Poly(U)-Agarose Affinity Chromatography: Specific,Sensitivity Selectivity,and Affinity of Binding

These studie s were done to determine four basic intrinsic properties of poly(U)-agarose affinity columns. Specificity of binding studies demonstrated that binding to these columns is highly specific with >90% complementary binding and 3% noncomplementary binding. Sensitivity of binding studies indicated that a minimum sequence of 10 adenylates is required for detectable complementary binding. Selectivity of binding studies revealed that nonsequential adenylates in native RNAs and randomly distribut edadenylates in synthetic poly(A)-poly(C) co-polymers did not bind to poly(U)-agarose affinity columns. Whereas, affinity of binding studies demonstrated that A=U complementary base pairing is independent of chain-lengths of 25 a denylates and dependent of chain-lengths of <25 adenylates. Thus the data demonstrates that poly(U)-agarose affinity chromatography is scientifically sound and expedient for thedetection and isolation of poly(A)-containing cellular and viral RNAs.     

Purification of Avian Myeloblastosis Virus DNA Polymerase by Affinity Chromatography on Polycytidylate-Agarose

Polycytidylic acid [poly(rC)] covalently linked to cyanogen bromide-activated agarose is an effective affinity matrix for the RNA-dependent DNA polymerase from avian myeloblastosis virus. Poly(rC)-agarose is capable of binding large quantities of avian myeloblastosis DNA polymerase, which is then eluted by using a linear KCl gradient of increasing concentration. The DNA polymerase isolated from crude, detergent-disrupted virions by a single pass through columns of poly(rC)-agarose appears nearly homogeneous (approximately 90% pure) as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complete recovery of input enzymatic activity was obtained. Results suggest that polyribonucleotide columns may provide a high-yield, rapid method for the purification of oncornaviral DNA polymerase.     

Hydrophobic affinity chromatography of nucleic acids and proteins.

5' tritylated oligonucleotides binding hydrophobically to low trityl cellulose/sepharose (< 15 microMTr/ml) retain their hydrogen-bonding specificities for complementary sequences. This, constitutes a novel mode of attaching affinity ligands to solid supports, is more convenient than existing methods, and proceeds with 100% yield. The salt, dielectric constant and temperature dependence of these non-covalently anchored ligands permits the isolation of a variety of RNAs including fibroin mRNA. Medium trityl sepharose (15-40 microM Tr/ml) has a high binding specificity for poly A and poly A containing mRNA, equivalent to dT cellulose. Most proteins, including nucleic acid enzymes, bind to these columns and retain enzymatic activity, thus mimicking enzymes attached covalently to solid phases. A number of in vivo counterparts to this hydrophobically determined specificity are noted, as are homologies to nitro-cellulose filters.     

A basic isozyme of yeast glyceraldehyde-3-phosphate dehydrogenase with nucleic acid helix-destabilizing activity

A nucleic acid helix-destabilizing protein has been purified from Saccharomyces cerevisiae using affinity chromatographic techniques. Crude protein extracts at low ionic strength (approx. 0.05 M) were applied sequentially to tandem columns of native DNA-cellulose, aminophenyl-phosphoryl-UMP-agarose, poly(I . C)-agarose, poly(U)-cellulose and denatured DNA-cellulose. The 2 M NaCl eluant of the poly(U)-cellulose column was dialyzed to low ionic strength and recycled through native DNA-cellulose, poly(I . C)-agarose and poly(U)-cellulose. Purified helix-destabilizing protein eluted from the poly(U)-cellulose between 0.1 and 0.5 M NaCl. On the basis of enzymatic activity, immunological cross-reactivity, mobility on SDS gels, amino acid analysis and preliminary peptide mapping experiments, this material was identified as an isozymic fraction of glyceraldehyde-3-phosphate dehydrogenase. The major crystallizable isozyme of this enzyme from yeast is, however, considerably more acidic than the helix-destabilizing protein, and displays significantly lower helix-destabilizing activity. Stoichiometric levels of the isolated protein at low (approx. 0.01) ionic strength depress the Tm of poly(A-U) and poly [d(A-T)] by as much as 28 and 22 degrees C, respectively. Longer double helices, poly(A . U) and Clostridium perfringens DNA are also denatured by the helix-destabilizing protein, but at relatively slow rates. The binding of this protein to [3H]-poly(U) on nitrocellulose filters in [Na+]-dependent, with a 50% reduction at 0.09 M NaCl. Based on its effect on the circular dichroism spectrum of poly(A), the protein was shown to distort the conformation of the polynucleotide chain. An analogous protein from mammalian cells, P8, was also shown to depress poly(A-U) Tm.     

Synthesis of poly(ADP-ribose)-agarose beads: an affinity resin for studying (ADP-ribose)n-protein interactions.

