Laboratori dEnginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus Montilivi, 17071 Girona, Spain
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Abstract |
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Keywords: helicity/human pancreatic ribonuclease/site-directed mutagenesis/thermal stability
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Introduction |
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Some members of the ribonuclease superfamily are cytotoxic against tumoral cells. Cytotoxicity is exerted because these molecules can reach the cellular cytosol where they degrade RNA by evading the action of the ribonuclease inhibitor (RI). Paradigmatic examples are bovine seminal ribonuclease (BS-RNase) and onconase [for a review, see Youle and DAlessio (Youle and DAlessio, 1997)]. Although human pancreatic ribonuclease (HP-RNase, EC 3.1.27.5) is inhibited by RI, engineered forms of HP-RNase which could be able either to evade RI or to reach the cytosol, have been proposed as promising candidates for antitumoral drugs, because these variants would probably be more tolerated by the human immune system. In order to ensure cytotoxicity, HP-RNase mutants should maintain their enzymatic activity and stability at physiological temperature (Leland et al., 2001
). However, since amino acid replacements may alter protein stability, a ribonuclease template with increased stability would increase the chances of producing cytotoxic variants. Correlation between the conformational stability of a protein and its catabolism and consequently its biological actions has been suggested. In recent work (Klink and Raines, 2000
), the cytotoxicity of bovine pancreatic ribonuclease (RNaseA) variants was linked to its conformational stability.
In this work, we investigated two sets of mutations in highly exposed residues of HP-RNase that have previously been described as increasing enzyme stability. Characterizing this higher degree of stability at the molecular level would be a valuable tool for engineering cytotoxic ribonucleases that maintain or even increase their stability.
On the one hand, we investigated a variant, named PM5, which has been described as increasing the midpoint transition temperature (T1/2) by 5°C with respect to the parental enzyme (Canals et al., 1999). PM5 is an HP-RNase variant whose N-terminal sequence (residues 121) was replaced by that of BS-RNase. Because of the high degree of sequence homology between both ribonucleases (Beintema et al., 1984
), only five residues are different between PM5 and the wild-type enzyme. These differences are shown in Figure 1A
. We produced and characterized five different N-terminal HP-RNase variants with the aim of dissecting the individual contribution of each of the amino acid changes to the increase in protein stability. It is well known, from the three-dimensional structures of RNase A (Wlodawer et al., 1988
), BS-RNase (Mazarella et al., 1993
) and two HP-RNase variants very similar to PM5 (Canals et al., 2001
; Pous et al., 2000
), that this peptide contains an
-helix that spans residues 34 to residue 12. Our results suggest that the increase in conformational stability correlates with the helical propensity of the N-terminal edge and that global stabilization of HP-RNase may be achieved by local stabilization of secondary structure segments.
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Materials and methods |
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Construction of PM5 and PM9 has been described elsewhere (Canals et al., 1999). PM9 corresponds to the wild-type HP-RNase carrying an additional methionine at the N-terminus of the protein. PM5 corresponds to a PM9 variant with five additional mutations located in the N-terminus: Arg4 to Ala, Lys6 to Ala, Gln9 to Glu, Asp16 to Gly and Ser17 to Asn. PM7 is a PM5 variant in which Pro50 has been substituted by Ser. For clarity, a capital letter P is used to refer to the protein variant and a lower case letter p to refer to the plasmid carrying the mutated gene.
Construction of N-terminal mutants.
