Effects of K+ concentration on inhibition of protein synthesis by trimer and tetramer triphosphates of 2',5'-oligoadenylates in rabbit reticulocyte lysates

The inhibition of protein synthesis by trimer or tetramer triphosphates of 2',5'-oligoadenylates (2-5A) in rabbit reticulocyte lysates is dependent on the concentration of K+ in the assay mixture. Based on studies using mRNA-dependent rabbit reticulocyte lysates and the degradation of [3H] polyadenylated mRNA, the greater inhibitory effects of trimer or tetramer triphosphates at the higher K+ is shown to be due to a more active 2-5A-dependent endoribonuclease (RNase L).     

Cordycepin Analogs of 2–5 a as Activators of RNase L: Study of the Structural Requirements for RNase L Activation

Abstract

Enzymatically and chemically synthesized cordycepin analogs of 2–5A^? trimer and tetramer were found to be biologically active as protein synthesis inhibitors in intact cultured human fibroblast and murine L929 cells ^1,2. In rabbit reticulocyte lysates, the cordycepin tetramer analog of 2–5A inhibits protein synthesis through binding to and activation of RNase L³. Our present results using L929 cell extracts provide direct evidence that the cordycepin analogs of 2–5A can bind to and activate RNase L.     

Functional analysis of 2-5A-dependent RNase and 2-5a using 2',5'-oligoadenylate-cellulose

2-5A is an intracellular effector that has been implicated in interferon action, hormonal regulation, and cell growth control. 2-5A action is mediated through its activation of 2-5A-dependent RNase (RNase L, RNase F). Affinity resins [2-5A-cellulose and core (2-5A)-cellulose] were chemically synthesized for purification and immobilization of 2-5A-dependent RNase from mouse L cells and rabbit reticulocyte lysates. The breakdown of poly(U)-[3'-32P]Cp to acid-soluble fragments was demonstrated using the 2-5A-dependent RNase:2-5A -cellulose complex; this activity was enhanced by adding (free) 2-5A. In contrast, RNase activity was measured from the 2-5A-dependent RNase:core (2-5A)-cellulose complex only after the addition of free 2-5A. The rabbit reticulocyte 2-5A-dependent RNase is activated only by tetramer or higher oligomers of 2-5A; therefore there was breakdown of poly(U)-[3'-32P]Cp using core (2-5A)-cellulose-bound reticulocyte 2-5A-dependent RNase after addition of tetramer 2-5A but there was no poly(U) degradation in the presence of trimer 2-5A. The absence of significant general nuclease in the assays was demonstrated by the resistance to breakdown of poly(C)-[3'-32P]Cp (not susceptible to 2-5A-dependent RNase). Moreover, core (2-5A)-cellulose was used to develop a sensitive (subnanomolar) assay for the detection of authentic 2-5A. 2-5A, or the material to be tested, was added to mouse L-cell 2-5A-dependent RNase:core (2-5A)-cellulose complex in the presence of poly(U)-[3'-32P]Cp. The concentration of 2-5A in the sample could be measured from the amount of poly(U) degradation. Several closely related analogs of 2-5A were tested and found to be completely inactive. The technology described herein may be applied to the study of the regulation of 2-5A-dependent RNase, the detection of 2-5A from cells and tissues, and other aspects of the 2-5A system.     

2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)     

2 and 8-Azido ATP: Photoaffinity Labeling of 2-5A Synthetase,Enzymatic Synthesis of 2 and 8-Azido Anamgs of 2-5A for Use as Photoaffinity Probes of Rnase L

