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• W2023607888 abstract "Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA. α-Sarcin, the best characterized member of this family, is a potent cytotoxin that promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with nontoxic ribonucleases of the RNase T1 family. However, significant structural differences between these two groups of proteins are related to the presence of a long amino-terminal β-hairpin in ribotoxins and to the different length of their unstructured loops. The amino-terminal deletion mutant Δ(7–22) of α-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type protein to degrade rRNA in intact ribosomes. The results indicate that α-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the β-hairpin of the protein. In addition, this latter protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human rhabdomyosarcoma cells. Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA. α-Sarcin, the best characterized member of this family, is a potent cytotoxin that promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with nontoxic ribonucleases of the RNase T1 family. However, significant structural differences between these two groups of proteins are related to the presence of a long amino-terminal β-hairpin in ribotoxins and to the different length of their unstructured loops. The amino-terminal deletion mutant Δ(7–22) of α-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type protein to degrade rRNA in intact ribosomes. The results indicate that α-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the β-hairpin of the protein. In addition, this latter protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human rhabdomyosarcoma cells. Fungal extracellular ribonucleases are a diverse group of proteins, with RNase T1 being its best known representative (1Steyaert J. Eur. J. Biochem. 1997; 247: 1-11Crossref PubMed Scopus (94) Google Scholar). They show different substrate specificity, but most of them degrade RNA in a nonspecific manner. However, among this group of RNases, there is a family displaying an exquisite specificity because they are capable of cleaving a single phosphodiester bond of those present in the ribosome. These enzymes are named ribotoxins (2Lamy B. Davies J. Schindler D. Genetically Engineered Toxins. Marcel Dekker, New York1992: 237-257Google Scholar), and their target bond is located in the larger rRNA at a sequence known as the sarcin-ricin loop conserved in all prokaryota and eukaryota (3Correl C.C. Munishkin A. Chan Y.L. Ren Z. Wool I.G. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13436-13441Crossref PubMed Scopus (170) Google Scholar, 4Correll C.C. Wool I.G. Munishkin A. J. Mol. Biol. 1999; 292: 275-287Crossref PubMed Scopus (134) Google Scholar). This cleavage releases a characteristic oligonucleotide, the α-fragment (the oligonucleotide released from the 3′ end of the 28 S rRNA in the large ribosomal subunit by the action of α-sarcin). As a result ribosomes are inactivated, and protein biosynthesis is inhibited (5Schindler D.G. Davies J.E. Nucleic Acids Res. 1977; 4: 1097-1100Crossref PubMed Scopus (135) Google Scholar,6Endo Y. Wool I.G. J. Biol. Chem. 1982; 257: 9054-9060Abstract Full Text PDF PubMed Google Scholar), placing ribotoxins among the most potent inhibitors of translation (2Lamy B. Davies J. Schindler D. Genetically Engineered Toxins. Marcel Dekker, New York1992: 237-257Google Scholar, 7Kao R. Martı́nez-Ruiz A. Martı́nez del Pozo A. Crameri R. Davies J. Methods Enzymol. 