Comp. Biochem. Physiol. Vol. 101B,No. I/2, pp. 289-297, 1992 Printed in Great Britain

0305-0491/92 $5.00+ 0.00 © 1992PexgamonPress pie

PORCINE THYROID CYTOSOLIC, LATENT ALKALINE RIBONUCLEASE: RESISTANCE TO PROTEIN DENATURANTS BARBARAE. CRUTE,*JOSEPH D. KAY/f ELIZARETHS. GRACE~ and FREDRICKJ. KULL*§ *Department of Biological Sciences, State University, Center at Binghamton, NY 13902-6000, USA (Tel: 607 777-2649); tSUNY, School of Medicine, Buffalo, NY 14214, USA; and ~Denver Department of Health and Hospitals, CityCare, 605 Bannock Street, Denver, CO 80204-4507, USA (Received 22 April 1991)

Abstract--1. A ribonuclease isolated from porcine thyroid cytosol using phenol:sodium dodecylsulfate treatment was associated with RNA and identical to latent alkaline ribonuclease. 2. Distribution of activity between aqueous and phenolic phases depended on pH, RNA, and ribonuclease inhibitor. 3. The ribonuciease was totally resistant to urea, guanidinium:HCl, chloroform:isoamyl alcohol, ethanol, heating at 100°C for 10 rain or at 80°C plus 100 mM NaC1. It was highly resistant to hydrolysis by proteinase K except in the presence of detergent. 4. The extreme stability and other properties of latent alkaline ribonuclease could be the result of its association with RNA.

INTRODUCTION Intracellular alkaline ribonucleases have been isolated from a variety of mammalian tissues using both denaturing and non-denaturing methods of purification (Okazaki et al., 1975; Bartholeyns and Baudhuin, 1977; Davies et al., 1980; Button et al., 1982; Rutherford et al., 1983). In vivo some, if not all, alkaline ribonuclease is associated with ribonuclease inhibitor, an endogenous protein (Roth, 1956; Turner et al., 1983), such that homogenates contain negligible (liver; Specht and Kull, unpublished) or significant ribonucleolytic activity (thyroid; Button et al., 1982). However, when sulfhydryl-active agents [e.g. p-chloromercuriphenylsulfonate (pCI-HgPhS)] are added to homogenates, ribonucleolytic activity substantially increases in the case of thyroid and large amounts appear in extracts from liver. These agents irreversibly inactivate ribonuclease inhibitor by interacting with an essential sulfhydryl group (Roth, 1958; Shortman, 1962; Turner et al., 1983). Thus, intracellular alkaline ribonuclease activity that is masked because of ribonuclease inhibitor but expressed upon addition ofpCI-HgPhS is referred to as latent alkaline ribonuclease activity and the enzyme, even when separated from ribonuclease inhibitor, as latent alkaline ribonuclease. Because the alkaline ribonuclease:ribonuclease inhibitor system has been proposed to be involved with many important cellular functions including

§Author to whom correspondence should be addressed. Abbreviations used--BD-ceHulose, benzoyl-diethyl-aminoethyl-cellulose; SDS, sodium dodecylsulfate; PS-, phenol:sodium dodecylsulfate-resistant; pCI-HgPhS, parachloromercuriphenylsulfonate; PAGE, polyacrylamide gel electrophoresis. 289