Abstract

The 2-5A/RNase L system is widely accepted to be part of the antiviral mechanism of interferon^{1, 2}an and may also regulate cell growth3, where 2-5A exerts its biological effects by activating RNase L. Numerous 2-5A analogs have been synthesized with the goal of binding to, but not activating, RNase L. However, these analogs have had limitations when studied In vitrQ. We have reported on the unique properties of 2-5A molecules in which Rp and Sp chirality have been introduced into the 2-5A backbone to form the phosphorothioate analogs of 2-5A^4-6. By chiral modification of the 2-5A backbone, we have examined the stereochemical requirements for binding to and activation of RNase L. In order to elucidate the mechanism by which 2-5A binds to and activates RNase L, it is essential to ascertain the interactions in the nucleotide binding domain of RNase L and/or other 2-5A binding proteins. By employing photoaffinity labeling using enzymatically synthesized 2 and 8 azido photoprobes of 2-5A, we have characterized the 2- and 8-azido trimer 5′-triphoshate photoprobes of 2-5A and described the biological properties of these photoprobes (Figure 1) of 2-5A and their application in photolabeling of RNase L and/or other 2-5A binding proteins? have been reported. 2- and 8-azidoATP are substrates for the 2-5A synthetase from IFN-8-treated HeLa cell extracts and from rabbit reticulocyte lysates, but not for highly purified 2-5A synthetase from rabbit reticulocyte lysates'. W irradiation results in the photoinsertion of 2- and 8-azidoATP into the catalytic site of the 2-5A synthetase. Analysis of Scatchard plots of the 2-5A synthetase suggests the presence of high affinity and low affinity binding sites that may correspond to the acceptor and the 2′-adenylation sites of the enzyme.     

Phosphorothioate Analogs OF 2-5A: Activation / Inhibition of Rnase L and Inhibition of HIV-1 Reverse Transcriftase

Abstract

Chemically and enzymatically synthesizeddiastereomeric 2′,5′- phosphorothioate dimer, trimer and tetramer cores and their 5′ - mono- and triphosphates demonstrate marked differences in their ability to bind to and activate RNase L from L929 cell extracts in radiobinding, core-cellulose and rRNA cleavage assays¹,² (Fig. 1). These are the first 2-5A cores that are able to bind to and activate Mase L. The enzymatically synthesized 2′,5′- phosphorothioate dimer and trimer 5′-triphosphates can also bind to and activate mase L¹.     

2',5'-Oligoadenylates chiral at phosphorus: enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioate trimer and tetramer analogues synthesized from (SP)-ATP alpha S

The enzymatic synthesis and characterization of (RP)-2',5'-AMPS trimer and tetramer (SP)-5'-O-(1-thiotriphosphates) from chirally substituted (SP)-[alpha-35S]ATP alpha S by 2',5'-oligoadenylate synthetase from interferon-treated L cell extracts are described. The (RP)-ATP alpha S isomer is not a substrate for the synthetase. The identification of the trimer and tetramer analogues (molar ratio 70:30) was accomplished by high-performance liquid chromatography and subsequent separation by charge using DEAE-cellulose thin-layer chromatography. The digestion of the analogue by snake venom phosphodiesterase I (SVPD) to [alpha-35S]ATP alpha S and [35S]AMPS but not by T2 RNase demonstrated the presence of the 2',5' linkage. The assignment of RP configuration of the 2',5'-phosphorothiodiester linkage was based on the highly specific stereoselectivity of SVPD for RP diastereomers [Burgers, P. M. J., & Eckstein, F. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4978-4800; Bryant, F. R., & Benkovic, S. J. (1979) Biochemistry 18, 2825-2828; Nelson, P. S., Bach, C. T., & Verheyden, J. P. H. (1984) J. Org. Chem. 49, 2314-2317]. This suggests that the synthesis of the phosphorothioate analogues proceeded via inversion of configuration at the chiral phosphorus of (SP)-ATP alpha S. The putative (RP)-2',5'-AMPS tetramer (SP)-5'-O-(1-thiotriphosphate) displaced the 2',5'-p3A4[32P]pCp analogue from 2',5'-oligoadenylate-dependent endonuclease 5 times more efficiently than did equimolar concentrations of authentic 2',5'-adenylate tetramer triphosphate. Furthermore, in studies using the calcium phosphate coprecipitation technique, the 2',5'-phosphorothioate trimer and tetramer analogues inhibited protein synthesis better than did 2',5'-adenylate trimer and tetramer triphosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

5′-O-Dephosphorylated 2′,5′-oligoadenylate (2-5A) with 8-methyladenosine at the 2′-terminus activates human RNase L

Human ribonuclease L (RNase L), an interferon-induced endoribonuclease, becomes enzymatically active after binding to 2-5A. The 5′-phosphoryl group of 2-5A is reportedly necessary for the conformational change leading to RNase L activation. However, we found that 5′-O-dephosphorylated 2-5A tetramer analogs with 8-methyladenosine at the 2′-terminus were more effective as an activator of RNase L than the parent 2-5A tetramer. Introduction of 8-methyladenosine is thought to induce a dramatic shift of 2-5A in the binding site of RNase L.     

