scholarly journals Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis upon binding nucleotide

2020 ◽  
Author(s):  
Tessa R. Young ◽  
Maria Alessandra Martini ◽  
Deenah Osman ◽  
Richard J. Morton ◽  
Evelyne Deery ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Vitamin B ◽  
Free Energies ◽  
High Affinity ◽  
High Affinity Site ◽  
Metal Sensors ◽  
Intracellular Milieu ◽  
In Cells

Protein metal-occupancy (metalation) in vivo has been elusive. Here we develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. The calculations are based on available free-energies of metals determined from the responses of metal sensors. We use the calculator to understand the function and mechanism of CobW, a predicted CoII-chaperone for vitamin B12. CobW is calculated to acquire negligible metal alone: But, upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator reveals how CobW acquires its metal and is made available for use with other proteins.

scholarly journals Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis while related proteins prefer ZnII

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tessa R. Young ◽  
Maria Alessandra Martini ◽  
Andrew W. Foster ◽  
Arthur Glasfeld ◽  
Deenah Osman ◽  
...  
Keyword(s):  
Free Energy ◽  
Vitamin B12 ◽  
Vitamin B ◽  
Free Energies ◽  
High Affinity ◽  
High Affinity Site ◽  
Metal Sensors ◽  
Related Proteins ◽  
Intracellular Milieu

AbstractProtein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.

scholarly journals Roles of Two ATPase-Motif-containing Domains in Cyanobacterial Circadian Clock Protein KaiC

2004 ◽  
Vol 279 (50) ◽  
pp. 52331-52337 ◽  
Author(s):  
Fumio Hayashi ◽  
Noriyo Itoh ◽  
Tatsuya Uzumaki ◽  
Ryo Iwase ◽  
Yuka Tsuchiya ◽  
...  
Keyword(s):  
Reaction Mechanism ◽  
Circadian Clock ◽  
Binding Sites ◽  
Wild Type ◽  
High Affinity ◽  
High Affinity Site ◽  
Clock Protein

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing thein vitroactivity of ATPγS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and byin vivorhythm assays both in wild type KaiC (KaiCWT) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.

The 22.5kDa spectrin-binding domain of ankyrinR binds spectrin with high affinity and changes the spectrin distribution in cells in vivo

2008 ◽  
Vol 60 (2) ◽  
pp. 157-164 ◽  
Author(s):  
Adam Kolondra ◽  
Michal Grzybek ◽  
Anna Chorzalska ◽  
Aleksander F. Sikorski
Keyword(s):  
Binding Domain ◽  
High Affinity ◽  
In Cells

Heterogeneity of Hepatic Vitamin B12 in the Rat after Parenteral Cyanocobalamin

1975 ◽  
Vol 49 (3) ◽  
pp. 257-264
Author(s):  
W. G. E. Cooksley ◽  
A. S. Tavill
Keyword(s):  
Vitamin B12 ◽  
Rat Liver ◽  
Intramuscular Injection ◽  
Isolated Perfused Rat Liver ◽  
Vitamin B ◽  
Double Labelling ◽  
Time Intervals ◽  
Rapid Release ◽  
Biphasic Release