2001; 341: 324-335Crossref PubMed Scopus (42) Google Scholar). In addition, ribotoxins are cytotoxic proteins because they are able to gain entry into some cells (8Turnay J. Olmo N. Jiménez J. Lizarbe M.A. Gavilanes J.G. Mol. Cell. Biochem. 1993; 122: 39-47Crossref PubMed Scopus (59) Google Scholar, 9Olmo N. Turnay J. González de Buitrago G. López de Silanes I. Gavilanes J.G. Lizarbe M.A. Eur. J. Biochem. 2001; 268: 2113-2123Crossref PubMed Scopus (130) Google Scholar). The best characterized member of this family of highly specific RNases is α-sarcin, which is a single polypeptide chain protein composed of 150 amino acid residues and secreted by the mold Aspergillus giganteus (10Olson B.H. Goerner G.L. Applied Microbiol. 1965; 13: 314-321Crossref PubMed Google Scholar, 11Sacco G. Drickamer K. Wool I.G. J. Biol. Chem. 1983; 258: 5811-5818Abstract Full Text PDF PubMed Google Scholar). This ribotoxin has been shown to be cytotoxic for many human tumor cells (8Turnay J. Olmo N. Jiménez J. Lizarbe M.A. Gavilanes J.G. Mol. Cell. Biochem. 1993; 122: 39-47Crossref PubMed Scopus (59) Google Scholar, 9Olmo N. Turnay J. González de Buitrago G. López de Silanes I. Gavilanes J.G. Lizarbe M.A. Eur. J. Biochem. 2001; 268: 2113-2123Crossref PubMed Scopus (130) Google Scholar, 10Olson B.H. Goerner G.L. Applied Microbiol. 1965; 13: 314-321Crossref PubMed Google Scholar) by producing the α-fragment, which leads to cell death via apoptosis (9Olmo N. Turnay J. González de Buitrago G. López de Silanes I. Gavilanes J.G. Lizarbe M.A. Eur. J. Biochem. 2001; 268: 2113-2123Crossref PubMed Scopus (130) Google Scholar). Endocytosis is the mechanism responsible for the cell internalization of α-sarcin (9Olmo N. Turnay J. González de Buitrago G. López de Silanes I. Gavilanes J.G. Lizarbe M.A. Eur. J. Biochem. 2001; 268: 2113-2123Crossref PubMed Scopus (130) Google Scholar), which is probably related to its ability to interact with phospholipid bilayers (12Gasset M. Martı́nez del Pozo A. Oñaderra M. Gavilanes J.G. Biochem. J. 1989; 258: 569-575Crossref PubMed Scopus (58) Google Scholar, 13Gasset M. Oñaderra M. Thomas P.G. Gavilanes J.G. Biochem. J. 1990; 265: 815-822Crossref PubMed Scopus (51) Google Scholar, 14Mancheño J.M. Gasset M. Lacadena J. Ramón F. Martı́nez del Pozo A. Oñaderra M. Gavilanes J.G. Biophys. J. 1994; 67: 1117-1125Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 15Mancheño J.M. Gasset M. Albar J.P. Lacadena J. Martı́nez del Pozo A. Oñaderra M. Gavilanes J.G. Biophys. J. 1995; 68: 2387-2395Abstract Full Text PDF PubMed Scopus (33) Google Scholar, 16Oñaderra M. Mancheño J.M. Lacadena J. De los Rı́os V. Martı́nez del Pozo A. Gavilanes J.G. Mol. Membr. Biol. 1998; 15: 141-144Crossref PubMed Scopus (11) Google Scholar). The three-dimensional structures of α-sarcin (17Pérez-Cañadillas J.M. Santoro J. Campos-Olivas R. Lacadena J. Martı́nez del Pozo A. Gavilanes J.G. Rico M. Bruix M. J. Mol. Biol. 2000; 299: 1061-1073Crossref PubMed Scopus (64) Google Scholar) and restrictocin (18Yang X.J. Moffat K. Structure. 1996; 4: 837-852Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), another member of the ribotoxin family, have been solved. This information has revealed that ribotoxins share a common structural core with the family of fungal extracellular nontoxic RNases, represented by RNase T1 (1Steyaert J. Eur. J. Biochem. 