methyl donor needed for epigenetic mechanisms. Objectives This study aimed to evaluate whether the availability of vitamin B-12 and folate and the expression or activity of the target enzymes of the remethylation pathway are involved in NTD risk. Methods We studied folate and vitamin B-12 concentrations and activity, expression, and gene variants of the 3 enzymes in liver from 14 NTD and 16 non-NTD fetuses. We replicated the main findings in cord blood from pregnancies of 41 NTD fetuses compared with 21 fetuses with polymalformations (metabolic and genetic findings) and 375 control pregnancies (genetic findings). Results The tissue concentration of vitamin B-12 (P = 0.003), but not folate, and the activity (P = 0.001), transcriptional level (P = 0.016), and protein expression (P = 0.003) of MS were decreased and the truncated inactive isoforms of MS were increased in NTD livers. SAM was significantly correlated with MS activity and vitamin B-12. A gene variant in exon 1 of GIF (Gastric Intrinsic Factor gene) was associated with a dramatic decrease of liver vitamin B-12 in 2 cases. We confirmed the decreased vitamin B-12 in cord blood from NTD pregnancies. A gene variant of GIF exon 3 was associated with NTD risk. Conclusions The decreased vitamin B-12 in liver and cord blood and decreased expression and activity of MS in liver point out the impaired remethylation pathway as hallmarks associated with NTD risk. We suggest evaluating vitamin B-12 in the nutritional recommendations for prevention of NTD risk beside folate fortification or supplementation.

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