1. The rate of radioactive vitamin B12 excretion into plasma and bile from the isolated perfused rat liver and into bile in vivo has been measured after the intramuscular injection of radioactive cyanocobalamin at various time-intervals before study. 2. With an interval of labelling of 10 or more days, the release of radioactive vitamin B12 over 4 h by the isolated perfused liver was linear and con-stituted approximately 35% and 1% of the hepatic radioactivity into plasma and bile respectively. 3. In contrast, after a shorter period of prelabelling (less than 7 days), there was a biphasic release of radioactive vitamin Blz: an initial rapid rate followed by a slower rate after about 1 h of perfusion. The total radioactive vitamin Blz was considerably increased (e.g. 25% and 5% of hepatic radioactivity into plasma and bile respectively during a 4 h perfusion of a liver labelled 18 h previously). 4. Confirmation of coexisting stable and labile pools of intrahepatic vitamin B12 was provided by: (a) the patterns of hepatic release ifter double labelling with 57Co-labelled and 58Co-labelled cyanocobalamin at different times before liver per-fusion; (b) the rates of biliary excretion of 57Co-labelled and 58Co-labelled vitamin B12 in vivo after different periods of prelabelling. 5. The rate and pattern of release were not altered by changes in the quantity of precursor cyanocobala-min, by phenobarbitone treatment or by the addition of cycloheximide to the perfusion. 6. The injection into rats of subcellular preparations of rat liver labelled in viuo with radioactive vitamin B12 demonstrated that hepatic heterogeneity did not depend on physical compartmentation. 7. Despite the rapid release rate from the liver recently administered radioactive cyanocobalamin, the hepatic radioactivity increased progressively with time after labelling in vivo, in contrast to the other tissues, where it decreased. In the presence of rapid bidirectional fluxes the ability of the liver to store vitamin B can be largely explained by the reduction in the rate of hepatic release that continues for about 10 days after parenteral administration of cyano-cobalamin.

scholarly journals In vivo PET Imaging of [11C]CIMBI-5, a 5-HT2AR Agonist Radiotracer in Nonhuman Primates

2019 ◽  
Vol 22 ◽  
pp. 352-364
Author(s):  
Jaya Prabhakaran ◽  
Christine DeLorenzo ◽  
Francesca Zanderigo ◽  
Gitte M Knudsen ◽  
Nic Gilling ◽  
...  
Keyword(s):  
Pet Imaging ◽  
Regional Distribution ◽  
Brain Regions ◽  
Binding Potential ◽  
Vervet Monkeys ◽  
Reference Tissue ◽  
High Affinity ◽  
Logan Plot ◽  
High Affinity Site

Purpose: 5-HT2AR exists in high and low affinity states. Agonist PET tracers measure binding to the active high affinity site and thus provide a functionally relevant measure of the receptor. Limited in vivo data have been reported so far for a comparison of agonist versus antagonist tracers for 5-HT2AR used as a proof of principle for measurement of high and low affinity states of this receptor. We compared the in vivo binding of [11C]CIMBI-5, a 5-HT2AR agonist, and of the antagonist [11C]M100907, in monkeys and baboons. Methods: [11C]CIMBI-5 and [11C]M100907 baseline PET scans were performed in anesthetized male baboons (n=2) and male vervet monkeys (n=2) with an ECAT EXACT HR+ and GE 64-slice PET/CT Discovery VCT scanners. Blocking studies were performed in vervet monkeys by pretreatment with MDL100907 (0.5 mg/kg, i.v.) 60 minutes prior to the scan. Regional distribution volumes and binding potentials were calculated for each ROI using the likelihood estimation in graphical analysis and Logan plot, with either plasma input function or reference region as input, and simplified reference tissue model approaches. Results: PET imaging of [11C]CIMBI-5 in baboons and monkeys showed the highest binding in 5-HT2AR-rich cortical regions, while the lowest binding was observed in cerebellum, consistent with the expected distribution of 5-HT2AR. Very low free fractions and rapid metabolism were observed for [11C]CIMBI-5 in baboon plasma. Binding potential values for [11C]CIMBI-5 were 25-33% lower than those for [11C]MDL100907 in the considered brain regions. Conclusion: The lower binding potential of [11C]CIMBI-5 in comparison to [11C]MDL100907 is likely due to the preferential binding of the former to the high affinity site in vivo in contrast to the antagonist,  [11C]MDL100907, which binds to both high and low affinity sites.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

Bioavailability of Vitamin B12

2010 ◽  
Vol 80 (45) ◽  
pp. 330-335 ◽  
Author(s):  
Lindsay Helen Allen
Keyword(s):  
Vitamin B12 ◽  
Vitamin B12 Deficiency ◽  
The Elderly ◽  
Radioactive Isotopes ◽  
Animal Source Foods ◽  
Dietary Requirements ◽  
In Vivo Labeling ◽  
B12 Deficiency ◽  
Vitamin B12 Malabsorption