1997; 247: 1-11Crossref PubMed Scopus (94) Google Scholar), composed of a central antiparallel β-sheet packed against a small α-helix and a conserved active site located on the other side (see Fig. 1). Although proteins from the RNase T1 family display a low degree of sequence similarity with ribotoxins, RNase U2 is the member more closely related to α-sarcin (11Sacco G. Drickamer K. Wool I.G. J. Biol. Chem. 1983; 258: 5811-5818Abstract Full Text PDF PubMed Google Scholar, 19Mancheño J.M. Gasset M. Lacadena J. Martı́nez del Pozo A. Oñaderra M. Gavilanes J.G. J. Theor. Biol. 1995; 172: 259-267Crossref PubMed Scopus (28) Google Scholar). This RNase is a nontoxic protein secreted by the smut fungus Ustilago sphaerogena and composed of a single polypeptide chain of 104 amino acid residues (20Yoshida H. Methods Enzymol. 2001; 341: 28-41Crossref PubMed Scopus (62) Google Scholar). Whereas α-sarcin, a basic protein (pI > 8) (11Sacco G. Drickamer K. Wool I.G. J. Biol. Chem. 1983; 258: 5811-5818Abstract Full Text PDF PubMed Google Scholar), hydrolyzes a single bond in ribosomal RNA and interacts with membranes, RNase U2, a highly acidic protein (pI = ∼3) (21Satto S. Uchida T. Biochem. J. 1975; 145: 353-360Crossref PubMed Scopus (37) Google Scholar, 22Kanaya S. Uchida T. J. Biochem. (Tokyo). 1995; 118: 681-682Crossref PubMed Scopus (6) Google Scholar), causes extensive degradation of the RNA but does not interact with lipid bilayers and is not cytotoxic (23Martı́nez-Ruiz A. Garcı́a-Ortega L. Kao R. Lacadena J. Oñaderra M. Mancheño J.M. Davies J. Martı́nez del Pozo A. Gavilanes J.G. Methods Enzymol. 2001; 341: 335-351Crossref PubMed Scopus (48) Google Scholar). Nevertheless, both share a common three-dimensional structure pattern (17Pérez-Cañadillas J.M. Santoro J. Campos-Olivas R. Lacadena J. Martı́nez del Pozo A. Gavilanes J.G. Rico M. Bruix M. J. Mol. Biol. 2000; 299: 1061-1073Crossref PubMed Scopus (64) Google Scholar, 24Noguchi S. Satow Y. Uchida T. Sasaki C. Matsuzaki T. Biochemistry. 1995; 34: 15583-15591Crossref PubMed Scopus (41) Google Scholar) (see Fig. 1). In vitro assays have revealed that nanomolar concentrations of α-sarcin result in the specific hydrolysis of the large rRNA or a synthetic oligoribonucleotide mimicking the SRL (25Endo Y. Glück A. Chan Y.-L. Tsurugi K. Wool I.G. J. Biol. Chem. 1990; 265: 2216-2222Abstract Full Text PDF PubMed Google Scholar), but micromolar concentrations result in a less specific cleavage of the substrates (25Endo Y. Glück A. Chan Y.-L. Tsurugi K. Wool I.G. J. Biol. Chem. 1990; 265: 2216-2222Abstract Full Text PDF PubMed Google Scholar), showing only a preference for the 3′-side of purines. α-Sarcin can also cleave A/GpA dinucleotides, although with low specific activity (26Lacadena J. Martı́nez del Pozo A. Lacadena V. Martı́nez-Ruiz A. Mancheño J.M. Oñaderra M. Gavilanes J.G. FEBS Lett. 1998; 424: 46-48Crossref PubMed Scopus (37) Google Scholar, 27Lacadena J. Martı́nez del Pozo A. Martı́nez-Ruiz A. Pérez-Cañadillas J.M. Bruix M. Mancheño J.M. Oñaderra M. Gavilanes J.G. Proteins. 1999; 37: 474-484Crossref PubMed Scopus (53) Google Scholar, 28Pérez-Cañadillas J.M. Campos-Olivas R. Lacadena J. Martı́nez del Pozo A. Gavilanes J.G. Santoro J. Rico M. Bruix M. Biochemistry. 1998; 37: 15865-15876Crossref PubMed Scopus (65) Google Scholar). RNase U2 displays a low specificity beyond a strong preference for 3′-linked purine nucleotide phosphodiester bonds (A > G ≫ C > U) (29Rushizky G.W. Mozejko J.H. Rogerson D.J., Jr. Sober H. Biochemistry. 