Structural Requirements For Binding Research Articles

Pentosan Polysulfate Inhibits Attachment and Infection by SARS-CoV-2 In Vitro: Insights into Structural Requirements for Binding

Two years since the outbreak of the novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic, there remain few clinically effective drugs to complement vaccines. One is the anticoagulant, heparin, which in 2004 was found able to inhibit invasion of SARS-CoV (CoV-1) and which has been employed during the current pandemic to prevent thromboembolic complications and moderate potentially damaging inflammation. Heparin has also been shown experimentally to inhibit SARS-CoV-2 attachment and infection in susceptible cells. At high therapeutic doses however, heparin increases the risk of bleeding and prolonged use can cause heparin-induced thrombocytopenia, a serious side effect. One alternative, with structural similarities to heparin, is the plant-derived, semi-synthetic polysaccharide, pentosan polysulfate (PPS). PPS is an established drug for the oral treatment of interstitial cystitis, is well-tolerated, and exhibits weaker anticoagulant effects than heparin. In an established Vero cell model, PPS and its fractions of varying molecular weights inhibited invasion by SARS-CoV-2. Intact PPS and its size-defined fractions were characterized by molecular weight distribution and chemical structure using nuclear magnetic resonance spectroscopy and liquid chromatography–mass spectrometry, then employed to explore the structural basis of interactions with SARS-CoV-2 spike protein receptor-binding domain (S1 RBD) and the inhibition of Vero cell invasion. PPS was as effective as unfractionated heparin, but more effective in inhibiting cell infection than low-molecular-weight heparin (on a weight/volume basis). Isothermal titration calorimetry and viral plaque-forming assays demonstrated size-dependent binding to S1 RBD and inhibition of Vero cell invasion, suggesting the potential application of PPS as a novel inhibitor of SARS-CoV-2 infection.

Affinity and Specificity for Binding to Glycosaminoglycans Can Be Tuned by Adapting Peptide Length and Sequence.

Although glycosaminoglycan (GAG)–protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound to cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs.

Use of Diverse Chemometric and Validation Methods to Accurately Predict Human Urotensin-II Receptor Antagonist Activity.

Despite being identified as the most potent receptor related to vasoconstriction, human urotensin-II receptor (hUT) has not been fully explored as a target for the treatment of cardiovascular diseases. In view of this and with an aim to identify precise structural requirements for binding of hUT antagonists, we endeavoured to develop, for the first time, multivariate QSAR models using chemometric methods like partial least squares (PLS) and feed-forward neural network (FFNN). A set of 48 pyrrolidine derivatives having hUT binding affinity was used for multivariate model development. The accuracy and predictability of the developed models was evaluated using crossvalidation. The PLS model showed good correlation between selected descriptors and Ki values (r(2) =0.745 and r(2) (CV) =0.773). However, the predictive performance of FFNN was better than the PLS technique with r(2) =0.810. The study clearly suggests the role of lipophilic and steric descriptors in the ligand-hUT interactions. The QSAR models generated can be successfully extended to predict the binding affinities and for the effective design of novel hUT antagonists.

Structural Basis of the Stereospecificity of Bacterial B12-dependent 2-Hydroxyisobutyryl-CoA Mutase

Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile(A90) and Asp(A117). Asp(A117) determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.

Insights into the Importance of DFD-Motif and Insertion I1 in Stabilizing the DFD-Out Conformation of Mnk2 Kinase.

Human mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1/2) are promising anticancer targets. Mnks possess special insertions and a DFD-motif that are distinct from other kinases. Crystallographic studies of Mnk1/2 have revealed that the DFD-motif adopts the DFG/D-out conformation in which residue F227 flips into the ATP binding pocket. This is rarely observed in other kinases. Although the DFG-out conformation has attracted great interest for designing selective inhibitors, structural requirements for binding and the mechanism governing the DFG-out conformation remain unclear. This work presents for the first time the applicability of 3D models of Mnk2 protein in studying conformational changes by utilizing homology modeling and molecular dynamics simulations. The study reveals that the interactions between residue K234 of insertion I1 and D226 of the DFD motif play a key role in inducing and stabilizing the DFD-out conformation. The structural features will aid in the rational design of Mnk2 inhibitors.