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at 相似文献   

Synthesis and biological activities of beta-alanyltyrosine derivative of 2',5'-oligoadenylate, and its use in radiobinding assay for 2',5-oligoadenylate

beta-Alanyltyrosine derivative of 2',5'-tetraadenylate 5'-triphosphate, pppA2'p5'A2'-p5'A2'p5'A-beta-Ala-Tyr was prepared by coupling of periodate-oxidized pppA2'p5'-A2'p5'A2'p5'A with beta-alanyltyrosine methyl ester, followed by reduction with sodium cyanoborohydride. Its stability to 2',5'-phosphodiesterase and phosphatase was investigated in mouse L cell extract. The 5'-triphosphate of the compound was cleaved gradually to form the 5'-dephosphorylated derivative, A2'p5'A2'p5'A2'p5'A-beta-Ala-Tyr, followed by slow degradation of the 2',5'-phosphodiester bond. On the other hand, pppA2'p5'A2'p5'A2'p5'A was hydrolyzed very quickly under the same conditions. The tetramer derivative bound tightly to the 2',5'-oligoadenylate-dependent endoribonuclease in rabbit reticulocyte lysate or mouse L cell extract and inhibited protein synthesis of mouse L cells more effectively than the unmodified 2',5'-tetraadenylate 5'-triphosphate. The corresponding trimer derivative had slightly weaker activities than the unmodified trimer for binding to the endoribonuclease and for inhibition of protein synthesis. The compound, pppA2'p5'A2'p5'-A2'p5'A-beta-Ala-Tyr, was iodinated easily at the tyrosine residue with 125I, giving a high-specific-radioactivity derivative which was used as a radio-labeled probe in a radiobinding assay for 2',5'-oligoadenylate.     

3'-Fluoro-3'-deoxy analogs of 2-5A 5'-monophosphate: binding to 2-5A-dependent endoribonuclease and susceptibility to (2'-5')phosphodiesterase degradation

Analogs of 2-5A trimer 5'-monophosphate (2'-5')pA3,p5'A2'p5'A2'p5'A containing 9-(3-fluoro-3-deoxy-c-D-xylofuranosyl)adenine (AF) or 3'-fluoro-3'- deoxyadenosine (AF) at different positions of the chain have been synthesized. All of them were compared with (2'-5')pA3 and (2'-5')pA2 (3'dA) by (i) their ability to bind to 2-5A-dependent endoribonuclease(RNase L) of mouse L cells and of rabbit reticulocyte lysates and (ii) their susceptibility to the degradation by the (2'-5')phosphodiesterase activity. The results of this study suggest that the oligonucleotide conformation is important for its biochemical properties.     

Only one 3'-hydroxyl group of ppp5' A2'p5'A2'p5' A (2-5A) is required for activation of the 2-5A-dependent endonuclease

To investigate the relative importance of each of the ribose 3'-hydroxyl groups of 2-5A (ppp5' A2'p5'A2'-p5' A) in determining binding to and activation of the 2-5A-dependent endonuclease (RNase L), the 3'-hydroxyl functionality of each adenosine moiety of 2-5A trimer triphosphate was sequentially replaced by hydrogen. The analog in which the 5'-terminal adenosine was replaced by 3'-deoxyadenosine (viz. ppp5'(3'dA)-2'p5' A2'p5' A) was bound to RNase L as well as 2-5A itself and was only 3 times less potent than 2-5A as an activator of RNase L. On the other hand, when the second adenosine unit was replaced by 3'-deoxyadenosine (viz. ppp5' A2'p5'(3'dA)2'p5' A), binding to RNase L was decreased by a factor of eight relative to 2-5A trimer and, even more dramatically, there was a 500-1000-fold drop in ability to activate the 2-5A-dependent endonuclease. Finally, when the 3'-hydroxyl substituent was converted to hydrogen in the 2'-terminal residue of 2-5A, a significant increase in both binding and activation ability occurred. We conclude that only the 3'-hydroxyl group of the second (from the terminus) nucleotide residue of 2-5A is needed for effective activation of RNase L.     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatically synthesized 2',5'-phosphorothioate dimer and trimer: unequivocal structural assignment and activation of 2',5'-oligoadenylate-dependent endoribonuclease

