Retinol uptake from retinol-binding protein (RBP) by liver parenchymal cells in vitro does not specifically depend on its binding to RBP

Biochemistry ◽  
1993 ◽  
Vol 32 (7) ◽  
pp. 1727-1733 ◽  
Author(s):  
Ariette M. Van Bennekum ◽  
William S. Blaner ◽  
Ingrid Seifert-Bock ◽  
Maria Moukides ◽  
Adriaan Brouwer ◽  
...  
Keyword(s):  
Binding Protein ◽  
Retinol Binding Protein ◽  
Liver Parenchymal ◽  
Parenchymal Cells

scholarly journals Receptor-mediated endocytosis of retinol-binding protein by liver parenchymal cells: interference by radioactive iodination

1993 ◽  
Vol 291 (1) ◽  
pp. 187-191 ◽  
Author(s):  
L Malaba ◽  
G M Kindberg ◽  
K R Norum ◽  
T Berg ◽  
R Blomhoff
Keyword(s):  
Binding Protein ◽  
The Other ◽  
Retinol Binding Protein ◽  
Lag Phase ◽  
Steady Increase ◽  
Release Process ◽  
Liver Parenchymal ◽  
Cell Association ◽  
Higher Temperature ◽  
Parenchymal Cells

Retinol-binding protein (RBP) was iodinated directly by radio-iodine substitution on the tyrosyl residues by the sodium hypochlorite (NaOCl) or the Enzymobead (EB) methods, or indirectly by linkage of 125I-tyramine-cellobiose (TC) or 125I-N-succinimidyl-3-(4- hydroxyphenyl)propionic acid ester (SHPP) adduct on to free amino residues of RBP. Binding, uptake and degradation of iodinated RBP were studied in isolated rat and rabbit liver parenchymal cells. The amount of ligand bound to cells at 4 degrees C was dependent on the type of labelling, in that the 125I-TC ligand was bound to a lesser extent than NaClO-labelled 125I-RBP, EB-labelled 125I-RBP and 125I-SHPP-RBP. At 37 degrees C, the 125I-SHPP-RBP and the EB-labelled 125I-RBP became cell-associated more rapidly than the other two ligands. The higher cell association at 37 degrees C than at 4 degrees C suggests that internalization of the ligand occurred at the higher temperature. The degradation of the ligands was also different. The EB-labelled 125I-RBP, the 125I-TC-RBP and the 125I-SHPP-RBP showed an apparent lag phase before a steady increase in acid-soluble radioactivity was observed. Much less of EB-labelled 125I-RBP and 125I-TC-RBP were degraded (about 6%) than of the other two ligands (about 16%) after 120 min. About 50% of the acid-soluble radioactivity in these experiments could be accounted for by degradation in the medium, suggesting that about half of the degradation observed was intracellular. The present study therefore shows that the different labelling techniques yield varying estimates of the cellular handling of RBP. In addition, a rapid release of RBP was observed in experiments where cells were pulsed with radioactive RBP at 4 degrees C, washed and incubated further at 37 degrees C. Between 50% and 70% was released after 5 min of incubation. By increasing the temperature during the pulse to 37 degrees C, or by lowering the temperature during the chase to 4 degrees C, much less RBP was released from the cells. These data suggest that the release process represents recycling of internalized ligand from an early endosome.

Retinoids: in vitro interaction with retinol‐binding protein and influence on plasma retinol

1993 ◽  
Vol 7 (12) ◽  
pp. 1179-1184 ◽  
Author(s):  
Rodolfo Berni ◽  
Monica Clerici ◽  
Giorgio Malpeli ◽  
Loredana Cleris ◽  
Franca Formelli
Keyword(s):  
Binding Protein ◽  
Retinol Binding Protein ◽  
Plasma Retinol ◽  
In Vitro Interaction

scholarly journals Preparative Scale Production of Recombinant Human Transthyretin for Biophysical Studies of Protein-Ligand and Protein-Protein Interactions

2020 ◽  
Vol 21 (24) ◽  
pp. 9640
Author(s):  
Ellen Y. Cotrina ◽  
Marta Vilà ◽  
Joan Nieto ◽  
Gemma Arsequell ◽  
Antoni Planas
Keyword(s):  
Binding Protein ◽  
Amyloid Β ◽  
Retinol Binding Protein ◽  
Familial Amyloidotic Polyneuropathy ◽  
Main Role ◽  
Scale Production ◽  
In Vitro Screening ◽  
Preparative Scale ◽  
Screening Assays

Human transthyretin (hTTR), a serum protein with a main role in transporting thyroid hormones and retinol through binding to the retinol-binding protein, is an amyloidogenic protein involved in familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and central nervous system selective amyloidosis. hTTR also has a neuroprotective role in Alzheimer disease, being the major Aβ binding protein in human cerebrospinal fluid (CSF) that prevents amyloid-β (Aβ) aggregation with consequent abrogation of toxicity. Here we report an optimized preparative expression and purification protocol of hTTR (wt and amyloidogenic mutants) for in vitro screening assays of TTR ligands acting as amyloidogenesis inhibitors or acting as molecular chaperones to enhance the TTR:Aβ interaction. Preparative yields were up to 660 mg of homogenous protein per L of culture in fed-batch bioreactor. The recombinant wt protein is mainly unmodified at Cys10, the single cysteine in the protein sequence, whereas the highly amyloidogenic Y78F variant renders mainly the S-glutathionated form, which has essentially the same amyloidogenic behavior than the reduced protein with free Cys10. The TTR production protocol has shown inter-batch reproducibility of expression and protein quality for in vitro screening assays.

scholarly journals Endocytosis and degradation of interstitial retinol-binding protein: differential capabilities of cells that border the interphotoreceptor matrix.

1985 ◽  
Vol 100 (5) ◽  
pp. 1676-1681 ◽  
Author(s):  
J G Hollyfield ◽  
H H Varner ◽  
M E Rayborn ◽  
G I Liou ◽  
C D Bridges
Keyword(s):  
Acid Phosphatase ◽  
Binding Protein ◽  
Pigment Epithelium ◽  
Retinol Binding Protein ◽  
Apical Plasma Membrane ◽  
Multivesicular Bodies ◽  
Cone Photoreceptors ◽  
Interphotoreceptor Matrix ◽  
Rod And Cone Photoreceptors

Between the pigment epithelium and the outer limiting membrane of the retina is an extracellular compartment filled with the interphotoreceptor matrix (IPM). A prominent component of the IPM is a glycoprotein known as interstitial retinol-binding protein (IRBP). Using in vitro techniques, we compared the ability of the cells that border this compartment to internalize colloidal gold (CG) coated with either IRBP or ovalbumin, a glycoprotein not found in the IPM. Neither IRBP-CG nor ovalbumin-CG was internalized by the Muller's cells. Both rod and cone photoreceptors take up IRBP-CG, which is observed in small vesicles and multivesicular bodies. Neither photoreceptor type takes up ovalbumin-CG. Acid phosphatase cytochemistry indicates that acid phosphatase reaction product in the multivesicular bodies co-localizes with IRBP-CG, which suggests that this molecule is degraded by rod and cone photoreceptors and is not recycled. The pigment epithelium internalizes IRBP-CG and ovalbumin-CG, both of which remain in small cytoplasmic vesicles near the apical plasma membrane. There is no indication that vesicles that contain either IRBP-CG or ovalbumin-CG fuse with the lysosomal system in the pigment epithelial cells during the incubation.

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