Polymers of ADP-ribose bind chromatosomal histones in solution and may play a role in chromatin accessibility in vivo. We have enzymatically synthesized a poly(ADP-ribose) affinity resin to further characterize binding of nuclear proteins to ADP-ribose polymers. NAD+- and (ADP-ribose)-derivatized agarose beads were recognized as polymer acceptors by the nuclear enzyme poly(ADP-ribose) polymerase. This polymerase elongated the existing ligands by successive addition of exogenously available ADP-ribose residues to form polymers covalently linked to the agarose beads. Poly(ADP-ribose) formation on the beads was dependent on incubation time and the mode of ligand attachment to the agarose. The resulting poly(ADP-ribose)-derivatized agarose beads possessed polymers which closely resembled those modifying the ADP-ribose polymerase by the automodification reaction. Fractionation of rat liver nuclear lysate over the poly(ADP-ribose) resin revealed a strong affinity of H1 for ADP-ribose polymers, thereby supporting a role for poly(ADP-ribose) in chromatin functions. Poly(ADP-ribose)-agarose beads are extremely stable and will be useful not only for affinity studies, but also for mechanistic studies involving polymer elongation and catabolism.     

Sequence content of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

Oligo(uridylic acid)-containing [oligo(U+)] RNA was isolated from poly(adenylic acid)-containing [poly(A+)] mRNA from HeLa cells by using either formaldehyde pretreatment or poly(A) removal, both of which resulted in increased accessibility of oligo(U)-rich sequences to a poly(A)-agarose affinity column. In this report, we compared the sequence content of oligo(U+) RNA with that of molecules lacking oligo(U) [oligo(U-) RNA] by their relative hybridization to cDNA reverse-transcribed from poly(A+) mRNA and by comparison of their in vitro translation products synthesized in a rabbit reticulocyte lysate. Formaldehyde-modified poly(A+) RNA, treated to remove the formol adjuncts, was inactive as a template for in vitro protein synthesis; consequently, only depolyadenylated RNA, which retains its translatability, could be used in the translation studies. The hybridization kinetic experiments revealed that oligo(U+) RNA contained most of the sequence information present in oligo(U-) RNA but at a reduced level (ca. 25%), the majority of the oligo(U+) RNA sequences being poorly represented in the cDNA. This result was supported by one- and two-dimensional gel analysis of their in vitro translation products which showed that oligo(U+) RNA, although less effective as a template for translation than oligo(U-) RNA, coded for proteins, the most abundant of which were encoded by rare messages not highly represented in oligo(U-) RNA or the total poly(A+) RNA. Although some minor products were synthesized by both oligo(U+) and oligo(U-) RNA, at least 33 proteins were unique to or highly enriched in the pattern of products directed by oligo(U+) RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand-receptor interactions: detailed evaluation of occupancy-dependent affinity

The exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy-dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte-leukoagglutinin (LPHA) system, which closely mimics the IM9-insulin model. Reliable estimates of total binding capacity (728 ng/10(6) cells) essential to our report were calculated from a wide database by the least-squares model. At occupancies greater than or equal to 0.085, receptors are associated with low and fixed affinity (1.5 X 10(6) M-1), whereas at occupancies less than or equal to 0.085, affinity is high and fixed (1.8 X 10(8) M-1) or high but variable (1 X 10(7) M-1 to 1.5 X 10(6) M-1) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor-ligand complex dissociation velocity over a wide range of occupancies (0.01-0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of approximately equal to 0.004 and is magnified as ligand binding to high affinity receptors increases up to approximately equal to 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulating ligand-induced cell responses.     

Translation of mRNA for glutamate dehydrogenase and spectrophotometric procedure to follow the enzyme biosynthesis.

Heterogeneous poly (A)-mRNA fraction was isolated from rat liver microsomes using phenol-chloroform extraction, millipore filtration and poly (U)-agarose affinity chromatography. Obtained fractions were characterized with respect to their secondary structure and poly (A) content. Isolated poly (A)-mRNA fraction contained high template activity for glutamate dehydrogenase in cell-free systems with microsomes or polysomes. A spectrophotometric procedure to follow enzyme biosynthesis was also developed.     