HP-RNase variants carrying the substitutions Arg4 to Ala (R4A), Lys6 to Ala (K6A), Gln9 to Glu (Q9E), Asp16 to Gly (D16G) and Ser17 to Asn (S17N) were constructed using a PCR site-directed mutagenesis methodology described elsewhere (Juncosa-Ginestà et al., 1994). Oligonucleotides T7PROM and T7TERM (Canals et al., 1999
) were used to amplify the HP-RNase gene together with a specific oligonucleotide for each variant: M9R4A (5'-CTT TTT AGC TGC AGA TTC TTT C-3'), M9K6A (5'-GGA ACT TTG CAG CTC TAG ATT C-3'), M9Q9E (5'-GTG TTG TCT TTC GAA CTT TTT AG-3'), M9D16G (5'-GAT GGG GAG CTA CCA GAG TCC ATG TG-3') and M9S17N (5'-GCT CGA TGG GGA GTT ATC AGA GTC-3'). The PCR products were digested with NdeI and SalI restriction enzymes and ligated to pM9, previously digested with the same restriction enzymes. The resulting clones were confirmed by sequence analysis.
Construction of variant pM5-124.
The gene fragment of PM5 coding for amino acids 1124 was amplified by PCR using oligonucleotides T7PROM and HPR-124 (5'-CCC TCG AGT TAA ACG CTA GCA TCA AAA TG-3'). Oligonucleotide HPR-124 codes for the complementary sequence of the HP-RNase gene from residues 119 to 124 followed by a STOP codon and the recognition sequence of the XhoI restriction enzyme. The amplified fragment was digested with NdeI and XhoI and ligated to pET17b(+) vector, previously digested with the same restriction enzymes. The resulting clone was confirmed by sequence analysis.
Expression and purification
HP-RNase expression and purification have been described elsewhere in detail (Canals et al., 1999; Ribó et al., 2001
). Briefly, overnight cultures of BL21(DE3) cells carrying the desired vector were reinoculated 1:100 and allowed to grow at 37°C with vigorous agitation until protein expression was induced at late log phase by the addition of IPTG to a final concentration of 1 mM. After a further 34 h of growth, the cells were harvested by centrifugation and the resulting pellets were stored at 20°C until protein processing. Frozen pellets were thawed and resuspended in 50 mM TrisHCl, pH 8.0, 10 mM EDTA, disrupted by French press and centrifuged to isolate inclusion bodies. The aggregated protein was dissolved in 6 M guanidinium chloride, 100 mM Trisacetate, 2 mM EDTA, pH 8.5 and reduced by the addition of reduced glutathione to a final concentration of 0.1 M. The pH was adjusted to 8.5 and the sample was incubated at room temperature under a nitrogen atmosphere for 2 h. After removal of insoluble material by centrifugation, the solubilized protein was diluted 100-fold into 0.1 M Trisacetate, 0.5 M L-arginine, 1 mM oxidized glutathione, 2 mM EDTA, pH 8.5 and incubated at 10°C for at least 24 h. The sample was then adjusted to pH 5.0, concentrated by tangential ultrafiltration and dialysed exhaustively against 50 mM sodium acetate, pH 5.0. Precipitated or insoluble material was discarded by centrifugation. Refolded ribonucleases were then purified by cation-exchange chromatography on a Mono-S HR 5/5 fast protein liquid chromatography (FPLC) column equilibrated with 50 mM sodium acetate, pH 5.0 and eluted with a linear gradient from 0 to 600 mM NaCl. Fractions containing the pure and homogeneous ribonucleases were dialyzed against ultra-pure water, lyophilized and stored at 20°C until further analysis.
Determination of steady-state kinetic parameters
Spectrophotometric assays (Boix et al., 1994) were used to determine the kinetic parameters for the cleavage of poly(cytidylic acid) [poly(C)] and the hydrolysis of cytidine 2',3'-cyclic monophosphate (C>p) by the HP-RNase variants. In the C>p assays, the concentration of enzyme was 0.1 mM, the initial concentration of C>p ranged from 0.1 to 5.5 mM and the activity was measured by recording the increase in absorbance at 296 nm. For assays with poly(C), the concentration of enzyme was 5 nM, the initial concentration of poly(C) ranged from 0.1 to 2.5 mg/ml and the decrease in absorbance at 294 nm was used to monitor the cleavage. All assays were carried out at 25°C in 0.2 M sodium acetate buffer, pH 5.5, using 1 cm pathlength quartz cells for C>p and 0.2 cm pathlength quartz cells for poly(C). Steady-state kinetic parameters were obtained by non-linear regression analysis using the program ENZFITTER (Leatherbarrow, 1987
). The values in Table I
are the averages of three determinations, with a standard error of <10%.