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate-dependent endoribonuclease (RNase L), chirality has been introduced into the 2',5'-oligoadenylate (2-5A, p3An) molecule to give the Rp configuration in the 2',5'-internucleotide backbone and the Sp configuration in the alpha-phosphorus of the pyrophosphoryl moiety of the 5'-terminus. This was accomplished by the enzymatic conversion of (Sp)-ATP alpha S to the 2',5'-phosphorothioate dimer and trimer by the 2-5A synthetase from lysed rabbit reticulocytes. The most striking finding reported here is the ability of the 2',5'-phosphorothioate dimer 5'-triphosphate (i.e., p3A2 alpha S) to bind to and activate RNase L. p3A2 alpha S displaces the p3A4[32P]pCp probe from RNase L with an IC50 of 5 X 10(-7) M, compared to an IC50 of 5 X 10(-9) M for authentic p3A3. Further, p3A2 alpha S activates RNase L to hydrolyze poly(U)-3'-[32P]pCp (20% at 2 X 10(-7) M), whereas authentic p3A2 is unable to activate the enzyme. Similarly, the enzymatically synthesized p3A2 alpha S at 10(-6) M activated RNase L to degrade 18S and 28S rRNA, whereas authentic p3A2 was devoid of activity. p3A3 alpha S was as active as authentic p3A3 in the core--cellulose and rRNA cleavage assays. The absolute structural and configurational assignment of the enzymatically synthesized p3A2 alpha S and p3A3 alpha S was accomplished by high-performance liquid chromatography, charge separation, enzymatic hydrolyses, and comparison to fully characterized chemically synthesized (Rp)- and (Sp)-2', 5'-phosphorothioate dimer and trimer cores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the activation site of ribonuclease L with new N6-substituted 2',5'-adenylate trimers

2-5A trimer [5'-monophosphoryladenylyl(2'-5')adenylyl(2'-5')adenosine] activates RNase L. While the 5'-terminal and 2'-terminal adenosine N(6)-amino groups play a key role in binding to and activation of RNase L, the exocyclic amino function of the second adenylate (from the 5'-terminus) plays a relatively minor role in 2-5A's biological activity. To probe the available space proximal to the amino function of the central adenylate of 2-5A trimer during binding to RNase L, a variety of substituents were placed at that position. To accomplish this, the convertible building block 5'-O-dimethoxytrityl-3'-O-(tert-butyldimethylsilyl)-6-(2,4-dinitrophenyl)thioinosine 2'-(2-cyanoethylN,N-diisopropylphosphoramidite) was prepared as a synthon to introduce 6-(2,4-dinitrophenyl)thioinosine into the middle position of the 2-5A trimer during automated synthesis. Post-synthetic treatment with aqueous amines transformed the (2,4-dinitrophenyl)thioinosine into N(6)-substituted adenosines. Assays of these modified trimers for their ability to bind and activate RNase L showed that activation activity could be retained, albeit with some sacrifice compared to unmodified p5'A2'p5'A2'p5'A. Thus, the spatial domain about this N(6)-amino function could be available for modifications to enhance the biological potency of 2-5A analogues and to ligate 2-5A to targeting vehicles such as antisense molecules.     

Doxifluridine-conjugated 2-5A analog shows strong RNase L activation ability and tumor suppressive effect

RNase L is activated by 2′,5′-oligoadenylates (2-5A) at subnanomolar levels to cleave single-stranded RNA. We previously reported the hypothesis that the introduction of an 8-methyladenosine residue at the 2′-terminus of the 2-5A tetramer shifts the 2-5A binding site of RNase L. In this study, we synthesized various 5′-modified 2-5A analogs with 8-methyladenosine at the 2′-terminus. The doxifluridine-conjugated 8-methyladenosine-substituted 2-5A analog was significantly more effective as an activator of RNase L than the parent 5′-monophophorylated 2-5A tetramer and showed a tumor suppressive effect against human cervical cancer cells.     

