The use of poly(L-proline)-Sepharose in the isolation of profilin and profilactin complexes

In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin_β and profilin-actin_γ. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg²⁺, and can be crystallized. From the isolated profilin-actin complexes the β- and γ-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin.     

Lipid rafts constrain basal α1A-adrenergic receptor signaling by maintaining receptor in an inactive conformation

We have reported that the α_1A-adrenergic receptor (α_1AAR) in rat-1 fibroblasts is a lipid raft protein. Here we examined whether disrupting lipid rafts by methyl-β-cyclodextrin (MCD) sequestration of cholesterol affects α_1AAR signaling. Unexpectedly, MCD increased α_1AAR-dependent basal inositol phosphate formation and p38 mitogen-activated protein kinase activation in a cholesterol-dependent manner. It also initiated internalization of surface α_1AAR, which was partially blocked by receptor inhibition. Binding assays revealed MCD-mediated increases in receptor agonist affinity as well as reciprocal decreases in inverse agonist affinity, a behavior that is usually interpreted as a shift toward the active receptor conformation. In untreated cells a fraction of the receptor was found to be present in preassociated receptor/G protein complexes, which rapidly dissociate upon receptor stimulation. Consistent with MCD-induced signaling, raft disruption resulted in an increase in receptor/G protein complexes. These results strongly suggest that lipid rafts constrain basal α_1AAR activity; however, preassembled receptor/G protein complexes could still provide a mechanism for accelerating α_1AAR signaling following stimulation.     

Production of an Anti-Prostate-Specific Antigen Single-Chain Antibody Fragment from Pichia pastoris

Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. Because PSA levels are normally quite low, an antibody-based assay must be used to detect PSA. However, not all PSA-specific antibodies bind equally well to PSA or to its different isoforms. Therefore, a better understanding of how PSA interacts with PSA-specific antibodies is of considerable clinical interest. B80.3 is a widely used murine monoclonal anti-PSA antibody (IgG), which has very high affinity for both free and α-anti-chymotrypsin complexed PSA. More importantly, its gene sequence is known—making it one of only two anti-PSA antibodies that has been fully cloned and sequenced. To better elucidate the interaction between PSA and B80.3, a single-chain antibody fragment, derived from the variable domain of B80.3 (scFvB80), was cloned into a pPIC9 vector and expressed in Pichia pastoris. The secreted protein was purified using a three-step protocol beginning with a 50% ammonium sulfate precipitation step, followed by a T-gel thio-affinity step and concluding with a simple anion-exchange (DE52) filtration step. NMR studies indicate the protein is correctly folded while competitive enzyme-linked immunosorbant assays show that the purified scFvB80 has approximately 20% of the activity of the full-length B80.3 antibody. The protocol described here provides a quick and convenient route to prepare large quantities of very pure anti-PSA antibody fragments (15–20 mg/L culture medium) for detailed structural and biophysical characterization.     

Recognition of ribonuclease A by 3'-5'-pyrophosphate-linked dinucleotide inhibitors: a molecular dynamics/continuum electrostatics analysis