Molecular Dynamics Simulation and Free Energy Calculation Studies of the Binding Mechanism of Allosteric Inhibitors with p38α MAP Kinase

p38 MAP kinase is a promising target for anti-inflammatory treatment. The classical kinase inhibitors imatinib and sorafenib as well as BI-1 and BIRB-796 were reported to bind in the DFG-out form of human p38α, known as type II or allosteric kinase inhibitors. Although DFG-out conformation has attracted great interest in the design of type II kinase inhibitors, the structural requirements for binding and mechanism of stabilization of DFG-out conformation remain unclear. As allosteric inhibition is important to the selectivity of kinase inhibitor, herein the binding modes of imatinib, sorafenib, BI-1 and BIRB-796 to p38α were investigated by molecular dynamics simulation. Binding free energies were calculated by molecular mechanics/Poisson-Boltzmann surface area method. The predicted binding affinities can give a good explanation of the activity difference of the studied inhibitors. Furthermore, binding free energies decomposition analysis and further structural analysis indicate that the dominating effect of van der Waals interaction drives the binding process, and key residues, such as Lys53, Gly71, Leu75, Ile84, Thr106, Met109, Leu167, Asp168, and Phe169, play important roles by forming hydrogen bond, salt bridge, and hydrophobic interactions with the DFG-out conformation of p38α. Finally, we also conducted a detailed analysis of BI-1, imatinib, and sorafenib binding to p38α in comparison with BIRB-796 exploited for gaining potency as well as selectivity of p38 inhibitors. These results are expected to be useful for future rational design of novel type II p38 inhibitors.

Effect of polyvalencies of glycotopes on the binding of a lectin from the edible mushroom, Agaricus bisporus.

Agaricus bisporus agglutinin (ABA) isolated from edible mushroom has a potent anti-proliferative effect on malignant colon cells with considerable therapeutic potential as an anti-neoplastic agent. Since previous studies on the structural requirement for binding were limited to molecular or submolecular levels of Galbeta1-3GalNAc (T; Thomsen-Friedenreich disaccharide glycotope; where Gal represents D-galactopyranose and GalNAc represents 2-acetamido-2-deoxy-D-galactopyranose) and its derivatives, the binding properties of ABA were further investigated using our collection of glycans by enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay. The results indicate that polyvalent Galbeta1-related glycotopes, GalNAcalpha1-Ser/Thr (Tn), and their cryptoforms, are the most potent factor for ABA binding. They were up to 5.5x10(5) and 4.7x10(6) times more active than monomeric T and GalNAc respectively. The affinity of ABA for ligands can be ranked as: multivalent T (alpha) (Galbeta1-3GalNAcalpha1-), Tn and I / II (Galbeta1-3GlcNac/Galbeta1-4GlcNAc, where GlcNAc represents 2-acetamido-2-deoxy-D-glucopyranose)>>>>monomeric T (alpha) and Tn > I >>GalNAc>>> II, L (Galbeta1-4Glc, where Glc represents D-glucopyranose) and Gal (inactive). These specific binding features of ABA establish the importance of affinity enhancement by high-density polyvalent (versus multiantennary I / II) glycotopes and facilitate our understanding of the lectin receptor recognition events relevant to its biological activities.

The origin of anti-GM1 antibodies in neuropathies: the "binding site drift" hypothesis.

