Tracing the history of the ubiquitin proteolytic system: The pioneering article

A series of findings made by several researchers during a two-decade period between the mid-1950s and mid-1970s raised the suspicion that the lysosome might not be the organelle that degrades the bulk of cellular proteins under basal conditions. These findings predicted the existence of a nonlysosomal, adenosine triphosphate (ATP)-dependent proteolytic system. Yet, following the initial discovery of such activity in a crude cell extract, it was a single article published in this journal [A. Ciechanover, Y. Hod, A. Hershko, A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes, Biochem. Biophys. Res. Commun. 81 (1978) 1100-1105], my first study as a graduate student of Avram Hershko, that made it clear that the system that catalyzes the activity is novel and complex, and does not follow the paradigm in the field of proteolysis where a single protease typically cleaves its substrate; here at least two components were required to carry out this activity, and one of them was an unusual, small, and heat-stable protein later identified as ubiquitin.     

细胞内一种耗能蛋白质降解途径的发现——2004年诺贝尔化学奖工作介绍

2004年诺贝尔化学奖授予Aaron Ciechanover,Avram Hershko和Irwin Rose三位科学家,以表彰他们在上世纪80年代发现了泛素介导的蛋白质降解过程。文章简单介绍了该现象的科学发现历程,并讨论了该科学发现历程给予我们的启示。     

The Ever Expanding Role of Ubiquitin and SUMO in Plant Biology

When it was discovered in the 1970s as a ubiquitous protein,ubiquitin, a 76-aminoacid peptide, had no assigned function. It was not until later that ubiquitin was found to be a necessary cofactor in a vital cellular process: the degradation of proteins.Work by Avram Hershko, Aaron Ciechanover, and Irwin Rose     

Functional heterogeneity of ubiquitin carrier proteins

In the formation of covalent ubiquitin-protein conjugates that occurs during ATP- and ubiquitin-dependent proteolysis in reticulocyte extracts, ubiquitin (Ub) is activated to a thiol ester of the activating enzyme E1 (via the Ub carboxyl terminus), transferred to low-molecular weight "carrier proteins" (E2s) to form E2-Ub thiol esters, and then transferred by a third enzyme (E3) to amino groups on target proteins (Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983) J. Biol. Chem. 258, 8206-8214). We report here the fractionation of Ub carrier proteins by molecular weight, and their characterization with respect to several activities. The Ub thiol ester forms of at least four of the five E2s catalyze Ub transfer to a number of small amines, in a reaction that does not require E3; only primary amines on primary carbons can serve as Ub acceptors. E3-independent Ub transfer to the small, basic proteins histones H2A and H2B, and cytochrome c, is also observed. The Ub thiol ester forms of two of the E2s were found to catalyze Ub transfer to cytochrome c. Only a single E2 functions in E3-dependent conjugate formation (with the substrates creatine phosphokinase, reduced/carboxymethylated serum albumin, and oxidized RNase) and in E3-dependent protein breakdown (with the substrate serum albumin). This E2 has a subunit molecular weight of 14,000 and migrates as a dimer on Sephacryl 200.     

Post-translational addition of an arginine moiety to acidic NH2 termini of proteins is required for their recognition by ubiquitin-protein ligase

Recent studies have shown that selection of proteins for degradation by the ubiquitin system occurs most probably by binding to specific sites of the ubiquitin-protein ligase, E3. A free alpha-NH2 residue of the substrate is one important determinant recognized by the ligase. Selective binding sites have been described for basic and bulky-hydrophobic NH2 termini (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698) and for alanine, serine, and threonine at the NH2-terminal position (Gonda, D. K., Bachmair, A., Wünning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). Proteins with acidic NH2-terminal residues are degraded by the ubiquitin system only following conversion of the acidic residue to a basic residue by the addition of an arginine moiety (Ferber, S., and Ciechanover, A. (1987) Nature 326, 808-811). Although the enzymes involved in this post-translational modification have been characterized, the underlying mechanism has been obscure. By using a chemical cross-linking technique, we demonstrate that proteins with acidic NH2 termini do not bind to E3 without prior modification of this residue by the addition of arginine. In contrast, proteins with a basic NH2-terminal residue bind to the ligase without any modification. The recognition of acidic NH2-terminal substrates by E3 is dependent upon the addition of all the components of the modifying machinery, arginyl-tRNA-protein transferase, arginyl-tRNA synthetase, tRNA, and arginine. The ligase-bound modified proteins are converted to ubiquitin conjugates in a "pulse-chase" experiment, indicating that the binding is functional and that the enzyme-substrate complex is an obligatory intermediate in the conjugation process. Chemical modification of the carboxyl groups, which results in their neutralization, generates substrates that bind to E3 without modification. This finding suggests that the amino-terminal binding site of E3 is negatively charged, and only positively charged amino-terminal residues may bind to it. Negatively charged (acidic) NH2-terminal residues will bind only following neutralization or reversal of the charge.     

Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents

Growing HepG2 cells contain 50,000 functional surface transferrin-binding sites (Ciechanover, A., Schwartz, A.L., and Lodish, H.F. (1983) Cell 32,267-275) and 100,000 intracellular sites. At saturating concentrations of [59Fe]transferrin, and under conditions in which protein synthesis is blocked, iron uptake is linear for several hours at a rate of 9,500 transferrin molecules/cell/min. Thus, each receptor must recycle a ligand, on the average, each 15.8 min. Surface-bound transferrin is rapidly endocytosed (t1/2 = 3.5 min). All of the iron remains within the cell, while the apotransferrin is rapidly (t1/2 = 5.0 min) secreted into the medium. Previously, we showed (Dautry-Varsat, A., Ciechanover, A., and Lodish, H.F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2258-2262) that exposure of a ferrotransferrin-receptor complex to medium of pH less than 5.0 results in dissociation of iron, but that apotransferrin remains bound to its receptor. If the pH is raised to 7.0, such as would occur when an acidic intracellular vesicle fuses with the plasma membrane, apotransferrin is very rapidly dissociated (t1/2 = 17 s at 37 degrees C) from its receptor. Taken together, these results indicate that transferrin remains bound to its receptor throughout the endocytic cycle. In the present study, we have directly measured all the kinetic parameters involved in the transferrin receptor cycle. They are similar to those of the asialoglycoprotein receptor in the same cell line, and can be described by a simple kinetic model. In the presence of lysosomotropic agents, ferrotransferrin binds to its surface receptor and is internalized normally. However, iron is not dissociated from transferrin, and ferrotransferrin recycles back to the cell surface and is secreted into the medium. We conclude that the low pH in endocytic vesicles is essential for the dissociation of iron from transferrin and its delivery to the cell, but is not required for recycling of transferrin, and presumably of its receptor.     

Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates

Ubiquitin-lysozyme conjugates have been used as substrates to identify an ATP-dependent protease from rabbit reticulocyte lysates. The enzyme, which has been partially purified by DEAE chromatography and glycerol gradient centrifugation, has an apparent molecular weight greater than 600,000 based on sedimentation and gel filtration. Whereas it degrades conjugated lysozyme molecules in the presence of ATP, the protease does not degrade free lysozyme molecules even upon addition of ubiquitin, lysozyme-ubiquitin conjugates, and ATP. Degradation of lysozyme conjugates is independent of added ubiquitin and occurs in fractions incapable of ubiquitin conjugation. Proteolysis is maximal at pH 7.8, inhibited by hemin, N-ethylmaleimide, or aurintricarboxylic acid, and proceeds with an apparent Arrhenius activation energy in the range of 27 +/- 5 kcal/mol. These properties are similar to those observed for the degradation of lysozyme conjugates in lysates indicating that the partially purified protease catalyzes the "second" ATP-utilizing reaction identified previously (Hough, R., and Rechsteiner, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 90-94; Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1619-1623; Tanaka, K., Waxman, L., and Goldberg, A. L. (1983) J. Cell Biol. 96, 1580-1585).     

The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins.