The proteins of the pancreatic ribonuclease A (RNase A) family catalyze the cleavage of the RNA polymer chain. The development of RNase inhibitors is of significant interest, as some of these compounds may have a therapeutic effect in pathological conditions associated with these proteins. The most potent low molecular weight inhibitor of RNase reported to date is the compound 5′-phospho-2′-deoxyuridine-3-pyrophosphate (P→5)-adenosine-3-phosphate (pdUppA-3′-p). The 3′,5′-pyrophosphate group of this compound increases its affinity and introduces structural features which seem to be unique in pyrophosphate-containing ligands bound to RNase A, such as the adoption of a syn conformation by the adenosine base at RNase subsite B₂ and the placement of the 5′-β-phosphate of the adenylate (instead of the α-phosphate) at subsite P₁ where the phosphodiester bond cleavage occurs. In this work, we study by multi-ns molecular dynamics simulations the structural properties of RNase A complexes with the ligand pdUppA-3′-p and the related weaker inhibitor dUppA, which lacks the 3′ and 5′ terminal phosphate groups of pdUppA-3′-p. The simulations show that the adenylate 5′-β-phosphate binding position and the adenosine syn orientation constitute robust structural features in both complexes, stabilized by persistent interactions with specific active-site residues of subsites P₁ and B₂. The simulation structures are used in conjunction with a continuum-electrostatics (Poisson-Boltzmann) model, to evaluate the relative binding affinity of the two complexes. The computed relative affinity of pdUppA-3′-p varies between −7.9 kcal/mol and −2.8 kcal/mol for a range of protein/ligand dielectric constants (ε_p) 2–20, in good agreement with the experimental value (−3.6 kcal/mol); the agreement becomes exact with ε_p = 8. The success of the continuum-electrostatics model suggests that the differences in affinity of the two ligands originate mainly from electrostatic interactions. A residue decomposition of the electrostatic free energies shows that the terminal phosphate groups of pdUppA-3′-p make increased interactions with residues Lys⁷ and Lys⁶⁶ of the more remote sites P₂ and P₀, and His¹¹⁹ of site P₁.     

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

The DnaX complex (DnaX₃δδ′χψ) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β₂, onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β₂ binding (determined functionally) is diminished 12–30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β₂. DNA synthesis activity can be restored by increased concentrations of β₂. In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β₂ loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.     

Internalization of α2 macroglobulin in receptosomes. Studies with monovalent electron microscopic markers

Previous studies using multivalent, peroxidase-labeled antibody for localization of α₂ M have demonstrated that the binding of α₂-macroglobulin (α₂ M) to randomly distributed receptors on the surfaces of fibroblasts initiates the accumulation of α₂ M-receptor complexes in clathrin-coated pits. The α₂ M-receptor complexes are then internalized into a specialized vesicle termed the receptosome. In the present study we have used three different monovalent ligands to localize α₂ M and show that the endocytosis of α₂ M-receptor complexes by the receptosomal pathway is not initiated as a result of antibody-induced cross-linking of the α₂ M-receptor complexes. To perform these studies the following monovalent markers of α₂ M were prepared for electron microscopic visualization: (1) a monovalent hybrid antibody directed against α₂ M and ferritin; (2) a monovalent hybrid antibody directed against α₂ M-peroxidase; and (3) a direct 1:1 conjugate of α₂ M-peroxidase. We find that all three of the markers are internalized by the ligand pathway previously described using multivalent labels. The steps involved are clustering of α₂ M receptor complexes in coated regions of the plasma membrane followed by endocytosis of α₂ M into receptosomes. Our results are contrasted with previous studies on lymphocytes in which antibody induced cross-linking of membrane antigens was necessary for triggering their pinocytosis. The methods described in this paper are applicable for visualizing at the electron microscopic level the internalization of other ligands and hormones.     

Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α_IIbβ₃, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β₃-Endonexin is a novel 111–amino acid protein that binds selectively to the β₃ tail. Since β₃-endonexin is present in platelets, we asked whether it can affect α_IIbβ₃ function. When β₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α_IIbβ₃ affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α_IIbβ₃ bound little or no PAC1. However, those transfected with GFP/β₃-endonexin and α_IIbβ₃ bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β₃-endonexin did not affect levels of surface expression of α_IIbβ₃ nor did it modulate the affinity of an α_IIbβ₃ mutant that is defective in binding to β₃-endonexin. Affinity modulation of α_IIbβ₃ by GFP/β₃-endonexin was inhibited by coexpression of either a monomeric β₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β₃-endonexin can modulate the affinity state of α_IIbβ₃ in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of α_IIbβ₃.     