1970; 9: 4966-4971Crossref PubMed Scopus (17) Google Scholar, 30Uchida T. Egami F. The Enzymes. Academic Press, New York1971: 205-250Google Scholar). Both proteins, α-sarcin and RNase U2, are cyclizing RNases because they produce a 2′,3′-cyclic intermediate as a result of the cleavage reaction (26Lacadena J. Martı́nez del Pozo A. Lacadena V. Martı́nez-Ruiz A. Mancheño J.M. Oñaderra M. Gavilanes J.G. FEBS Lett. 1998; 424: 46-48Crossref PubMed Scopus (37) Google Scholar, 30Uchida T. Egami F. The Enzymes. Academic Press, New York1971: 205-250Google Scholar). However, the catalytic efficiency of RNase U2 against naked RNA, homopolynucleotides, or dinucleotides is several orders of magnitude higher (26Lacadena J. Martı́nez del Pozo A. Lacadena V. Martı́nez-Ruiz A. Mancheño J.M. Oñaderra M. Gavilanes J.G. FEBS Lett. 1998; 424: 46-48Crossref PubMed Scopus (37) Google Scholar, 30Uchida T. Egami F. The Enzymes. Academic Press, New York1971: 205-250Google Scholar, 31Martı́nez-Ruiz A. Garcı́a-Ortega L. Kao R. Oñaderra M. Mancheño J.M. Davies J. Martı́nez del Pozo A. Gavilanes J.G. FEMS Microbiol. Lett. 2000; 189: 165-169Crossref PubMed Scopus (9) Google Scholar). Thus, α-sarcin specifically cleaves a phosphodiester bond in ribosomes, whereas RNase U2 causes extensive digestion of the RNA and is a more efficient ribonucleolytic enzyme. It is therefore of interest to find out which portions of α-sarcin, absent in related fungal RNases, account for its ribonuclease specificity and cytotoxicity. Comparison of the three-dimensional structures of RNase U2 and α-sarcin (17Pérez-Cañadillas J.M. Santoro J. Campos-Olivas R. Lacadena J. Martı́nez del Pozo A. Gavilanes J.G. Rico M. Bruix M. J. Mol. Biol. 2000; 299: 1061-1073Crossref PubMed Scopus (64) Google Scholar, 24Noguchi S. Satow Y. Uchida T. Sasaki C. Matsuzaki T. Biochemistry. 1995; 34: 15583-15591Crossref PubMed Scopus (41) Google Scholar) reveals that the greatest differences are present in both the unstructured loops and the amino-terminal region (see Fig. 1). In α-sarcin there is a NH2-terminal β-hairpin (residues 1–26) that forms a solvent exposed protuberance and shows a complex topology that can be considered as two consecutive minor β-sheets connected by a hinge region (17Pérez-Cañadillas J.M. Santoro J. Campos-Olivas R. Lacadena J. Martı́nez del Pozo A. Gavilanes J.G. Rico M. Bruix M. J. Mol. Biol. 2000; 299: 1061-1073Crossref PubMed Scopus (64) Google Scholar) (see Fig. 1). The second minor β-sheet of this amino-terminal hairpin is composed of two short strands (Asp9–Asn12 and Lys17–Thr20) connected by a type I β-turn (Pro13–Asn16). This structural component is absent in RNase U2 (see Fig. 1), although residues 7–14 constitute a shorter β-hairpin structure. The K11L mutant of α-sarcin shows both decreased ability to interact with lipid bilayers and reduced cytotoxicity (32Garcı́a-Ortega L. Lacadena J. Mancheño J.M. Oñaderra M. Kao R. Davies J. Olmo N. Martı́nez del Pozo A. Gavilanes J.G. Protein Sci. 2001; 10: 1658-1668Crossref PubMed Scopus (28) Google Scholar). This led to the proposal that the absence of the second minor β-sheet at the NH2-terminal of RNases U2 or T1 could explain why they are not cytotoxic (32Garcı́a-Ortega L. Lacadena J. Mancheño J.M. Oñaderra M. Kao R. Davies J. Olmo N. Martı́nez del Pozo A. Gavilanes J.G. Protein Sci. 2001; 10: 1658-1668Crossref PubMed Scopus (28) Google Scholar). We have prepared, isolated, and characterized α-sarcin Δ(7–22), a