Studies on 125I-labeled proteins in rat plasma using an air-driven ultracentrifuge: protein-protein interactions and nonideality

The molecular weights of a number of ¹²⁵I-labeled plasma proteins have been determined from an analysis of their sedimentation equilibrium behavior in an air-driven ultracentrifuge. The values obtained agree well with results obtained by other methods. Molecular weights obtained for ¹²⁵I-labeled bovine serum albumin and the rat serum proteins albumin, α₁-acid glycoprotein, and major acute-phase α₁-protein were unaffected by the addition of 7% rat plasma. Direct evidence for protein-protein interactions was obtained for mixtures of ¹²⁵I-labeled rat α₁-acid glycoprotein and the plant lectin concanavalin A and for mixtures of ¹²⁵I-labeled protein A from Staphylococcus aureus and 7% rat plasma. Interactions of a different type were observed when the sedimentation equilibrium profiles of ¹²⁵I-labeled proteins were determined in concentrated solutions of other proteins. Under these conditions the effects of molecular exclusion or nonideality became significant and low estimates were obtained for the molecular weights of the labeled proteins. Analysis of the data obtained for ¹²⁵I-labeled bovine serum albumin in concentrated solutions of bovine serum albumin (20–80 mg/ ml) yielded nonideality coefficients in good agreement with literature values. Analysis of the behavior of ¹²⁵I-labeled rat serum albumin, transferrin, and α₁-acid glycoprotein yielded nonideality coefficients and hence activities of these proteins in undiluted rat plasma.     

Interaction of [125I]β nerve growth factor with acidic proteins

We have demonstrated that the nerve growth factor will interact with various acidic proteins apparently nonspecifically. When¹²⁵I-labeled nerve growth factor at a concentration of 3.8×10^–10 M is incubated with an acidic protein at 2 mg/ml (4.5×10^–6–4.4×10^–5 M), a complex is formed. This complex changes the isoelectric point of the¹²⁵I-labeled nerve growth factor sufficiently so that the¹²⁵I-labeled nerve growth factor migrates anomalously in polyacrylamide gel electrophoresis. The interaction between nerve growth factor and bovine serum albumin, which appears to be complex, may be the cause of the previously reported activation of the nerve growth factor when bovine serum albumin is present in a typical bioassay.A preliminary report of this work was presented at the American Society of Biological Chemists, 71st Annual Meeting, in June 1980.     

Interaction of Cholera Toxin B Subunit with Rat Intestinal Epithelial Cells

We prepared ¹²⁵I-labeled cholera toxin B subunit (¹²⁵I-labeled CT-B, specific activity 98 Ci/mmol) and found that its binding to rat IEC-6 intestinal epithelial cells was high-affinity (K_d 1.9 nM). The binding of labeled protein was completely inhibited by unlabeled thymosin-α₁ (TM-α₁), interferon-α₂ (IFN-α₂), and synthetic peptide LKEKK, which corresponds to residues 16–20 in TM-α₁ and 131–135 in IFN-α₂ (K_i 1.2, 0.9, and 1.6 nM, respectively), but was not inhibited by synthetic peptide KKEKL with inverted amino acid sequence (K_i > 10 μM). Thus, TM-α₁, IFN-α₂, and the LKEKK peptide bind with high affinity and specificity to CT-B receptor on rIEC-6 cells. It was found that CT-B and the LKEKK peptide at concentrations of 10–1000 nM increased nitric oxide production and soluble guanylate cyclase activity in the cells in a dose-dependent manner.     

Studies of albumin binding to rat liver plasma membranes: Implications for the albumin receptor hypothesis

To characterize a previously proposed hepatocyte albumin receptor, we examined the binding of native and defatted ¹²⁵I-labeled rat albumin to rat liver plasma membranes. After incubation for 30 min, binding was determined from the distribution of radioactivity between membrane pellet and supernatant following initial centrifugation (15 000 × g for 15 min), after repeated cycles of washing with buffer and re-centrifugation. ¹²⁵I-labeled albumin recovered in the initial membrane pellet averaged only 4% of that incubated. Moreover, this albumin was only loosely associated with the membrane, as indicated by recovery in the pellet of under 0.5% of the counts after three washes. Binding of ¹²⁵I-labeled albumin to the plasma membranes was no greater than to erythrocyte ghosts, was not inhibited by excess unlabeled albumin, and was not decreased by heat denaturation of the membranes, all suggestive of a lack of specific binding. Failure to observe albumin binding to the membranes was not due to a rapid dissociation rate or ‘off-time’, as incubations in the presence of sufficient ultraviolet light to promote covalent binding of ligands to receptors did not increase ¹²⁵I counts bound to the membrane. Finally, affinity chromatography over albumin/agarose gel of solubilized membrane proteins provided no evidence of a membrane protein with a high affinity for albumin. These studies, therefore, do not support the hypothesis that liver cell plasma membranes contain a specific albumin receptor.     