Vitamin B12 deficiency is common in people of all ages who consume a low intake of animal-source foods, including populations in developing countries. It is also prevalent among the elderly, even in wealthier countries, due to their malabsorption of B12 from food. Several methods have been applied to diagnose vitamin B12 malabsorption, including Schilling’s test, which is now used rarely, but these do not quantify percent bioavailability. Most of the information on B12 bioavailability from foods was collected 40 to 50 years ago, using radioactive isotopes of cobalt to label the corrinoid ring. The data are sparse, and the level of radioactivity required for in vivo labeling of animal tissues can be prohibitive. A newer method under development uses a low dose of radioactivity as 14C-labeled B12, with measurement of the isotope excreted in urine and feces by accelerator mass spectrometry. This test has revealed that the unabsorbed vitamin is degraded in the intestine. The percent bioavailability is inversely proportional to the dose consumed due to saturation of the active absorption process, even within the range of usual intake from foods. This has important implications for the assessment and interpretation of bioavailability values, setting dietary requirements, and interpreting relationships between intake and status of the vitamin.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Biologic Aspects of Expression of Stably Integrated Transgenes in Cells of the Skin In Vitro and In Vivo

1999 ◽  
Vol 111 (3) ◽  
pp. 198-205 ◽  
Author(s):  
Gerald G. Krueger ◽  
Jeffery R. Morgan ◽  
Marta J. Petersen
Keyword(s):  
In Cells

A High-Affinity Human Antibody That Targets Tumoral Blood Vessels

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 192-198 ◽  
Author(s):  
Lorenzo Tarli ◽  
Enrica Balza ◽  
Francesca Viti ◽  
Laura Borsi ◽  
Patrizia Castellani ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Small Cell Lung Carcinoma ◽  
Antibody Fragment ◽  
Human Colon ◽  
Experimental Models ◽  
Human Antibody ◽  
Cell Lung Carcinoma ◽  
High Affinity ◽  
Biodistribution Studies

Angiogenesis is a characteristic feature of many aggressive tumors and of other relevant disorders. Molecules capable of specifically binding to new-forming blood vessels, but not to mature vessels, could be used as selective vehicles and would, therefore, open diagnostic and therapeutic opportunities. We have studied the distribution of the ED-B oncofetal domain of fibronectin, a marker of angiogenesis, in four different tumor animal models: the F9 murine teratocarcinoma, SKMEL-28 human melanoma, N592 human small cell lung carcinoma, and C51 human colon carcinoma. In all of these experimental models we observed accumulation of the fibronectin isoform containing the ED-B domain around neovascular structures when the tumors were in the exponentially growing phase, but not in the slow-growing phase. Then we performed biodistribution studies in mice bearing a subcutaneously implanted F9 murine teratocarcinoma, using a high-affinity human antibody fragment (L19) directed against the ED-B domain of fibronectin. Radiolabeled L19, but not an irrelevant anti-lysozyme antibody fragment (D1.3), efficiently localizes in the tumoral vessels. The maximal dose of L19 accumulated in the tumor was observed 3 hours after injection (8.2% injected dose per gram). By virtue of the rapid clearance of the antibody fragment from the circulation, tumor-to-blood ratios of 1.9, 3.7, and 11.8 were obtained at 3, 5, and 24 hours, respectively. The tumor-targeting performance of L19 was not dose-dependent in the 0.7 to 10 μg range of injected antibody. The integral of the radioactivity localized in tumoral vessels over 24 hours was greater than 70-fold higher than the integral of the radioactivity in blood over the same time period, normalized per gram of tissue or fluid. These findings quantitatively show that new-forming blood vessels can selectively be targeted in vivo using specific antibodies, and suggest that L19 may be of clinical utility for the immunoscintigraphic detection of angiogenesis in patients.

Sign in / Sign up

Export Citation Format

Share Document