deletion mutant in which residues 7–22 were replaced by two Gly residues. Thus, the hinge region, the second minor β-sheet, and the turn in the amino-terminal hairpin of α-sarcin were replaced by the Gly-Gly turn connecting the first minor β-sheet present in RNase U2 (see Fig. 1). All of the materials and reagents were molecular biology grade. Cloning procedures and bacteria manipulations were carried out according to standard methods (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), as described previously (27Lacadena J. Martı́nez del Pozo A. Martı́nez-Ruiz A. Pérez-Cañadillas J.M. Bruix M. Mancheño J.M. Oñaderra M. Gavilanes J.G. Proteins. 1999; 37: 474-484Crossref PubMed Scopus (53) Google Scholar, 34Lacadena J. Martı́nez del Pozo A. Barbero J.L. Mancheño J.M. Gasset M. Oñaderra M. López-Otı́n C. Ortega S. Garcı́a J.L. Gavilanes J.G. Gene (Amst.). 1994; 142: 147-151Crossref PubMed Scopus (57) Google Scholar). Site-directed mutagenesis was used to obtain the deletion mutant as previously described (27Lacadena J. Martı́nez del Pozo A. Martı́nez-Ruiz A. Pérez-Cañadillas J.M. Bruix M. Mancheño J.M. Oñaderra M. Gavilanes J.G. Proteins. 1999; 37: 474-484Crossref PubMed Scopus (53) Google Scholar, 34Lacadena J. Martı́nez del Pozo A. Barbero J.L. Mancheño J.M. Gasset M. Oñaderra M. López-Otı́n C. Ortega S. Garcı́a J.L. Gavilanes J.G. Gene (Amst.). 1994; 142: 147-151Crossref PubMed Scopus (57) Google Scholar, 35Lacadena J. Mancheño J.M. Martı́nez-Ruiz A. Martı́nez del Pozo A. Gasset M. Oñaderra M. Gavilanes J.G. Biochem. J. 1995; 309: 581-586Crossref PubMed Scopus (29) Google Scholar, 36Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar). The mutagenic primer used was: 5′-GTGACCTGGACCTGCGGCGGCCTCCTCTACAACCAG-3′. The two codons that substitute the α-sarcin sequence stretch from Leu7 to Arg22 by Gly-Gly are underlined (Fig. 1). The Escherichia coli strains used were BW313 ((HfrKL16 pol45 (LysA (61Eftink M.R. Biltonen R.L. Biochemistry. 1983; 22: 5134-5140Crossref PubMed Scopus (43) Google Scholar, 62Egami F. Oshima T. Uchida T. Molecular Biology, Biochemistry and Biophysics. Springer-Verlag New York Inc., New York1980: 250-277Google Scholar) dut1 ung1 thi1 relA1) to obtain the uridine-rich single-stranded DNA, DH5αF′ (((F′) endA1 hsdR17 (r− K m− K)supE44 thi-1 recA1 gyrA(NaIR) relA1 Δ(lacZYA-argF) U169 deoR(f80 dlac Δ(lacZ) M15))) for the expression constructs, and BL21(DE3)(F′ ompT(lon) hsdB(r− B m− B)) for protein production. The thioredoxin producing plasmid (pT-Trx) (37Yasukawa T. Kanei-Ishii C. Mackaura T. Fujimoto J. Yamamoto T. Ishii S. J. Biol. Chem. 1995; 270: 25328-25331Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) was a generous gift of Dr. S. Ishii (Riken Tsukuba Life Science Center). BL21(DE3) cotransformed with pT-Trx and the corresponding α-sarcin mutant plasmid were used to produce and purify the mutant as described for the wild-type protein (34Lacadena J. Martı́nez del Pozo A. Barbero J.L. Mancheño J.M. Gasset M. Oñaderra M. López-Otı́n C. Ortega S. Garcı́a J.L. Gavilanes J.G. Gene (Amst.). 1994; 142: 147-151Crossref PubMed Scopus (57) Google Scholar, 38Garcı́a-Ortega L. Lacadena J. Lacadena V. Masip M. de Antonio C. Martı́nez-Ruiz A. Martı́nez del Pozo A. Lett. Appl. Microbiol. 2000; 30: 298-302Crossref PubMed Scopus (19) Google Scholar). Fungal wild-type α-sarcin was produced and purified according to methods previously reported (10Olson B.H. Goerner G.L. Applied Microbiol. 