Elevated titers of serum antibodies against GM1-ganglioside are associated with a variety of autoimmune neuropathies. The origin of these autoantibodies is still unknown, although there is evidence that they are produced by CD5+ B-lymphocytes and that antigen mimicry is involved. Anti-GM, IgM-antibodies in the normal human immunological repertoire are low affinity antibodies that cross-react with other glycoconjugates carrying Gal beta1-3GalNAc and probably do not have GM1-mediated biological activity. Other anti-GM1 IgM-antibodies with higher affinity and/or different fine specificity are present in patients with motor syndromes. Based on our studies of structural requirement for binding, we hypothesize that disease-associated anti-GM1 antibodies originate at random by mutations affecting the binding site of naturally-occurring ones. The hypothesis is conceptually similar to the established phenomenon of "genetic drift" in species evolutionary biology and is therefore termed "binding site drift".

Direct Comparison of NPPB and DPC as Probes of CFTR Expressed in Xenopus Oocytes

Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at -100 mV. The affinity for NPPB calculated from single-channel block, K(D) = 35 microm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type (WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB. We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects (i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes for overlapping regions in the pore.

Binding and Biological Activity of C-Terminally Modified Melanocortin Peptides: A Comparison Between Their Actions at Rodent MC1 and MC3 Receptors

PENG, P.-J., U. G. SAHM, R. V. M. DOHERTY, R. G. KINSMAN, S. H. MOSS AND C. W. POUTON.Binding and biological activity of C-terminally modified melanocortin peptides: A comparison between their actions at rodent MC1and MC3receptors.PEPTIDES 18(7) 1001–1008, 1997.—Five subtypes of melanocortin receptors have to date been identified, but to date little is known about the different structural requirements for binding and biological activity at these receptors. In this study, the role of C-terminal melanocortin peptide residues in imparting selectivity for the receptor subtypes was examined. C-terminally modified analogues of α-MSH and γ-MSH were synthesized and their interaction with MC1 and MC3 melanocortin receptors was investigated. This study provides further evidence for an important role of proline 12 (numbering with respect to α-MSH) for binding and activity at the MC1 receptor. Although the influence of C-terminal amino acids on binding and activity at MC3-R was less marked, some of them were nevertheless observed to be beneficial for the interaction with this receptor subtype.

Small Peptide Mimics of Nerve Growth Factor Bind TrkA Receptors and Affect Biological Responses

Small monomeric cyclic analogs that mimic the beta-turn regions of nerve growth factor (NGF) were designed and synthesized. Potent competitive antagonists were derived from the NGF beta-turn C-D, which inhibited [125I] NGF binding to TrkA receptors and specifically inhibited optimal NGF-mediated neurite outgrowth in PC12 cells. The cyclic beta-turn A'-A" analog also inhibited NGF binding to TrkA receptors but with lower potency. These data indicate that beta-turns C-D and A'-A" are critical for TrkA binding and may confer neurotrophin receptor specificity. Furthermore, structural requirements for binding are absolute, because unconstrained analogs derived from the same regions had no effect. Compounds that mimic NGF will be useful in deciphering the interactions of NGF and its receptors and in rational drug design.

P190 RhoGAP, the major RasGAP-associated protein, binds GTP directly.

In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.

P190 RhoGAP, the major RasGAP-associated protein, binds GTP directly.

In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.

Molecular structural requirements for binding and activation of L-alanine taste receptors

L-Alanine binds to and activates specific taste receptors ofIctalurus punctatus, the channel catfish. In order to determine the structural requirements for receptor binding and activation in this model system, a number of analogues of L-alanine were tested using a neurophysiological assay and a competitive ligand binding assay. These assays measured the ability of analogues to activate taste receptors and to displace L-[(3)H]alanine from L-alanine binding sites. Of those derivatives with modifications of the sidechain, L-serine, glycine,β-chloro-L-alanine and 1-amino-cyclopropane-1-carboxylic acid were the most potent analogues with IC50s similar to and neural responses slightly decremented from that of L-alanine. Derivatives containing branched sidechains or sidechains of otherwise increased volume were considerably less active. All modifications of theα-carboxylic acid and theα-amine, including amides, esters and various isosteres, led to substantial reduction in the analogues' ability to displace L-[(3)H]alanine and, in most cases, very weak stimulatory capability. However, L-lactic acid was a reasonably strong stimulus, but a poor competitor, suggesting that it acts at a different receptor site. Overall, these results indicate the importance of the charged amine and carboxylic acid groups for binding to and activation of the receptor for L-alanine. Moreover, modifications around the chiral center of L-alanine support the hypothesis that receptor binding and activation are separate processes in this model taste system.