The binding of spermine to polynucleotides and complementary oligonucleotides at near physiological ionic strength

The binding of [14C] spermine to polynucleotides has been studied by equilibrium dialysis and the data analysed by Scatchard plots. The binding of spermine to poly(A) shows a binding site for 1 spermine/140 nucleotides when measured in 0.2M NaCl at 5 degrees C. Poly(C) also has a similar sites; on the other hand poly(U) and poly(G) each have a binding site for 1 spermine/12 nucleotides. The addition of complementary di- or trinucleotides to either poly(A) or poly(U) affects their ability to bind spermine, in particular the high affinity site on poly(A) is no longer detectable. The effect of spermine, spermidine and putrescine on the binding of polynucleotides to complementary di- and trinucleotides was also studied. Spermine markedly increased the binding of both ApA and of ApApA to poly(U) whereas spermidine and putrescine had very little effect. In contrast spermine had little effect on the binding of either UpU or UpUpU to poly(A). These results suggest that spermine binding to oligo- and polynucleotides is dependent on the particular nucleotide combination involved and that spermine may therefore be able to act selectively within cells.     

The effect of the antibiotic edeine on tRNA interaction with A, P and E sites of Escherichia coli 70S ribosomes

Edeine inhibits poly(U)-dependent binding of tRNAPhe to the P and A sites simultaneously, both on 30S subunits and 70S ribosomes. Hence, edeine cannot be considered as antibiotic, "complementary" to tetracycline for selective adsorption of tRNA only to the P or to the A site. Further, edeine decreases the affinity constant of tRNAPhe for the P-site by more than two orders of magnitude, no matter poly(U) is present or not. Neither edeine nor tetracycline affect interaction of deacylated tRNAPhe with the E-site of E. coli 70S ribosomes.     

Separation of the high affinity insulin-like growth factor I receptor from low affinity binding sites by affinity chromatography

We have identified high and low affinity insulin-like growth factor I (IGF I)-binding sites with mean dissociation constants of 0.37 and 6.25 nM, respectively, in solubilized placental membranes. We have separated these sites and purified the high affinity IGF I receptor 1,300-fold, with an overall yield of 9.9%, using wheat germ agglutinin-Sepharose chromatography, insulin affinity chromatography, and IGF I affinity chromatography. The Scatchard plot of IGF I binding to the high affinity receptor is linear, suggesting the purification of a single homogeneous class of binding sites. Insulin is two orders of magnitude less effective than IGF I in competitively inhibiting IGF I binding to this receptor. The high affinity IGF I receptor is composed of alpha and beta subunits with apparent molecular weights of 135,500 and 96,200, respectively. IGF I at concentrations of greater than or equal to 50 ng/ml stimulates autophosphorylation of the beta subunit of the purified high affinity receptor 4.6-fold. Low affinity IGF I-binding sites run through the IGF I affinity column or are eluted from the insulin affinity column. The separation of IGF I receptors with different binding affinities by sequential affinity chromatography will make it possible to examine directly the determinants of receptor affinity.     

A method for isolation of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

When cytoplasmic polyadenylated ribonucleic acid [poly(A+)RNA] from HeLa cells was treated with ribonuclease H (RNase H) and oligodeoxythymidylate [oligo(dT)] to remove its 3'-poly(A) tail, an increased binding to poly(A)-agarose was observed. The bound material, which comprised 4-6% of the initial RNA, contained 65-80% of the oligo(uridylic acid) [oligo(U)] sequences generated by RNase T1 digestion. Oligo(U) isolated from the bound fraction was shown to be 83% U and to have a U/G ratio of 33. In contrast, oligo(U) from the unbound material was 77% U and had a U/G ratio of 13, suggesting that it is shorter and less U rich than the oligo(U) in the bound fraction. On sucrose gradients, oligo(U+)RNA consistently sedimented with a larger s value than oligo(U-) RNA. The oligo(U) content of oligo(U+) RNA suggests one oligo(U) tract of 33 nucleotides per RNA molecule of 2000-3000 residues.     

Cooperative, excluded-site binding and its dynamics for the interaction of gene 5 protein with polynucleotides

The binding of gene 5 protein to various single-stranded polynucleotides is investigated by fluorescence titrations and stopped-flow measurements. The association state of gene 5 protein itself is analyzed by equilibrium sedimentation: the monomer-dimer equilibrium found in the micromolar concentration range is described by a stability constant of 8 X 10(5) M-1. The fluorescence quenching upon binding to polynucleotides, studied over a broad concentration range and analyzed in terms of a cooperative excluded-site binding model, provides binding constants for "isolated" and for "cooperative" sites. The cooperativity for various ribo- and deoxyribopolymers is between 400 and 800 and is virtually independent of the ionic strength. The binding to isolated sites is strongly dependent upon the ionic strength; analysis in terms of polyelectrolyte theory indicates the compensation of 4 +/- 0.5 charges upon complex formation. The number of nucleotide residues covered by one protein molecule is also found to be 4 +/- 0.5 units. The affinity of gene 5 protein for polynucleotides increases in the series poly(C) less than poly(dA) less than poly(A) less than poly(U) much less than poly(dT); the binding constant for poly(dT) is roughly a factor of 1000 higher than that for the other polymers. Model studies with Lys-Tyr-Lys and Lys-Trp-Lys suggest that the preferential interaction with poly(dT) is not simply due to enhanced stacking interactions between the aromatic amino acids and the thymine residues. Stopped-flow reaction curves obtained by mixing of gene 5 protein with poly(dT) in the micromolar concentration range show three relaxation processes with time constants between 1 ms and 1 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloned human interferons alpha: differential affinities for polyinosinic acid and relationship between molecular structure and species specificity