3-Deazaadenosine analogues of p5'A2'p5'A2'p5'A: synthesis, stereochemistry, and the roles of adenine ring nitrogen-3 in the interaction with RNase L

Sequence-specific 3-deazaadenosine (c(3)A)-substituted analogues of trimeric 2',5'-oligoadenylate, p5'A2'p5'A2'p5'A, were synthesized and evaluated for their ability to activate human RNase L (EC 3.1.2.6) aiming at the elucidation of the nitrogen-3 role in this biochemical process. Substitution of either 5'-terminal or 2'-terminal adenosine with c(3)A afforded the respective analogues p5'(c(3)A)2'p5'A2'p5'A and p5'A2'p5'A2'p5'(c(3)A) that were as effective as the natural tetramer itself as activators of RNase L (EC(50)=1nM). In contrast, p5'A2'p5'(c(3)A)2'p5'A showed diminished RNase L activation ability (EC(50)=10nM). The extensive conformational analysis of the c(3)A-substituted core trimers versus the parent natural core trimer by the (1)H and (13)C NMR, and CD spectroscopy displayed close stereochemical similarity between the natural core trimer and (c(3)A)2'p5'A2'p5'A and A2'p5'A2'p5'(c(3)A) analogues, thereby strong evidences for the syn base orientation about the glycosyl bond of the c(3)A residue of the latter were found. On the contrary, an analogue A2'p5'(c(3)A)2'p5'A displayed rather essential deviations from the spatial arrangement of the parent natural core trimer.     

Microwave exposure alters the expression of 2-5A-dependent RNase

The effects of 2.45-GHz continuous-wave microwaves (SAR = 130 mW/g) on the expression of the interferon-regulated enzymes 2'-5'-oligoadenylate (2-5A) synthetase(s) and 2-5A-dependent endoribonuclease (RNase L) were studied in murine L929 cells. Cells growing as monolayers were removed from the substratum and placed in suspension culture for a 4-h sham or microwave exposure. The cells were returned to monolayer growth for 18 h, and then harvested and assayed to determine the amount of RNase L protein (via [32P]2-5A binding) and the specific activities of RNase L and 2-5A synthetase. Binding of radioactive 2-5A to RNase L for sham- and microwave-exposed samples was 14.5 and 36.4% above control, respectively (the microwave-exposed bound 19.0% more probe than the sham-exposed). The increases in 2-5A binding were accompanied by corresponding elevations of RNase L specific activity. In contrast, sham or microwave irradiation produced no alterations in 2-5A synthetase specific activity. No detectable differences were noted in the postexposure cell viability, plating efficiency, or proliferation rate. Also, there were no detectable differences in cell viability or plating efficiency between controls and cultures irradiated for 2 h when the temperature was simultaneously increased to above normal physiological limits (39 to 45 degrees C). The SAR (130 mW/g) and the power density (95 mW/cm2) used for the greater part of this study were nearly 20 times higher than the ANSI limit of 8 mW/g and 5 mW/cm2 for any 1 g of exposed human tissue.     

8-Azido double-stranded RNA photoaffinity probes. Enzymatic synthesis, characterization, and biological properties of poly(I,8-azidoI).poly(C) and poly(I,8-azidoI).poly(C12U) with 2',5'-oligoadenylate synthetase and protein kinase