ts85, a cell line that harbors a mutant thermolabile ubiquitin-activating enzyme, E1, fails to degrade short lived proteins at the restrictive temperature (Ciechanover, A., Finley, D., and Varshavsky, A. (1984) Cell 37, 57-66). However, the involvement of the ubiquitin system in the degradation of long lived proteins (most cellular proteins fall in this category) has not been addressed. In the present study we show that upon shifting the mutant cells to the restrictive temperature, there is no change in the rate of degradation of long lived proteins. In contrast, shifting the wild-type cells (FM3A) to the high temperature is accompanied by a 2-fold increase in the rate of proteolysis of this group of proteins. This heat-induced accelerated degradation can be inhibited completely by NH4Cl and chloroquine. Similarly, exposure of the cells to starvation, a stimulus that activates the autophagic-lysosomal pathway, has no effect on the degradation of long lived proteins in the mutant cells after inactivation of E1. Under the same conditions, the degradation rate in the wild-type cells increases almost 4-fold. Analogous results were obtained using a different cell line that also harbors a thermolabile E1 (ts20 (Kulka, R. G., Raboy, B., Schuster, R., Parag, H. A., Diamond, G., Ciechanover, A., and Marcus, M. (1988) J. Biol. Chem. 263, 15726-15731)). Cycloheximide and 3-methyladenine, known inhibitors of formation of autophagic vacuoles, inhibit the heat-induced accelerated degradation of long lived proteins in wild-type cells. Taken together, the results suggest that 1) heat stress induces enhanced degradation of intracellular proteins; 2) the process occurs most probably in autophagic vacuoles; and 3) activation of ubiquitin is required for the formation of these vacuoles. As there is no change in the basal rate of degradation of intracellular proteins in the mutant cells at the restrictive temperature, it appears that the ubiquitin system is not involved in their breakdown.     

Purification, characterization, and rapid inactivation of thermolabile ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85

Conjugation of ubiquitin to certain proteins can trigger their degradation in the in vitro reticulocyte system. In order to determine whether ubiquitin conjugation serves as an intermediate step in the turnover of cellular proteins in vivo, it is necessary to isolate proteolytic intermediates, i.e. ubiquitin-protein adducts of specific cellular proteins. While the steady-state level of conjugates of rapidly turning over proteins is relatively high, that of long-lived proteins is presumably extremely low, and therefore undetectable. Therefore, mutant cell lines with conditionally altered function(s) of the ubiquitin system can serve as powerful tools in studying the degradation of stable cellular proteins. We have characterized a temperature sensitive cell cycle arrest mutant cell (ts85) with a thermolabile ubiquitin-activating enzyme (E1; Finley, D., Ciechanover, A., and Varshavsky, A. (1984) Cell 37, 43-55). Following incubation at the restrictive temperature (39.5 degrees C), these cells fail to degrade short-lived proteins (Ciechanover, A., Finley, D., and Varshavsky, A. (1984) Cell 37, 57-66). However, involvement of the ubiquitin system in the turnover of long-lived proteins has not been addressed in these cells. A slow rate of inactivation of E1 in vivo, and significant rate of cell death following long incubation periods at the restrictive temperature, make this question difficult to address experimentally. In the present study we show that incubation of the cells for 1 h at 43 degrees C leads to rapid inactivation of ubiquitin conjugation in the intact mutant cell. Following heat treatment, the cells can be incubated at 39.5 degrees C for at least 6 h in order to study the possible involvement of the system in the turnover of long-lived cellular proteins. The viability of the cells is excellent at the end of the incubation. Following extraction, we have shown that inactivation occurs much more rapidly in the cell lysate in vitro than in the intact cell (t1/2 of 10 min compared to 4 h at 39.5 degrees C). The enzyme from both the mutant cell and the wild-type cell was purified to homogeneity. The molecular mass of the native enzyme from both cells is approximately 220 kDa with a subunit molecular mass of about 108 kDa. The structure of the enzyme is therefore very similar to that purified from rabbit reticulocytes. At the permissive temperature, the enzymes from both cells catalyze ATP-PPi and ATP-AMP exchange in similar kinetics. However, at the high temperature, the mutated enzyme is at least 7-fold less stable than the wild-type enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ubiquitin-lysozyme conjugates. Purification and susceptibility to proteolysis

To produce ubiquitinated substrates for studies on ATP-dependent proteolysis, 125I-lysozyme was incubated in hemin-inhibited rabbit reticulocyte lysates. A portion of the labeled molecules became linked to ubiquitin in large covalent complexes. When these were partially purified and returned to uninhibited lysates containing ATP, the conjugated lysozyme molecules were degraded 10 times faster than free lysozyme. Purification of covalently modified lysozyme from hemin-inhibited lysates containing 125I-ubiquitin and 131I-lysozyme confirmed that both molecules were present in the complexes. The doubly labeled conjugates also permitted us to determine the fate of each molecule in uninhibited lysates. Besides degradation of lysozyme, there was a progressive release of intact lysozyme molecules from the complexes. This disassembly, which was the only fate of the complexes in the absence of ATP, proceeded through a series of smaller intermediates, several having molecular weights expected for ubiquitin-lysozyme conjugates, and eventually free lysozyme was regenerated. The behavior of labeled ubiquitin was similar, though not identical, to that of lysozyme. Even in lysates containing ATP ubiquitin emerged from the complex undegraded. Furthermore, ubiquitin was present in a greater number of species than was lysozyme. The demonstration that ubiquitin-lysozyme conjugates are rapidly degraded provides support for the hypothesis of Hershko, Rose, Ciechanover, and their colleagues that a key function of ubiquitin is to modify the proteolytic substrate. Further support for the hypothesis is presented in the following paper where we show that the conjugated lysozyme molecules are substrates for an ATP-dependent protease that does not degrade free lysozyme.     

Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system

Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341-1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed.     

Arsenite inhibits two steps in the ubiquitin-dependent proteolytic pathway

Eukaryotic cells possess a multienzyme ATP-dependent proteolytic pathway in which the small, highly conserved protein ubiquitin (Ub) acts as a cofactor. In this pathway, formation of a covalent Ub-substrate conjugate precedes ATP-dependent degradation of the substrate. Inorganic arsenite (AsO2-) inhibited Ub-dependent protein degradation in a concentration-dependent fashion, both in intact rabbit reticulocytes and in a reticulocyte lysate (fraction II). Concentrations of arsenite causing half-maximal inhibition in fraction II varied with the substrate, ranging from 0.025 mM (bovine alpha-lactalbumin) to 3.3 mM (reduced/carboxymethylated bovine serum albumin). Inhibition was rapidly reversed upon addition of dithiothreitol. Arsenite inhibited the Ub-dependent proteolytic pathway at one or both of two steps, depending on the substrate. 1) Proteins with acidic amino termini must be amino terminally arginylated, in a tRNA-dependent reaction, prior to becoming conjugated to Ub (Ferber, S., and Ciechanover, A. (1987) Nature 326, 808-811). Arsenite inhibited substrate arginylation, and therefore also inhibited Ub conjugation. The inhibited species appeared to be arginyl aminoacyl-tRNA transferase, since arsenite was without strong effect on the rate or extent of arginyl-tRNA formation in fraction II, yet almost completely inhibited arginine transfer from arginyl-tRNA to reduced/carboxymethylated bovine serum albumin. 2) Arsenite also inhibited Ub-substrate conjugate turnover, as shown in pulse-chase experiments. For a given substrate, degradative (protease-dependent) and Ub regenerative (isopeptidase-dependent) components of conjugate turnover were similarly inhibited by arsenite. The potency of this inhibition varied for different substrates. Monosubstituted trivalent arsenicals such as arsenite typically interact with sites containing vicinal sulfhydryl groups. Inhibition by arsenite of two steps in the Ub-dependent proteolytic pathway suggests that the relevant pathway components could possess this kind of structural/catalytic feature.     

Affinity purification of ubiquitin-protein ligase on immobilized protein substrates. Evidence for the existence of separate NH2-terminal binding sites on a single enzyme

Previous studies have indicated the existence of separate binding sites of ubiquitin-protein ligase, E3, specific for basic (Type I) or bulky hydrophobic (Type II) NH2-terminal amino acid residues of proteins. Another class (Type III) of protein substrates appeared to interact with E3 at regions other than the NH2 terminus (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698). In the present study we have used affinity chromatography on immobilized protein substrates to examine the question of whether the different binding sites belong to one E3 enzyme, or to different E3 species. Another objective was to develop a procedure for the extensive purification of E3. When a crude extract of reticulocytes is applied to Type I or Type II protein substrates linked to Sepharose, E3 becomes strongly bound to the affinity columns and is not eluted with salt at high concentration. However, the enzyme can be specifically eluted by a dipeptide that has an NH2-terminal residue similar to that of matrix-bound protein substrate. A 350-fold purification is obtained in this single step. Preparations of E3 purified on either Type I or Type II protein substrate affinity columns act on both types of protein substrates, indicating that the separate binding sites for basic and hydrophobic NH2-terminal residues belong to one enzyme. Another species of E3 that acts strongly on some Type III protein substrates does not bind to Type I or Type II protein substrate affinity columns.     

Characterization of the Taxol binding site on the microtubule. Identification of Arg(282) in beta-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol

Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically beta-tubulin. Our previous studies have indicated that [(3)H]3'-(p-azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. 269, 3132-3134) and [(3)H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-233 of beta-tubulin (Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, D. G. I., and Horwitz, S. B. (1995) J. Biol. Chem. 270, 20235-20238). The site of photoincorporation of a third photoaffinity analogue of Taxol, [(3)H]7-(benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [(3)H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues 277-293. Amino acid sequence analysis indicated that the photoaffinity analogue cross-links to Arg(282) in beta-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin dimer have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in beta-tubulin as determined by photoaffinity labeling and electron crystallography.     