Uptake and degradation of α2-macroglobulin-protease complexes in human cells in culture

Serum components, present intracellularly in cultured human fibroblasts, were identified as α₂-macroglobulin (α₂M), albumin, α₁-trypsin inhibitor, hemopexin and transferrin, among others. These components were shown to be taken up from the culture medium. Kinetic analysis of the uptake of α₂M-trypsin complexes by the cells showed the uptake to be of a high affinity mechanism (K_M = 6 × 10⁻⁸ M α₂M in the medium), with a high rate of internalization (V_max=1.03 × 10⁶ molecules α₂M/cell and α₂M per hour). The intracellular degradation of α₂M is rapid, as judged by the half-life of 1.6 h. Virus-transformed or tumor-derived cell lines showed low or undetectable levels of α₂M. The possible physiological significance of the described phenomena is discussed in relation to the in vivo situation.     

Complementary Roles for Receptor Clustering and Conformational Change in the Adhesive and Signaling Functions of Integrin αIIbβ3

Integrin α_IIbβ₃ mediates platelet aggregation and “outside-in” signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to α_IIbβ₃ function, α_IIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified α_IIb subunits were expressed with β₃ in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25–50% of that observed when α_IIbβ₃ affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of α_IIbβ₃ also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72^Syk and fibrinogen-dependent phosphorylation of pp125^FAK, even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in α_IIbβ₃ function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of α_IIbβ₃ in platelets.     

Neurotoxicity of acetylcholinesterase amyloid β-peptide aggregates is dependent on the type of Aβ peptide and the AChE concentration present in the complexes

Alzheimer’s disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid β-peptide (Aβ) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Aβ_1–40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Aβ complexes were found to be more toxic than those formed without the enzyme, for Aβ_1–40 and Aβ_1–42, but not for amyloid fibrils formed with Aβ_Val18→Ala, a synthetic variant of the Aβ_1–40 peptide. Of all the AChE-Aβ complexes tested the one containing the Aβ_1–40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Aβ_1–40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Aβ_1–40 aggregates are more toxic than those of AChE-Aβ_1–42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.     

Gel-based oligonucleotide microarray approach to analyze protein–ssDNA binding specificity

Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to single-stranded DNA (ssDNA). To demonstrate additional capabilities of this method, we analyzed the binding specificity of ribonuclease (RNase) binase from Bacillus intermedius (EC 3.1.27.3) to ssDNA using generic hexamer oligodeoxyribonucleotide microchip. Single-stranded octamer oligonucleotides were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5′-{N}N₁N₂N₃N₄N₅N₆{N}-3′ consisted of a fixed hexamer motif N₁N₂N₃N₄N₅N₆ in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096 pads with a complete set of hexamer sequences. The affinity was determined by measuring dissociation of the RNase–ssDNA complexes with the temperature increasing from 0°C to 50°C in quasi-equilibrium conditions. RNase binase showed the highest sequence-specificity of binding to motifs 5′-NNG(A/T/C)GNN-3′ with the order of preference: GAG > GTG > GCG. High specificity towards G(A/T/C)G triplets was also confirmed by measuring fluorescent anisotropy of complexes of binase with selected oligodeoxyribonucleotides in solution. The affinity of RNase binase to other 3-nt sequences was also ranked. These results demonstrate the applicability of the method and provide the ground for further investigations of nonenzymatic functions of RNases.     

Identification of α2 adrenergic receptors in human frontal cortex membranes by binding of [H]RX 821002, the 2-methoxy analog of [H]idazoxan

The antagonist [³H]idazoxan binds with comparable affinity to α₂ adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-³H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300–1200 times lower affinity for the non-stereoselective sites. Their affinity for the α₂ receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [³H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the α₂ receptors (K_D = 2.8 nM than [³H]idazoxan (K_D = 24 nM) and about equal affinity as [³H]rauwolscine (K_D = 3.6 nM).[³H]Rauwolscine binds with comparable affinity to α₂ receptors and to 5-HT_1A receptors, and competition studies indicate that the K_i value of unlabelled RX 821002 for the 5-HT_1A receptors (30 nM) is about one order in magnitude above its K_i value for the α₂ receptors (4.1 nM). Labelling of the 5-HT_1A receptors by [³H]RX 821002 and by [³H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 μM). Under these conditions, specific binding of [³H]RX 821002 to the α₂ receptors represents 84% of total binding (at its K_D), as compared to 77% for [³H]rauwolscine and 20% for [³H]idazoxan.[³H]RX 821002 labels the α₂ receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37°C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [³H]RX821002. About 60% of the α₂ receptors possess high agonist affinity.     