The technique of photoaffinity labeling has been applied to the double-stranded RNA (dsRNA)-dependent enzyme 2',5'-oligoadenylate (2-5A) synthetase to provide a means for the examination of RNA-protein interaction(s) in the dsRNA allosteric binding domain of this enzyme. The synthesis, characterization, and biological properties of the photoaffinity probe poly[( 32P]I,8-azidoI).poly(C) and its mismatched analog poly[( 32P]I,8-azidoI).poly(C12U), which mimic the parent molecules poly(I).poly(C) and poly(I).poly(C12U), are described. The efficacy of poly[( 32P]I,8-azidoI).poly(C) and poly[( 32P]I,8-azidoI).poly(C12U) as allosteric site-directed activators is demonstrated using highly purified 2-5A synthetase from rabbit reticulocyte lysates and from extracts of interferon-treated HeLa cells. The dsRNA photoprobes activate these two 2-5A synthetases. Saturation of 2-5A synthetase is observed at 6 x 10(-4) g/ml poly[( 32P]I,8-azidoI).poly(C) following photolysis for 20 s at 0 degrees C. The photoincorporation of poly[( 32P]I,8-azidoI).poly(C) is specific, as demonstrated by the prevention of photoincorporation by native poly(I).poly(C). DNA, poly(I), and poly(C) are not competitors of poly[( 32P]I,8-azidoI).poly(C). Following UV irradiation of 2-5A synthetase with poly[( 32P]I,8-azidoI).poly(C), the reaction mixture is treated with micrococcal nuclease to hydrolyze azido dsRNA that is not cross-linked to the enzyme. A radioactive band of 110 kDa (the same as that reported for native rabbit reticulocyte lysate 2-5A synthetase) is observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The specific photolabeling of the 2-5A synthetase suggests that the azido dsRNA is intrinsic to the allosteric binding domain. The utility of poly[( 32P]I,8-azidoI).poly(C) for the detection of dsRNA-dependent binding proteins and the isolation of peptides at or near the allosteric binding site is discussed.     

Macromolecular binding equilibria in the lac repressor system: studies using high-pressure fluorescence spectroscopy

High hydrostatic pressure coupled with fluorescence polarization has been used to investigate protein subunit interactions and protein-operator association in lac repressor labeled with a long-lived fluorescent probe. On the basis of observation of a concentration-dependent sigmoidal decrease in the dansyl fluorescence polarization, we conclude that application of high hydrostatic pressure results in dissociation of the lac repressor tetramer. The 2-fold decrease in the rotational relaxation time and the high-pressure plateau are consistent with a tetramer to dimer transition. The volume change for tetramer dissociation to dimer is -82 +/- 5 mL/mol. The dissociation constant calculated from the data taken at 4.5 degrees C is 4.3 +/- 1.3 nM. The tetramer dissociation constant increases by a factor of 3 when the temperature is raised from 4.5 to 21 degrees C. A very small effect of inducer binding on the subunit dissociation is observed at 4.5 degrees C; the Kd increases from 4.5 to 7.1 nM. At 21 degrees C, however, inducer binding stabilizes the tetramer by approximately 0.8 kcal/mol. Pressure-induced monomer formation is indicated by the curves obtained upon raising the pH to 9.2. The addition of IPTG shifts the pressure transition to only slightly higher pressures at this pH, indicating that the stabilization of the tetramer by inducer is not as marked as that observed at pH 7.1. From the decrease in the polarization of the dansyl repressor-operator complexes, we also conclude that the application of pressure results their dissociation and that the volume change is large in absolute value (approximately 200 mL/mol). The lac repressor-operator complex is more readily dissociated upon the application of pressure than the tetramer alone, indicating that operator binding destabilizes the lac repressor tetramer.     

Effect of ribonuclease on the physico-chemical properties of estrogen receptor

Estrogen receptors (ER) from rat and rabbit uterine cytosol were examined for their sensitivity to ribonuclease (RNase). After RNase treatment, a major part of rabbit uterine ER was converted from the 7S to 3-4S form, and its binding to DNA-cellulose was increased by 40%. Similar treatment on rat uterine ER showed a shift from 7S to 4.5S, and the DNA-cellulose binding was stimulated by 20%. Measurement of endogenous RNase levels showed that lower RNase concentration in rabbit uterine cytosol coincided with larger stimulation of DNA-cellulose binding by exogenous RNase. These results indicate that a major part of 7S ER is susceptible to RNase, and cleavage of bound RNA seems to uncover additional binding sites for DNA. In contrast to the general thinking that 4S to 5S transformation is essential for nuclear binding, we have observed that RNase-treated rat uterine ER did not undergo such a transformation by warming at 25 degrees C, while DNA-cellulose binding of the receptors increased. Thus, temperature activation could occur independent of 4S to 5S transformation.