Decay products of the S(3) state of the oxygen-evolving complex of photosystem II at cryogenic temperatures. Pathways to the formation of the S = 7/2 S(2) state configuration

The water-oxidizing complex of photosystem II cycles through five oxidation states, denoted S(i)() (i = 0-4), during water oxidation to molecular oxygen, which appears at the (transient) S(4) state. The recent detection of bimodal EPR signals from the S(3) state [Matsukawa, T., Mino, H., Yoneda, D., Kawamori, A. (1999) Biochemistry 38, 4072-4077] has drawn significant attention to this critical state. An interesting property of the S(3) state is the sensitivity to near-IR (NIR) light excitation. Excitation of the S(3) state by near-IR light at cryogenic temperatures induces among other signals a derivative-shaped EPR signal at g= 5 [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. The signal bears unexpected similarities to a signal observed earlier in samples that had undergone multiple turnovers and subsequently had been stored at 77 K for a week or longer [Nugent, J. H. A., Turconi, S., and Evans, M. C. W. (1997) Biochemistry 36, 7086-7096]. Recently, both signals were assigned to an S = 7/2 configuration of the Mn cluster [Sanakis, Y., Ioannidis, N., Sioros, G., and Petrouleas, V. (2001) J. Am. Chem. Soc. 123, 10766-10767]. In the present study, we employ bimodal EPR spectroscopy to investigate the pathways of formation of this unusual state. The following observations are made: (i) The g = 5 signal evolves in apparent correlation with the diminution of the S(3) state signals during the slow (tens of hours to several days range) charge recombination of S(3) with Q(A)(-) at 77 K. The tyrosyl radical D* competes with S(3) for recombination with Q(A)(-), the functional redox couple at cryogenic temperatures inferred to be D*/D(-). Transfer to -50 degrees C and above results in the relaxation of the g = 5 to the multiline and g = 4.1 signals of the normal S(2) state. (ii) The transition of S(3) to the state responsible for the g = 5 signal can be reversed by visible light illumination directly at -30 degrees C or by illumination at 4.2 K followed by brief (2 min) transfer to -50 degrees C in the dark. The latter step is required in order to overcome an apparent thermal activation barrier (charge recombination appears to be faster than forward electron transfer at 4.2 K). (iii) The "g = 5" state can be reached in a few tens of minutes at 4.2 K by near-IR light excitation of the S(3) state. This effect is attributed to the transfer of the positive hole from the Mn cluster to a radical (probably tyr Z), which recombines much faster than the Mn cluster with Q(A)(-). (iv) The above properties strongly support the assignment of the configuration responsible for the g = 5 signal to a modified S(2) state, denoted S(2)'. Evidence supporting the assignment of the S(2)' to a proton-deficient S(2) configuration is provided by the observation that the spectrum of S(2) at pH 8.1 (obtained by illumination of the S(1) state at -30 degrees C) contains a g = 5 contribution.     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioates from adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate)

The chiral and achiral phosphorothioate analogues of 2',5'-oligoadenylates (2-5A) have been enzymatically synthesized from the Sp and Rp isomers of adenosine 5'-O-(2-thiotriphosphate) [(Sp)-ATP beta S and (Rp)-ATP beta S, respectively] and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) by 2-5A synthetase from L929 cells and lysed rabbit reticulocytes. These 2',5'-phosphorothioate analogues were separated, purified, and structurally characterized. While ATP gamma S and (Sp)-ATP beta S were as efficient substrates for the 2-5A synthetase as was ATP, (Rp)-ATP beta S was more than 50-fold less efficient a substrate. The beta- and gamma-phosphorothioates were more resistant to enzymatic hydrolysis than was authentic 2-5A. Compared to 2-5A, there were marked differences in the biological activities of the 2',5'-phosphorothioates as determined by (i) binding to 2-5A-dependent endoribonuclease (RNase L), (ii) activation of RNase L to hydrolyze RNA, and (iii) inhibition of protein synthesis in intact L929 cells. These studies extend previous reports on the elucidation of the stereochemical requirements of 2-5A synthetase and RNase L [Karikó, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (first of three papers in this issue); Karikó, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (second of three papers in this issue)] with the phosphorothioate analogues of 2-5A.(ABSTRACT TRUNCATED AT 250 WORDS)