scholarly journals Solubilization of the ileal receptor for intrinsic factor–vitamin B-12 complex by digestion with papain

1981 ◽  
Vol 200 (2) ◽  
pp. 225-229
Author(s):  
R C Beesley
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border Membrane ◽  
Intrinsic Factor ◽  
Membrane Vesicles ◽  
Receptor Activity ◽  
Vitamin B ◽  
Isolation And Purification ◽  
Vitamin B 12 ◽  
Soluble Supernatant ◽  
Insoluble Pellet

Brush-border-membrane vesicles isolated from hamster ileum were incubated with either papain or Pronase P and subsequently centrifuged to obtain soluble (supernatant) and insoluble (pellet) fractions. Papain (4 units/ml) solubilized 95--100% of the sucrase and leucine naphthylamide-hydrolysing activities but only 30% of the alkaline phosphatase. Digestion with papain also resulted in the solubilization of more than 75% of the ileal receptor for intrinsic factor-vitamin B-12 complex with a corresponding decrease in receptor activity in the pellet. Essentially 100% of the receptor activity was recovered. In contrast, digestion with Pronase P resulted in a decrease in total receptor activity. Papain-solubilized receptor was not sedimented by centrifugation at 105 000 g for 90 min and was eluted in the included volume of Sepharose 6B. Like the binding to more intact preparations, binding of intrinsic factor-vitamin B-12 complex to papain-solubilized receptor was rapid, reaching 50% of maximum in 8 min, and required Ca2+. Although Mg2+ could not completely substitute for Ca2+, Mg2+ did stimulate Ca2+-dependent binding at low Ca2+ concentrations. These results demonstrate that the ileal receptor for intrinsic factor-vitamin B-12 complex can be solubilized with papain, and suggest that papain solubilization may be a useful first step in the isolation and purification of this receptor.

Divalent cations and vitamin B12 uptake by intestinal brush-border membrane vesicles

1980 ◽  
Vol 239 (6) ◽  
pp. G452-G456
Author(s):  
R. C. Beesley ◽  
C. D. Bacheller
Keyword(s):  
Vitamin B12 ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Divalent Cations ◽  
Intrinsic Factor ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Tetraacetic Acid ◽  
Intestinal Brush Border Membrane ◽  
Intestinal Brush Border

Brush-border membrane vesicles from hamster intestine were employed to investigate uptake (binding) of vitamin B12 (B12). Ileal vesicles took up 25 times more B12 than did jejunal vesicles. Uptake of B12 by ileal vesicles was dependent on intrinsic factor (IF) and required Ca2+. Increasing the Ca2+ concentration caused an increase in uptake of B12 reaching a maximum at approximately 8 mM Ca2+. At high Ca2+ concentrations, 6–8 mM, Mg2+ had little effect on uptake of B12. At low Ca2+ concentrations, up to 2 mM, Mg2+ stimulated B12 uptake. Mg2+, Mn2+, and, to a lesser extent, Sr2+ stimulated Ca2+-dependent B12 uptake, but Zn2+, Ba2+, Na+, K+, and La3+ did not. B12 was apparently not metabolized and was bound as IF-B12 complex, which could be removed with (ethylenedinitrilo)tetraacetic acid (EDTA). Our results suggest that two types of divalent cation reactive sites are involved in binding of IF-B12. One is Ca2+ specific. The other is less specific reacting with Mg2+, Mn2+, Sr2+, and perhaps Ca2+ itself, thereby stimulating Ca2+-dependent binding of IF-B12 to its ileal receptor.

Role of Alkaline Phosphatase in Phosphate Uptake into Brush Border Membrane Vesicles from Human Intestinal Mucosa

1985 ◽  
Vol 97 (5) ◽  
pp. 1461-1466 ◽  
Author(s):  
Kazuyuki HIRANO ◽  
Yuichi IIIZUMI ◽  
Yukio MORI ◽  
Kazumi TOYOSHI ◽  
Mamoru SUGIURA ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Phosphate Uptake ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles

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

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.

scholarly journals Alkaline phosphatase activity does not mediate phosphate transport in the renal-cortical brush-border membrane

1980 ◽  
Vol 190 (2) ◽  
pp. 473-476 ◽  
Author(s):  
H S Tenenhouse ◽  
C R Scriver ◽  
E J Vizel
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Mouse Kidney ◽  
Brush Border Membrane Vesicles ◽  
Low Phosphorus