Synthesis of [I]iodoDPA-713: A new probe for imaging inflammation

[¹²⁵I]IodoDPA-713 [¹²⁵I]1, which targets the translocator protein (TSPO, 18 kDa), was synthesized in seven steps from methyl-4-methoxybenzoate as a tool for quantification of inflammation in preclinical models. Preliminary in vitro autoradiography and in vivo small animal imaging were performed using [¹²⁵I]1 in a neurotoxicant-treated rat and in a murine model of lung inflammation, respectively. The radiochemical yield of [¹²⁵I]1 was 44 ± 6% with a specific radioactivity of 51.8 GBq/μmol (1400 mCi/μmol) and >99% radiochemical purity. Preliminary studies showed that [¹²⁵I]1 demonstrated increased specific binding to TSPO in a neurotoxicant-treated rat and increased radiopharmaceutical uptake in the lungs of an experimental inflammation model of lung inflammation. Compound [¹²⁵I]1 is a new, convenient probe for preclinical studies of TSPO activity.     

Mapping by synthetic peptides of the binding sites for acetylcholine receptor on α-bungarotoxin

A set of seven peptides constituting the various loops and most of the surface areas of α-bungarotoxin (BgTX) was synthesized. In appropriate peptides, the cyclical (by a disulfide bond) monomers were prepared. In all cases, the peptides were purified and characterized. The ability of these peptides to bindTorpedo californica acetylcholine receptor (AChR) was studied by radiometric adsorbent titrations. Three regions, represented by peptides 1–16, 26–41, and 45–59, were able to bind¹²⁵I-labeled AChR and, conversely,¹²⁵I-labeled peptides were bound by AChR. In these regions, residues Ile-1, Val-2, Trp-28 and/or Lys-38, and one or all of the three residues Ala-45, Ala-46, and Thr-47, are essential contact residues in the binding of BgTX to receptor. Other synthetic regions of BgTX showed little or no AChR-binding activity. The specificity of AChR binding to peptides 1–16, 26–41, and 45–59 was confirmed by inhibition with unlabeled BgTX. It is concluded that BgTX has three main AChR-binding regions (loop I with N-terminal extension and loops II and III extended toward the N-terminal by residues 45–47).     

α-Neurotoxin binding to acetylcholine receptor: Localization of the full profile of the cobratoxin-binding regions on the α-chain ofTorpedo californica acetylcholine receptor by a comprehensive synthetic strategy

Eighteen consecutive uniform overlapping synthetic peptides that spanned the entire extracellular part (residues 1–210) of the α-chain ofTorpedo californica acetylcholine receptor were screened for binding activity of¹²⁵I-labeled cobratoxin. Five toxin-binding regions were localized within residues 1–10, 32–41, 100–115, 122–150, and 182–198. The five toxin-binding regions may be distinct sites or, alternatively, different faces in one or more sites.     

Interaction of cholera toxin B-subunit with human T-lymphocytes

In this work, ¹²⁵I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (K _d = 3.3 nM) was determined. The binding of the ¹²⁵I-labeled CT-B was inhibited by unlabeled interferon-α₂ (IFN-α₂), thymosin-α1 (TM-α₁), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α₁ and 131-135 of IFN-α₂ (K _i 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (K _i > 1 μM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.     

Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.     