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Circular dichroism (CD) spectra were recorded at 10°C using a Jasco-J715 spectropolarimeter equipped with a thermostated cell holder from Jasco (Tokyo, Japan). The instrument was calibrated with an aqueous solution of d10-(1)-camphorsulfonic acid (Yang et al., 1986). Proteins were dissolved in 10 mM sodium cacodylate, pH 5.0 and filtered using a 0.2 µm filter (Millipore). A quartz cell of 0.01 cm optical pathlength was used to record the spectra of proteins in the far-UV region (178260 nm) at a protein concentration of 1 mg/ml. For measurements in the near-UV region (250310 nm), a 0.2 cm optical pathlength cell and a protein concentration of 0.5 mg/ml were used. CD spectra were acquired at a scan speed of 20 nm/min, using a 2 nm bandwidth and a response time of 1 s. Spectra were signal-averaged over four scans. The solvent dichroic absorbance contribution was subtracted using the Jasco software.
Thermal denaturation experiments
Thermal denaturation of the enzyme variants was followed spectrophotometrically at a protein concentration of 0.75 mg/ml in 50 mM sodium acetate, pH 5.0. Absorption spectra were recorded between 260 and 305 nm as a function of temperature, from 20 to 80°C, in 23°C steps, using a Perkin-Elmer spectrometer with a thermostatted cell holder. Temperature increments were measured with a thermocouple thermometer (Hanna) placed in another quartz cell filled with 50 mM sodium acetate also located in the cell holder. Following each temperature increment, the system was equilibrated during a 2 min pause before each measurement. Each spectrum was corrected for the temperature-dependent change in volume and enzyme concentration before calculating the fourth derivative as described elsewhere (Lange et al., 1996). A method that exploits the whole spectral region and determines an average transition (Torrent et al., 1999
) was used to calculate a parameter for each spectrum named the cumulative difference amplitude (CDA). The CDA values were plotted against temperature and the unfolding transition curves were fitted to a two-state thermodynamic model combined with sloping linear functions for the native and denatured states as described elsewhere (Torrent et al., 1999
). The standard errors of T1/2 were <1%.
Calculation of helix propensity
The residue sequences from 1 to 17 of each variant were introduced into the helix/coil transition algorithm AGADIR1s-2 (Lacroix et al., 1998), which can be found at http://www.embl-heidelberg.de/services/serrano/agadir/agadir-star.html . Each sequence was introduced with the C-terminus amidated in order to avoid the introduction of an additional negative charge not present in the protein. Although the
-helix 1 of HP-RNase extends only from residues 4 to 12 (Pous et al., 2000
), the flanking residues were included to take into consideration local motifs. The program provides the global helical propensity of the whole peptide.
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Results |
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PM5 is an HP-RNase variant whose N-terminal sequence (residues 121) is that of BS-RNase. As can be seen in Figure 1A, only five residues are different between PM5 and PM9 (wild-type enzyme). Temperature-induced unfolding experiments showed that these changes were enough to render PM5 more resistant to thermal denaturation than the wild-type enzyme, the difference in T1/2 being nearly 5°C (Canals et al., 1999
) (see also Table I
).
CD spectra at 10°C and steady-state kinetic parameters for the cleavage of poly(C) and the hydrolysis of C>p were determined for both enzymes. The shapes of the near- and far-UV CD spectra were almost overlapping (Figure 2). The
-helical content, calculated according to Chen et al. (Chen et al., 1972
), was 19.9 and 20.4% for PM5 and PM9, respectively. These values are very similar to those described in the literature, obtained from the crystal structure of the PM7 HP-RNase variant (20.3%) (Pous et al., 2000
) or from the secondary structure of the wild-type enzyme solved by a 1H/15N NMR study (23.4%) (El-Joubary et al., 1999
). The steady-state kinetic parameters shown in Table I
are very similar for both enzymes, as described elsewhere (Canals et al., 1999
). All these results suggest that the broad structures of wild-type HP-RNase and its PM5 variant are similar, and therefore differences in stability should arise from differences in specific atom-to-atom interactions.