Interactions between Immunogenic Peptides and MHC Proteins

The MHC class-I and class-II molecules are highly polymorphic membrane proteins, which bind and transport to the surface of cells peptide fragments of intact proteins. The peptide-MHC complexes are recognized by the antigen-specific receptor of T lymphocytes and are the basis by which the cellular immune system distinguishes self from nonself. In order to perform this function, MHC proteins simultaneously display a large spectrum of structurally divergent peptides for a sufficiently long period of time for the T cell repertoire to scan the cell effectively. Consistent with the protein's biological role, the rates of association and dissociation at physiological pH are very slow relative to other known receptor-ligand interactions. The mechanism by which the proteins do this is still poorly understood, but recent experimental results indicate that the rate determining step may be a conformational change that results in the entrapment of the peptide. A variety of binding assays have been developed that allow study of the detailed kinetics and specificity of the interaction. The optimal peptide length for binding is between 8 and 12 amino acids with the central 5-7 residues contributing the majority of the specific contacts. Determining the conformation of bound peptides has been hampered by the inherent ability of the receptor to bind manifold sequences. Consequently, strategies employing monosubstituted analogs have had only limited success. Approaches using biotinylated amino acids and other bulky substituents or multiple substitutions have generated more information. Recent experiments demonstrating that peptides with polyalanine, polyproline, or polyglycine bind well to MHC proteins have proven that the structural requirements for binding are quite minimal. In fact, a significant factor of the selectivity for binding appears to be the avoidance of deleterious contacts, rather than the need for a large number of critical interactions. Binding experiments also have shown that several peptides can bind a large number of MHC class-I and class-II alleles. The degenerate binding indicated that the binding site of MHC proteins must have a significant number of conserved features. Solution of the crystal structures of the MHC class-I alleles A2 and Aw68 has identified a putative antigen-combining site whose overall dimensions were quite similar between the two structures. The detailed surface topology of the site varied between the two alleles due to the size and chemical properties of the side chains of the polymorphic amino acids composing the cleft.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular modeling of antibody–antigen complexes between the Brucella abortus O-chain polysaccharide and a specific monoclonal antibody

A molecular model of the binding site of an anti-carbohydrate antibody (YsT9.1) has been developed using computer-assisted modeling techniques and molecular dynamics calculations. Sequence homologies among YsT9.1 and the Fv regions of McPC603, J539 and human Bence--Jones protein REI, all of which have solved crystal structures, provided the basis for the modeling. The groove-type combining site model had a topography which was complementary to low energy conformers of the polysaccharide, a Brucella O-antigen, and the site could be almost completely filled by a pentasaccharide epitope in either of two docking modes. Putative interactions between this epitope and the antibody are consistent with the known structural requirements for binding and lead to the design of oligosaccharide inhibitors that probe the veracity of the modeled docked complex. Ultimately both the Fv model and the docked complex will be compared with independent crystal structures of YsT9.1 Fab with and without pentasaccharide inhibitor, currently at the stage of refinement.

Indolealkylamine analogs share 5-HT 2 binding characteristics with phenylalkylamine hallucinogens