Structure, Properties, and Engineering of the Major Zinc Binding Site on Human Albumin

Most blood plasma zinc is bound to albumin, but the structure of the binding site has not been determined. Zn K-edge extended x-ray absorption fine structure spectroscopy and modeling studies show that the major Zn²⁺ site on albumin is a 5-coordinate site with average Zn-O/N distances of 1.98 Å and a weak sixth O/N bond of 2.48 Å, consistent with coordination to His⁶⁷ and Asn⁹⁹ from domain I, His²⁴⁷ and Asp²⁴⁹ from domain II (residues conserved in all sequenced mammalian albumins), plus a water ligand. The dynamics of the domain I/II interface, thought to be important to biological function, are affected by Zn²⁺ binding, which induces cooperative allosteric effects related to those of the pH-dependent neutral-to-base transition. N99D and N99H mutations enhance Zn²⁺ binding but alter protein stability, whereas mutation of His⁶⁷ to alanine removes an interdomain H-bond and weakens Zn²⁺ binding. Both wild-type and mutant albumins promote the safe management of high micromolar zinc concentrations for cells in cultures.Zinc is not only required for hundreds of essential extra- and intracellular proteins and enzymes but is also recruited by toxins such as anthrax lethal factor (1) and staphylococcal enterotoxin (2). There is a need to understand how zinc transport and distribution is controlled (3).Although considerable progress has been made in the identification and study of membrane-bound zinc transporters, the molecular mechanism of extracellular zinc transport is still obscure. The total concentration of zinc in blood is high, ∼15–20 μm (4), and plasma zinc concentrations are maintained at a relatively constant level, except during periods of dietary zinc depletion and acute responses to stress or inflammation, when they are depressed (5). In humans, ∼98% of so-called “exchangeable” zinc in blood plasma (9–14 μm) is bound to serum albumin (6). Studies on perfused rat intestine have implicated albumin in the transport of newly absorbed zinc in portal blood, from the intestine to the liver (5). Albumin has also been shown to promote zinc uptake by endothelial cells, with receptor-mediated endocytosis as the most likely mechanism (7).Albumin, the most abundant protein in blood plasma (∼40 mg ml⁻¹ and 0.6 mm), is synthesized in the liver and secreted into the blood stream as a 585-residue, single-chain protein after loss of a 24-residue propeptide (8). The protein is largely α-helical and folds into three structurally homologous domains (I, II, and III), each of which contains two subdomains (A and B) (see Fig. 1A) (9). There are 35 Cys residues that form six disulfide bridges in each domain, except for domain I, which contains only five bridges and a free thiol at Cys³⁴.Open in a separate windowFIGURE 1.A, domain structure of human serum albumin (9). Domain I (red), domain II (blue), and domain III (green) are held together solely by linker helices and weak interactions. B, the proposed interdomain zinc site on albumin is formed by two residues from domain I (red) and two residues from domain II (blue).Zinc binding to albumin has also been demonstrated in vitro, and the so-called high-affinity site for zinc on albumin displays a binding constant of K ≈ 10⁷ m⁻¹ (10–13). Although over 50 x-ray structures of albumin have been reported to date (9, 14–16), no experimental structural data are available for the zinc site on albumin. On the basis of ¹¹¹Cd NMR studies, site-directed mutagenesis, and molecular modeling, we have proposed that the major zinc site on albumin, termed site A, is located at the interface of domains I and II and is formed by the side chains of His⁶⁷ and Asn⁹⁹ from domain I and by His²⁴⁷ and Asp²⁴⁹ from domain II (Fig. 1B) (17, 18).Here, we report the first direct structural characterization of a zinc site on albumin, using Zn K-edge x-ray absorption fine structure (EXAFS)² spectroscopy. We also explored the possibility of engineering albumins with increased or decreased zinc-binding affinity by mutating the postulated zinc-binding ligands. NMR methods were used to identify mutated and zinc-binding histidines and to probe the effects of zinc binding on the conformational dynamics of the protein. Finally, we show that albumin at physiological concentrations promotes the culture of hepatocytes at otherwise toxic zinc concentrations.     

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

• 1.1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [¹²⁵I]insulin.
• 2.2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent.
• 3.3. [¹²⁵I]insulin was displaced by unlabelled insulin. A₁-B₂₉ dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity.
• 4.4. Brush-border membranes showed high insulin-degrading activity with an apparent K_m of 2.2 μM.
• 5.5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents.
• 6.6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.

     

α<Subscript>1</Subscript>-Thymosin, α<Subscript>2</Subscript>-interferon,and the LKEKK syntetic peptide inhibit the binding of the B subunit of the cholera toxin to intestinal epithelial cell membranes

The ¹²⁵I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (K _d = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α₂-interferon (IFN-α₂), α₁-thymosin, (TM-α₁), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α₁ and the 131–135 sequence of human IFN-α₂ (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (K _i > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α₁, IFN-α₂, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.