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In order to dissect the contribution of a particular residue change to the increase in stability, variants, each carrying a single one of the five replacements already present in PM5, were generated by site-directed mutagenesis on pM9. Variants carrying the substitutions R4A, K6A, Q9E, D16G and S17N were expressed and purified to homogeneity according to the procedures described in Canals et al. (Canals et al., 1999). Protein integrity was checked by MALDI-TOF mass spectrometry and homogeneity by reversed-phase chromatography (results not shown). Steady-state kinetic parameters for the hydrolysis of C>p and cleavage of poly(C) were very similar to those found for PM5 and PM9 variants (Table I
).
Temperature unfolding and refolding of each variant were followed spectrophotometrically. The unfolding transition curves obtained from the CDA analysis were nearly 100% reversible and the calculated T1/2 values are listed in Table I. Excluding the substitution of Ser17 to Asn, the other changes all led to an increased T1/2 relative to PM9. The T1/2 increase was more apparent in the case of the substitutions of Arg4 to Ala and Lys6 to Ala, being their
T1/2 of 2.7 and 2.2°C, respectively (
T1/2 is the difference between the T1/2 of PM9 and each of their variants). Positions 4, 6 and 9 are located in the
-helix 1 of the HP-RNase molecule, whereas positions 16 and 17 are located in the hinge loop that connects this helix with the rest of the protein (El-Joubary et al., 1999
; Pous et al., 2000
). The helical propensity of the N-terminal peptide (residues 117) of PM5 and PM9 proteins, and also of the single point variants of PM9: R4A, K6A and Q9E, were predicted using the program AGADIR1s-2 (Lacroix et al., 1998
).
A plot of the helical propensity predicted for each of the N-terminal peptides analyzed against T1/2 is shown in Figure 3
. A good correlation between the two parameters is found (r = 0.978), suggesting that the increase in conformational stability is directly related to the stabilization of
-helix 1.
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It has been described elsewhere (Bal and Batra, 1997) that deleting the last four residues of HP-RNase (GluAspSerThr) yields an enzyme (namely HPR1-124) with increased thermal stability. In the same work, the authors suggested that the last eight residues of the HP-RNase are in an
-helix structure. This result is not in agreement with the recently reported three-dimensional structure of two HP-RNase variants (Pous et al., 2000
; Canals et al., 2001
) or with the secondary structure of HP-RNase assigned from its 1H and 15N NMR spectra (El-Joubary et al., 1999
). Residues 116124 are in a ß-strand conformation, whereas the last four residues appear to be disordered.
In order to test whether this deletion could enhance the thermal stability of PM5 variant, we constructed a new variant of PM5 which lacked the four C-terminal residues of the sequence. This variant, named PM5-124, is different at its N-terminus from the HPR1-124 reported elsewhere (Bal and Batra, 1997). The former carries an extra Met together with the five internal substitutions, while the latter carries the extra dipeptide, AlaSer, at positions 2 and 1. In the structure of HP-RNase (Pous et al., 2000
), the N- and C-terminal edges of the protein are far apart one from the other (Figure 1B
). Hence it can be assumed that the amino acid differences located at the N-terminus have a negligible effect on the protein C-terminal end.