Twenty-one indolealkylamines, some of which are known to be psychoactive in man, were examined for their binding interactions with rat brain cortical 5-HT 2 receptors labeled with the antagonist radioligand [ 3H]ketanserin in order to develop structure-activity relationships for binding at these sites. Features investigated included aromatic, α-methyl and terminal amine substituents. 4-Methoxy and 5-methoxy substitution impart a higher affinity than 6- or 7-methoxy substitution; a 7-hydroxyl group essentially abolishes affinity whereas a 7-methyl or 7-bromo group enhances affinity. α-Methylation has little effect on affinity and, in the one case examined, the S(+) isomer of α-methyltryptamine was essentially equipotent with its racemate and twice as potent as its R(−) enentiomer. Terminal amine methylation results in a small but progressive decrease in affinity in the order: primary amine > dimethylamine > diethylamine. Similarities were noted between these structural requirements for binding and those of the phenalkylamines. Selected compounds (5-methoxytryptamine, N,N-dimethyltryptamine, 5-methoxy-N,N-diethyltryptamine and 5-methoxy-N,N-dimethyltryptamine) were further examined by two-site analysis of displacement studies for [ 3H]ketanserin specific binding. Hill co-efficients were significantly less than unity and computer-assisted analysis indicated that a two-site model better fit the data than a one-site model. In displacement studies using the putative agonist radioligand [ 3H]DOB to label 5-HT 2 receptors affinities were 10-100-fold higher than those using [ 3H]ketanserin. These results are also consistent with earlier findings using psychoactive phenalkylamines in competition studies for radiolabelled 5-HT 2 receptors. In summary, the interactions of indolealkylamines and phenalkylamines with 5-HT 2 receptors have many characteristics in common, supporting the hypothesis that 5-HT 2 receptors may mediate the psychoactive properties of indolealkylamines.

ChemInform Abstract: Structurally Specific Binding of Halogenated Biphenyls to Thyroxine Transport Protein.

Prealbumin is a major thyroxine binding protein in blood that has been well studied crystallographically and has also been proposed as a model for the thyroxine nuclear receptor in tissue. The high-affinity T4 binding site in prealbumin gave a linear plot on Scatchard analysis. The interactions of selected polychlorinated biphenyls (PCBs) with prealbumin have been studied with use of computer graphics and predictions made regarding relative binding affinities for such structures. These modeling predictions were tested by using competitive binding experiments involving selected PCBs and hydroxylated derivatives as soluble structural probes. The results are in excellent agreement with the modeling predictions and demonstrated that these compounds can be highly effective (3-8 times better than thyroxine itself) competitive binding ligands for thyroxine specific binding sites in prealbumin. Laterally (3,3',5,5'-) substituted PCBs show the highest binding activity and further substitution on nonlateral (2,2',6,6'-) positions lowers binding activity. Lateral chlorine substitution was common to all PCBs studied that showed high binding affinities. The binding model may also suggest a preference for a linear and symmetrical molecular shape. These structural requirements for binding are substantially consistent with the structure-toxicity relationship for closely related compounds of environmental interest. These specific binding interactions are likely to modulate the distribution of certain PCBs and related compounds and alter hormone-protein interactions that are responsible for the maintenance of normal thyroid status. Since prealbumin is also a model for the putative thyroxine nuclear receptor in tissue, our hypothesis that high toxicity of certain halogenated aromatic hydrocarbons is at least in part due to their thyromimetic properties is further supported.

Binding of rubratoxin B to mouse hepatic microsomes and in vitro effects of the mycotoxin on polysome binding to microsomal membranes as measured by the activity of an enzyme catalyzing disulphide interchange

The mycotoxin rubratoxin B binds to some component(s) of the microsomal fraction of mouse liver. The α-β unsaturated lactone ring was a structural requirement for binding. The rubratoxin B-microsomal adduct was not separated by Sephadex G-25 filtration. The binding occurred rapidly and persisted for at least 2 hr. Sulfhydryl reagents, dithiothreitol and cysteine partially protected from and/or reversed binding. Rubratoxin B inhibited the in vitro activity of an enzyme located in the endoplasmic reticulum associated with binding of the ribosomes to the membrane.

Structural requirements for binding of bovine tRNATrp with avian myeloblastosis virus DNA polymerase.

Avian DNA polymerases use host tRNATrp as the primer for transcription. Bovine tRNATrp has been previously shown to be a biologic substitute for the avian primer. A bovine tRNATrp fragment has been identified as having a high binding affinity for the polymerase. The fragment is assigned to be 67 nucleotides, and contains most of the elements required to maintain the secondary and tertiary structure of tRNATrp.