Photorespiration and Internal CO(2) Accumulation in Chara corallina as Inferred from the Influence of DIC and O(2) on Photosynthesis

An O₂ electrode system with a specially designed chamber for `whorl' cell complexes of Chara corallina was used to study the combined effects of inorganic carbon and O₂ concentrations on photosynthetic O₂ evolution. At pH = 5.5 and 20% O₂, cells grown in HCO₃⁻ medium (low CO₂, pH ≥ 9.0) exhibited a higher affinity for external CO₂ (K_½(CO₂) = 40 ± 6 micromolar) than the cells grown for at least 24 hours in high-CO₂ medium (pH = 6.5), (K_½(CO₂) = 94 ± 16 micromolar). With O₂ ≤ 2% in contrast, both types of cells showed a high apparent affinity (K_½(CO₂) = 50 − 52 micromolar). A Warburg effect was detectable only in the low affinity cells previously cultivated in high-CO₂ medium (pH = 6.5). The high-pH, HCO₃⁻-grown cells, when exposed to low pH (5.5) conditions, exhibited a response indicating an ability to fix CO₂ which exceeded the CO₂ externally supplied, and the reverse situation has been observed in high-CO₂-grown cells. At pH 8.2, the apparent photosynthetic affinity for external HCO₃⁻ (K_½[HCO₃⁻]) was 0.6 ± 0.2 millimolar, at 20% O₂. But under low O₂ concentrations (≤2%), surprisingly, an inhibition of net O₂ evolution was elicited, which was maximal at low HCO₃⁻ concentrations. These results indicate that: (a) photorespiration occurs in this alga and can be revealed by cultivation in high-CO₂ medium, (b) Chara cells are able to accumulate CO₂ internally by means of a process apparently independent of the plasmalemma HCO₃⁻ transport system, (c) molecular oxygen appears to be required for photosynthetic utilization of exogenous HCO₃⁻: pseudocyclic electron flow, sustained by O₂ photoreduction, may produce the additional ATP needed for the HCO₃⁻ transport.     

Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [μ-dppzip(phen)₄Ru₂]⁴⁺ (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)₂dppz) and the distal subunit (Ru(phen)₂ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δx_eq, and the dynamic DNA structural deformations during ligand association x_on and dissociation x_off. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a K_d of 27 ± 3 nM for (Δ,Λ)-Piz to a K_d of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δx_eq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and x_on from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited x_off changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Preparation and quantitation of urinary metabolites of prostaglandin F2α by radioimmunoassay

Radioimmunoassay systems are described which have been developed to quantitate two principle urinary metabolites of PGF_2α; 9α,11α-dihydroxy-15-oxo-2,3,4,5-tetranorprostanoic acid (I) and 9α-11α-dihydroxy-15-oxo-2,3,4,5-tetranorprosta-1,20-dioic acid (II). Preparation of the required metabolites was achieved by total synthesis (I) or by bioconversion (isolation from urine of animals treated with 15-keto-PGF_2α^*, II). These metabolites were used to prepare conjugates for immunization. Labeled metabolites, suitable as binding markers, were prepared by metabolism of ³H-PGF_2α (I) or (II). Specificity of the resulting antibodies was compared to an antibody to PGF_2α and to 13,14-dihydro-15-keto PGF_2α. Antisera of II had little or no affinity for 20-carbon precursors (PGF_2α or 13,14-dihydro-15-keto PGF_2α), but had nearly equal affinity for metabolite I. Antisera of I, however, had little or no affinity for antigen of II. Therefore, analysis of samples by both assay systems enables quantitation of these excretion products of PGF_2α. Other assay parameters (binding, affinity, recovery, precision and the repeatability of the assays) were similar to those previously described for other RIA systems, and were considered satisfactory for quanitation of compounds in biological fluids.Quantitation of 24 hour urinary excretion of di-acid metabolite in humans was in close agreement with previously published values determined by physical-chemical means. Greater quantity of di-acid metabolite was excreted by human males (42.0 μg/24 hr) than by females sampled either during the follicular (20.0) or luteal phase (21.2) of the menstrual cycle. The total quantity of C-16 metabolites (as approximated by system II) excreted/kg body weight by the rhesus monkey was similar to that excreted by the human. However, the ratio of di-acid to mono-acid was much nearer unity in the monkey than the human.     