PM5-124 was expressed and purified to homogeneity according to the procedures described in Canals et al. (Canals et al., 1999). Protein integrity was checked by MALDI-TOF mass spectrometry and its homogeneity by reversed-phase chromatography (results not shown). Steady-state kinetic parameters of PM5-124 for the cleavage of poly(C) and the hydrolysis of C>p were slightly lower than those found for PM5 (Table I
); however, the kcat/KM value was found to be identical for both proteins. Far- and near-UV CD spectra of PM5-124 were also measured at 10°C. The shape of the curves were very similar to those of PM5 (Figure 2
) with the
-helical content being 23.5%. Analysis of thermal stability was performed and the unfolding transition curve was 100% reversible (not shown). The PM5-124 truncated protein presents a T1/2 value equal to that found for PM5 (Table I
), indicating that this C-terminal deletion does not contribute to the stabilization of the protein.
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Discussion |
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We constructed variants carrying single point mutations in order to evaluate the importance of each change in the increase of thermal stability. The effect of the different mutations is nearly additive. The mutations introduced into the -helix give a more pronounced effect than those found in the hinge loop where they could be more easily accommodated. Substitutions at positions 4 and 6 probably enhance
-helix stability. Position 4 corresponds to the first residue of the
-helix in HP-RNase (El-Joubary et al., 1999
) and also in the PM7 HP-RNase variant (Pous et al., 2000
). In PM9 (Met-1 HP-RNase), this position is occupied by Arg. This residue, at this position, destabilizes the
-helix because the positively charged side chain makes unfavorable interactions with the partial helix dipole created by the impaired amide protons. When an Ala is found at this position, these unfavorable interactions are eliminated. The increase in thermal stability after the change of Lys6 to Ala can also be reasoned in terms of helical stability. Substitutions of internal
-helix residues by Ala in model peptides and proteins usually result in more stable
-helices [for a review, see Fersht (Fersht, 1998
)]. Substitution of Gln9 by Glu results in a higher helical propensity value for the peptide containing Glu residue (Figure 3
). Amino acid replacements that lead to the appearance or elimination of net charges (positions 4, 6 and 9) could alter chargecharge interactions, explaining the change in stability. However, analysis of the altered positions in the PM7 structure does not reveal chargecharge interactions with the rest of the protein. In addition, Figure 3
suggests that the increase in T1/2 is mainly a consequence of the increase in the stability of
-helix-1.
Designing new mutations that could increase this helical propensity even more, without altering non-local interactions, should produce even more stable HP-RNase variants. This strategy has been used elsewhere in the CheY protein (López-Hernández et al., 1997),
-spectrin SH3 domain (Blanco et al., 1999
) and the activation domain of human carboxypeptidase A (Villegas et al., 1996
), among others.
A previous study (Bal and Batra, 1997) indicated that a recombinant and truncated form of HP-RNase (HPR1-124) presented greater thermostability and also greater ribonuclease activity and a lower helical content than the corresponding recombinant non-truncated form. The possibility that a deletion in the HP-RNase could render a more thermostable enzyme would be of great interest in the design of cytotoxic HP-RNases because this increase in stability does not mean the appearance of new epitopes in the protein. Thus, a C-terminal-deleted ribonuclease could be a good template on which to introduce the desired mutations.
As we expected from the three-dimensional structure of PM7, our CD results do not indicate that the helical content of PM5-124 is less than that of PM5. In addition, the truncated enzyme has neither increased catalytic activity nor enhanced thermostability. HP-RNase and HPR1-124 differ from PM5 and PM5-124, respectively, in the N-terminal sequence: whereas the former has two additional residues at the N-terminus (AlaSer), the latter carries five mutations in the S-peptide and a Met residue at position 1. Analysis of the PM7 three-dimensional structure does not shed light on how the N-terminal changes could account for the different behavior of the two truncated forms.
In our studies, truncation of the C-terminal edge of PM5 did not appear to be a good strategy for increasing the stability of the enzyme. However, it could be concluded from our results that the engineering of the N-terminal -helix of HP-RNase in order to increase its helical propensity could be a valid strategy in constructing variants of HP-RNase with higher stability.
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Notes |
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Acknowledgments |
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References |
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Received March 8, 2002; revised August 7, 2002; accepted August 26, 2002.