The HuIFN-alpha A and HuIFN-alpha D interferons, produced by two independent recombinant bacterial clones, have different affinities for polyinosinic acid (poly I). The monomeric form HuIFN-alpha A (FMM), but not the HuIFN-alpha D, binds to poly (I)-agarose and is protected by poly (I) from thermal inactivation. Other subtypes of HuIFN-alpha A including the monomer SMM and oligomers have no affinity for this polynucleotide. In addition, these interferons show different target cell preferences in agreement with our previous suggestion (23) that the polynucleotide binding domain may be responsible for species specificity. Two significant observations are 1) the fractions of HuIFN-alpha D and HuIFN-alpha A unbound on poly (I)-agarose show higher antiviral inducing activity on heterologous (MDBK) than on homologous (WISH) cells, whereas they induce about the same activity of 2'5' oligoadenylate synthetase in these two cell lines. These fractions are also active on L929 cells. 2) The bound fraction of HuIFN-alpha A induces almost the same antiviral and 2'5' oligoadenylate synthetase activities in MDBK and in WISH cells but neither activity in L929 cells.     

Kinetic studies on the cleavage of oligouridylic acids and poly U by bovine pancreatic ribonuclease A

Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU greater than p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25 degrees C. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n greater than or equal to 2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1)+(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.     

Electrophoretic characterization of purified bovine, porcine, murine, rat, and human uterine estrogen receptors

The calf uterine estrogen receptor (E2R) in the presence of sodium molybdate has been purified, 7,000-fold by a single passage over an estradiol affinity column. A dominant 70,000-dalton band and two minor bands at 50,000 and 30,000 daltons were observed by electrophoretic analysis. These bands had been eluted using estradiol, sodium sulfocyanate, CHAPS, and HEPES (pH 7.4) with insulin as a carrier protein. The identities of the protein bands were initially confirmed by their failure to bind the affinity column when saturated with estradiol. This single step purification procedure was reproducible and rapid, with yields of 10-20%, providing 25% purity. Diffusion blot analysis, with specific 35S- and 125I-labeled monoclonal antibodies to E2R, confirmed that the 70,000-dalton band represented the estrogen receptor. Specificity was demonstrated by inhibition of binding of purified E2R by both estradiol and diethylstilbestrol but not testosterone, progesterone, corticosterone, aldosterone, or hydrocortisone. The relative binding affinity of the purified receptor was: ethynyl estradiol greater than 17 beta estradiol greater than estriol greater than or equal to estrone greater than or equal to 17 alpha-estradiol greater than mestranol. Pig, human, mouse, and rat uterine estrogen receptors were similarly purified with the affinity column. As with the calf uterine preparations, a dominant 70,000-dalton band with minor bands at 50,000 and 30,000 daltons was identified by diffusion blot analysis in all the species examined.     

The binding affinity of Ff gene 5 protein depends on the nearest-neighbor composition of the ssDNA substrate

The Ff gene 5 protein (g5p) is considered to be a nonspecific single-stranded DNA binding protein, because it binds cooperatively to and saturates the Ff bacteriophage single-stranded DNA genome and other single-stranded polynucleotides. However, the binding affinity Komega (the intrinsic binding constant times a cooperativity factor) differs by over an order of magnitude for binding to single-stranded polynucleotides such as poly[d(A)] and poly[d(C)]. A polynucleotide that is more stacked, like poly[d(A)], binds more weakly than one that is less stacked, like poly[d(C)]. To test the hypothesis that DNA base stacking, a nearest-neighbor property, is involved in the binding affinity of the Ff g5p for different DNA sequences, Komega values were determined as a function of NaCl concentration for binding to six synthetic sequences 48 nucleotides in length: dA48, dC48, d(AAC)16, d(ACC)16, d(AACC)12, and d(AAACC)9A3. The binding affinities of the protein for these sequences were indeed found to be related to the nearest-neighbor compositions of the sequences, rather than to simple base compositions. That is, the g5p binding site, which is spanned by four nucleotides, discriminates among these sequences on the basis of the relative numbers of nearest neighbors (AA, CC, and AC plus CA) in the sequence. The results support the hypothesis that the extent of base stacking/unstacking of the free, nonbound ssDNA plays an important role in the binding affinity of the Ff gene 5 protein.