1965; 13: 314-321Crossref PubMed Google Scholar, 34Lacadena J. Martı́nez del Pozo A. Barbero J.L. Mancheño J.M. Gasset M. Oñaderra M. López-Otı́n C. Ortega S. Garcı́a J.L. Gavilanes J.G. Gene (Amst.). 1994; 142: 147-151Crossref PubMed Scopus (57) Google Scholar). Recombinant RNase U2 was purified from the extracellular medium of Pichia pastoris cultures as described (31Martı́nez-Ruiz A. Garcı́a-Ortega L. Kao R. Oñaderra M. Mancheño J.M. Davies J. Martı́nez del Pozo A. Gavilanes J.G. FEMS Microbiol. Lett. 2000; 189: 165-169Crossref PubMed Scopus (9) Google Scholar). This protein retains the enzymatic and spectroscopic properties of the fungal natural RNase U2 (31Martı́nez-Ruiz A. Garcı́a-Ortega L. Kao R. Oñaderra M. Mancheño J.M. Davies J. Martı́nez del Pozo A. Gavilanes J.G. FEMS Microbiol. Lett. 2000; 189: 165-169Crossref PubMed Scopus (9) Google Scholar). Polyacrylamide gel electrophoresis of proteins, protein hydrolysis, and amino acid analysis were also performed according to standard procedures (34Lacadena J. Martı́nez del Pozo A. Barbero J.L. Mancheño J.M. Gasset M. Oñaderra M. López-Otı́n C. Ortega S. Garcı́a J.L. Gavilanes J.G. Gene (Amst.). 1994; 142: 147-151Crossref PubMed Scopus (57) Google Scholar). Absorbance measurements were carried out at room temperature in 1-cm optical path cells on a Uvikon 930 spectrophotometer (Kontron Instruments, Milan, Italy) at 100 nm/min scanning speed. Extinction coefficients E(0.1%, 1 cm, 280 nm) were calculated from the absorbance spectra of the proteins and amino acid analyses to determine concentration. Circular dichroism spectra were obtained on a Jasco 715 spectropolarimeter (Easton, MD) at 0.2 nm/s scanning speed; 0.1- and 1.0-cm optical path cells were used in the far and near UV, respectively. Mean residue weight ellipticities were expressed in units of degrees × cm2 × dmol−1. Thermal denaturation profiles were obtained by measuring the temperature dependence of the ellipticity at 220 nm in the range of 25–85 °C; the temperature was continuously changed at a rate of 0.5 °C/min. Tm values (temperature at the midpoint of the thermal transition) were calculated assuming a two-state unfolding mechanism. Fluorescence emission spectra were obtained on a SLM Aminco 8000 spectrofluorimeter (Urbana, IL) at 25 °C in 0.2-cm path cells. All of these determinations were made as described previously (27Lacadena J. Martı́nez del Pozo A. Martı́nez-Ruiz A. Pérez-Cañadillas J.M. Bruix M. Mancheño J.M. Oñaderra M. Gavilanes J.G. Proteins. 1999; 37: 474-484Crossref PubMed Scopus (53) Google Scholar). Mutant Δ(7–22) was dissolved at 1.5 mm concentration in 0.5 ml of H2O:D2O (9:1 v/v) at pH 6.0. The data were collected at 35 °C, using sodium 3-trimethylsilyl(2,2,3,3,–42H4) propionate as internal reference. NMR experiments were performed on a Bruker Avance 800 MHz spectrometer (Karlsruhe, Germany) equipped with a triple resonance probe and three axis pulsed field gradients. 1The abbreviations used are: HPLChigh performance liquid chromatographySRLsarcin-ricin loopWTwild-type H homonuclear total correlation spectra (39Bax A. Davies D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar) with a mixing time of 60 ms and nuclear Overhauser effect spectra (40Kumar A. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2030) Google Scholar) with a mixing time of 50 ms were recorded by standard methods with water suppression achieved by including the WATERGATE module (41Piotto M. Saudek V. Sklenár V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3527) Google Scholar) in the original pulse sequences. The size of the acquisition data matrix was 2048 × 512 words in f2 and f1, respectively. Before Fourier transformation, the two-dimensional data matrix was multiplied by a phase-shifted sine bell or square sine bell window function in both dimensions. The corresponding shift was optimized in every experiment. Base-line correction was applied in both dimensions. All of the spectra were processed and analyzed using the Bruker software package XWINNMR and ANSIG (42Kraulis P.J. Domaille P.J. Campbell-Burk S.L. van Aken T. Laue E.D. Biochemistry. 1994; 33: 3515-3531Crossref PubMed Scopus (289) Google Scholar) on an IRIS Indigo work station (Silicon Graphics, Mountain View, CA). 1H NMR resonances were assigned using standard sequential assignment procedures (43Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). Spin systems were identified by analysis and comparison of the total correlation spectroscopy spectra with those of the wild-type protein (44Campos-Olivas R. Bruix M. Santoro J. Martı́nez del Pozo A. Lacadena J. Gavilanes J.G. Rico M. Protein Sci. 1996; 5: 969-972Crossref PubMed Scopus (22) Google Scholar). The through-space connectivities were then determined using the nuclear Overhauser effect spectra. high performance liquid chromatography sarcin-ricin loop wild-type The specific ribonucleolytic activity of α-sarcin was followed by detecting the release of the 400-nucleotide α-fragment (5Schindler D.G. Davies J.E. Nucleic Acids Res. 1977; 4: 1097-1100Crossref PubMed Scopus (135) Google Scholar, 6Endo Y. Wool I.G. J. Biol. Chem. 1982; 257: 9054-9060Abstract Full Text PDF PubMed Google Scholar) from a cell-free reticulocyte lysate (Promega, Madison, WI) (34Lacadena J. Martı́nez del Pozo A. Barbero J.L. Mancheño J.M. Gasset M. Oñaderra M. López-Otı́n C. Ortega S. Garcı́a J.L. Gavilanes J.G. Gene (Amst.). 1994; 142: 147-151Crossref PubMed Scopus (57) Google Scholar, 35Lacadena J. Mancheño J.M. Martı́nez-Ruiz A. Martı́nez del Pozo A. Gasset M. Oñaderra M. Gavilanes J.G. Biochem. J. 1995; 309: 581-586Crossref PubMed Scopus (29) Google Scholar), which was visualized by ethidium bromide staining after electrophoresis on 2.4% (w/v) agarose. The activity of α-sarcin was also analyzed on naked rRNA extracted from E. coli with acid phenol-guanidinium thiocyanate-chloroform (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The culture was homogenized by sonication in a water bath after addition of the above denaturant solution. The integrity of the purified RNA was verified by electrophoresis, which confirmed the predominance of the 23 and 16 S rRNA species. The activity assay was performed with this RNA preparation under conditions identical to those described for the reticulocyte lysate. The reaction was stopped by addition of SDS to a final concentration of 1% (w/v), and the products were analyzed by electrophoresis on agarose gels. The specific cleavage of a synthetic 35-mer RNA by α-sarcin was also studied. The synthesis of this synthetic 35-mer RNA was carried out as described (7Kao R. Martı́nez-Ruiz A. Martı́nez del Pozo A. Crameri R. Davies J. Methods Enzymol. 