We studied (1) the effect of primary modulators of phosphate transport, namely the hypophosphataemic mouse mutant (Hyp) and low-phosphorus diet, on alkaline phosphatase activity in mouse renal-cortex brush-border membrane vesicles and (2) the effect of several primary inhibitors of alkaline phosphatase on phosphate transport. Brush-border membrane vesicles from Hyp-mouse kidney had 50% loss of Na+-dependent phosphate transport, but only 18% decrease in alkaline phosphatase activity. The low-phosphorus diet effectively stimulated Na+/phosphate co-transport in brush-border membrane vesicles (+ 118%), but increased alkaline phosphatase activity only slightly (+13%). Levamisole (0.1 mM) and EDTA (1.0 mM) inhibited brush-border membrane-vesicle alkaline phosphatase activity of 82% and 93% respectively, but had no significant effect on Na+/phosphate co-transport. We conclude that alkaline phosphatase does not play a direct role in phosphate transport across the brush-border membrane of mouse kidney.

scholarly journals C-Type Lectin-20 Interacts with ALP1 Receptor to Reduce Cry Toxicity in Aedes aegypti

Toxins ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 390 ◽  
Author(s):  
Khadija Batool ◽  
Intikhab Alam ◽  
Guohui Zhao ◽  
Junxiang Wang ◽  
Jin Xu ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Survival Rate ◽  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Western Blot ◽  
Brush Border Membrane ◽  
Mosquito Control ◽  
Membrane Vesicles ◽  
Cry Toxins ◽  
Far Western Blot

Aedes aegypti is a crucial vector for human diseases, such as yellow fever, dengue, chikungunya, and Zika viruses. Today, a major challenge throughout the globe is the insufficient availability of antiviral drugs and vaccines against arboviruses, and toxins produced by Bacillus thuringiensis (Bt) are still used as biological agents for mosquito control. The use of Cry toxins to kill insects mainly depends on the interaction between Cry toxins and important toxin receptors, such as alkaline phosphatase (ALP). In this study, we investigated the function of A. aegypti C-type lectin-20 (CTL-20) in the tolerance of Cry toxins. We showed that recombinant CTL-20 protein interacted with both Cry11Aa and ALP1 by the Far-Western blot and ELISA methods, and CTL-20 bound to A. aegypti larval brush border membrane vesicles (BBMVs). Binding affinity of CTL-20 to ALP1 was higher than that of Cry11Aa to ALP1. Furthermore, the survival rate of A. aegypti larvae fed with Cry11Aa toxin mixed with recombinant CTL-20 fusion protein was significantly increased compared with that of the control larvae fed with Cry11Aa mixed with thioredoxin. Our novel results suggest that midgut proteins like CTLs may interfere with interactions between Cry toxins and toxin receptors by binding to both Cry toxins and receptors to alter Cry toxicity.

scholarly journals Vitamin B12 absorption in the neonatal piglet

1985 ◽  
Vol 54 (1) ◽  
pp. 269-283 ◽  
Author(s):  
N. M. F. Trugo ◽  
J. E. Ford ◽  
D. N. Salter
Keyword(s):  
Small Intestine ◽  
Binding Sites ◽  
Protective Factor ◽  
Intrinsic Factor ◽  
Membrane Vesicles ◽  
Maximum Activity ◽  
Vitamin B ◽  
Uptake Mechanism ◽  
Ph Optimum ◽  
Lower Efficiency

1. The influence of the vitamin B12-binding protein isolated from sows' milk on the uptake of vitamin B12 was investigated using microvillus membrane vesicles prepared from the small intestine of 7- and 28-d-old piglets. Uptake of radioiodinated purified binder was also examined.2. The binder strongly promoted uptake of vitamin B12 at both ages; in the absence of the binder there was little uptake. The uptake mechanism was specific and operative in vesicles prepared from all regions of the small intestine. Uptake was a rapid process, exhibiting saturation kinetics, with a pH optimum at about 7.0, and dependent on the presence of magnesium or calcium ions for maximum activity. Atfinity constants of the binding sites for the milk binder were determined.3. Competition experiments using complexes of the binder with vitamin B12 and with non-cobalamin analogues (cobinamide and Co-α-[2-methyladenyl]cobamide) showed that the bound analogues competed with bound vitamin B12 for uptake but with lower efficiency.4. Intrinsic factor also promoted vitamin B12 uptake by the vesicles but it did not compete with the milk binder for the same binding sites. It promoted uptake only in microvilli isolated from the lower third of the small intestine, and was more effective with preparations from 28-d-old piglets, whereas the milk binder was more effective with the 7-d-old piglets. Porcine gastric cobalophilin competed with the milk cobalophilin, but with lower efficiency.5. It was concluded that a specific transport mechanism for absorption of vitamin B12, mediated by the vitamin B12-binder in milk, exists at the intestinal brush border of neonatal piglets and strongly reinforces the developing intrinsic factor-mediated mechanism during the early days or weeks of life.6. It is suggested that the binder in the milk has a wider physiological significance and acts also as a ‘host protective’ factor and as a scavenger of adventitious vitamin B12.

Sign in / Sign up

Export Citation Format

Share Document