Human Milk sIgA Molecules Contain Various Combinations of Different Antigen-Binding Sites Resulting in a Multiple Binding Specificity of Antibodies and Enzymatic Activities of Abzymes

In the classic paradigm, immunoglobulins are monospecific molecules that have stable structures and two or more identical antigen-binding sites. However, we show here for the first time that the sIgA pool of human milk contains, depending on the donor, only 35±5% λ-sIgAs, 48±7% κ-sIgAs, and 17±4% of chimeric λ-κ-sIgAs. sIgA preparations contained no traces of canonical enzymes. However, all sIgA fractions eluted from several specific affinity sorbents under the conditions destroying even strong immune complexes demonstrated high catalytic activities in hydrolysis of ATP, DNA, and oligosaccharides, and phosphorylation of proteins, lipids, and oligosaccharides. Sequential re-chromatographies of the sIgA fractions with high affinity to one affinity sorbents on the second, third and then fourth affinity sorbents bearing other immobilized antigens led to the distribution of Abs and all catalytic activities all over the profiles of these chromatographies; in all cases some fractions eluted from affinity sorbents only under the conditions destroying strong immune complexes. In vitro, only an addition of reduced glutathione and milk plasma containing no Abs to two sIgA fractions with different affinity for DNA-cellulose led to a transition of up to 11–20% of Ab from one fraction to the other. Our data are indicative of the possibility of half-molecule exchange between different IgA and sIgA molecules. In addition, it cannot be excluded that during the penetration of IgAs through the specific milk barrier, the secretory component (S) and the join chain (J) can combine molecules of dimeric H₂L₂ λ-IgAs and κ-IgAs against different antigens forming many different variants of H₄L₄SJ sIgA molecules. Therefore, some chimeric molecules of sIgA can contain from two to four HL-fragments to various antigens interacting with high affinity with different sorbents and catalyzing various chemical reactions. Our data essentially expand the ideas concerning explanation of the phenomenon of polyspecificity and cross-reactivity of Abs.     

Characterization of the interaction between alphaCP(2) and the 3'-untranslated region of collagen alpha1(I) mRNA

Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein–RNA interactions in the 3′-UTR of the collagen α1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is αCP₂. Recombinant αCP₂ is sufficient for binding to the 3′-UTR of collagen α1(I). To characterize the binding affinity of and specificity for αCP₂, we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3′-UTR of collagen α1(I) as probe. The binding affinity of αCP₂ for the 3′-UTR sequence is ~2 nM in vitro and the wild-type 3′ sequence binds with high specificity. Furthermore, we demonstrate a system for detecting protein–nucleotide interactions that is suitable for high throughput assays using molecular beacons. Molecular beacons, developed for DNA–DNA hybridization, are oligonucleotides with a fluorophore and quencher brought together by a hairpin sequence. Fluorescence increases when the hairpin is disrupted by binding to an antisense sequence or interaction with a protein. Molecular beacons displayed a similar high affinity for binding to recombinant αCP₂ to the wild-type 3′ sequence, although the kinetics of binding were slower.     

Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH₂-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γ_A/γ_A isoform of fibrin(ogen) and the γ_A/γ′ variant with an extended γ-chain. Thrombin binds to the γ′-chain and forms a higher affinity interaction with γ_A/γ′-fibrin(ogen) than γ_A/γ_A-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (K_d values of about 0.5 μm) even though it does not interact with the γ′-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γ_A/γ_A-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a K_d value of 2–5 μm, the α(17–51) and Bβ(1–42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.