2001; 341: 324-335Crossref PubMed Scopus (42) Google Scholar) by using synthetic and urea-PAGE purified DNA templates: (T7-promoter) 5′-TTCTAATACGACTCACTATAG-3′ and (template) 3′-AAGATTATGCTGAGTGATATCCCTTAGGACGAGTCATGCTCTCCTTGGCGTCCAA-5′ and the AmpliScribe T7 transcription kit (Epicentre Technologies; Madison, WI). The resulting product was purified by electrophoresis on 8% (w/v) polyacrylamide gel containing 7 m urea in 45 mm Tris-borate buffer, pH 8.3, containing 1 mmEDTA (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The assay was performed with 2 μm synthetic 35-mer RNA and a protein concentration (wild-type α-sarcin or deletion mutant) in the 6 nm to 6 μm range, after incubation for 20 min at 37 °C in 10 mm Tris-HCl buffer, pH 7.4 (7Kao R. Martı́nez-Ruiz A. Martı́nez del Pozo A. Crameri R. Davies J. Methods Enzymol. 2001; 341: 324-335Crossref PubMed Scopus (42) Google Scholar). The reaction products were detected by ethidium bromide staining after electrophoretic separation on a denaturing 19% (w/v) polyacrylamide gel. The specific action of α-sarcin produces both 21- and 14-mer fragments. The activity of the purified proteins against poly(A) was assayed in 15% (w/v) polyacrylamide gels containing 0.1% (w/v) SDS and 0.3 mg/ml of the homopolynucleotide. This zymogram method (34Lacadena J. Martı́nez del Pozo A. Barbero J.L. Mancheño J.M. Gasset M. Oñaderra M. López-Otı́n C. Ortega S. Garcı́a J.L. Gavilanes J.G. Gene (Amst.). 1994; 142: 147-151Crossref PubMed Scopus (57) Google Scholar, 35Lacadena J. Mancheño J.M. Martı́nez-Ruiz A. Martı́nez del Pozo A. Gasset M. Oñaderra M. Gavilanes J.G. Biochem. J. 1995; 309: 581-586Crossref PubMed Scopus (29) Google Scholar, 45Kao R. Davies J. J. Biol. Chem. 1999; 274: 12576-12582Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) was based on one previously described (46Blank A. Sugiyama R.H. Dekker C.A. Anal. Biochem. 1982; 120: 267-275Crossref PubMed Scopus (211) Google Scholar). After electrophoresis, the gel was incubated at 37 °C for 3 h and then stained with 0.2% (w/v) toluidine blue. The proteins exhibiting ribonuclease activity appear as colorless bands, because of degradation of the polynucleotide, after appropriate destaining. This assay, which was performed at two different pH values (4.5 and 7.0), is useful to detect the presence of other RNA degrading activities in the protein samples. Volumograms of these bands (based on integrating all of the pixel intensities composing the spot) were obtained with the photo documentation system UVI-Tec (Cambridge, UK) and the software facility UVIsoft UVI band Windows Application V97.04. These data were used to quantify the activity. The activity of the proteins against dinucleotides (ApA and ApG) was measured at pH 5.0 as described elsewhere (26Lacadena J. Martı́nez del Pozo A. Lacadena V. Martı́nez-Ruiz A. Mancheño J.M. Oñaderra M. Gavilanes J.G. FEBS Lett. 1998; 424: 46-48Crossref PubMed Scopus (37) Google Scholar) by analysis of the reaction products (ApA/G, adenosine or guanosine, 3′-AMP, and 2′,3′-cAMP) resolved by HPLC (26Lacadena J. Martı́nez del Pozo A. Lacadena V. Martı́nez-Ruiz A. Mancheño J.M. Oñaderra M. Gavilanes J.G. FEBS Lett. 1998; 424: 46-48Crossref PubMed Scopus (37) Google Scholar).1 RNase U2 was assayed at lower protein/substrate ratio and shorter incubation time than α-sarcin because of its considerably higher enzyme activity. All of the assays were performed with controls to test potential nonspecific degradation of the subs" @default.
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• W2023607888 title "Deletion of the NH2-terminal β-Hairpin of the Ribotoxin α-Sarcin Produces a Nontoxic but Active Ribonuclease" @default.
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