protein biosynthesis (Shortman, 1962; Blobel and Potter, 1966), m R N A metabolism (Kraft and Shortman, 1970; Levy, 1975; Button et al., 1982), and development (Aoki and Natori, 1981), there is considerable interest in the nature of these two proteins. In this paper we report a novel, efficient isolation of alkaline ribonuclease by means of phenol: sodium dodecylsulfate (SDS), show the alkaline ribonuclease to be undamaged latent alkaline ribonuclease, describe factors relating to latent alkaline ribonuclease's phase distribution, and document latent alkaline ribonuclease's unusual stability to other protein denaturants. MATERIALS AND METHODS Materials Porcine thyroids were obtained and maintained as previously described (Button et aL, 1982). BD-cellulose from Bovhringer Mannheim Biochemicals (Indianapolis, IN) and DEAE-cellulose (DE-32) from Whatman (Clifton, NJ) were precycied and pre-equilibrated as recommended by the manufacturers. Scphadexes were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Sodium dodc~yl sulfate (SDS; analytical grade) was from Scrva Fine Bit)chemicals (Westbury, NY). Synthetic polynucieotides were purchased from P-L Biochemicais (Milwaukee, WI) and Sigma Chemical Company (St Louis, MO). Protein standards used to calibrate Sephadex columns were purchased from Pharmacia Fine Chemicals or Sigma Chemical Company, as were N,N'-methylene-bisacrylamide, acrylamide, Coomassie Brilliant Blue G-250 and R-250, glycine, Trizma base, ammonium sulfate (Grade I), EDTA, urea, sucrose (Grade I) and pCI-HgPhS. The m4C-amino acid hydrolysate was from AmershamScarle (Arlington Heights, IL) and the protein reagent for Bradford protein assays was purchased from Bio-Rad (RockviUe Centre, NY). Solutions of urea (8 M) were treated with acid-washed charcoal (Norite) and then filtered through DEAE-cellulose before concentrations were adjusted to 7 M. Guanidinium: HC1

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BARnARAE. CRUTE et aL

was a product of Eastman Organics (Rochester, NY) and dialysis tubing (Spectra/Por) was from Spectrum Medical Industries of Los Angeles, CA. The 2-mercaptoethanol was bought from Baker Chemical Co., Phillipsburg, NJ. Proteinase K (Boehringer-Mannheim Biochemicals, Indianapolis, IN) and human placental ribonuclease inhibitor (RNasin, Promega Biotec, Madison, WI) were generous gifts of Dr William Patrie. Except for brewer's yeast tRNA, purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN), native nucleic acids were prepared according to Rutherford et al. (1983). All other chemicals were reagent grade.

Methods Preparation ofsubstrate. Brewer's yeast tRNA, aminoacylated with ~4C-amino acids, was prepared and analyzed according to the methods of Goldman et al. (1981) except mixtures were adjusted to 0.2 M sodium acetate, pH 5.5, prior to extraction using 85% aqueous phenol titrated with NaOH to pH 6.0. Assays. Ribonuclease activity was measured as described by Goldman et al. (1981) using the above aminoacyltRNA/tRNA substrate. Typical duplicate assays (180 #1) at room temperature (facility controlled at 23°C) were 50 mM Tris-HCI, pH8.1 and contained 200#g of substrate per milliliter. Portions (50-75 #1) were removed at appropriate times, and assayed according to Waters and Novelli (1971) using a Beckman Liquid Scintillation 7500 Spectrophotometer. One unit of ribonuclease activity is defined as the amount able to cleave 1 #g of substrate to acid soluble products per minute. Absorbances (260 and 280 nm) refer to a 1.0 cm path length. Absorbance at 280 nm was used as a rough measure of protein concentration. For more accurate measurements, the microassay method of Bradford (1976) was used with bovine serum albumin as standard. Absorbance at 260 nm and the A26o/A2so ratio were used to locate and estimate amounts of nucleic acids and their hydrolytic products eluting from columns. Analysis of activity in aqueous and organic phases. Alkaline ribonuclease activity present in the aqueous phases of samples extracted with phenol or other organic solvents was routinely recovered by addition of NaCI to 0.2 M and 3 vols of 95% ethanol followed by overnight precipitation at -20°C. Control experiments showed that little if any activity was present in ethanolic supernatants following centrifugation. Precipitates were dissolved in 10ram Tris-HC1, pH 8.1, 1 mM MgC12 and dialyzed at least 8 hr against greater than 200 vols of the same buffer. The alkaline ribonuclease activity present in organic phases was recovered either by precipitation and dialysis as just described or by direct dialysis of the organic phase. Control experiments in which tRNA was mixed with phenol and then dialyzed showed that the dialysis tubing we used was unaffected by the phenol. SDS is soluble in phenol and ethanol and in some instances crystallized out of solution during dialysis in the cold. Most could be removed by centrifugation and the remainder removed by addition of 3 vols of ethanol to supernatants. Electrophoresis. Polyacrylamide gels (10%) were prepared according to Davis (1964). The electrode buffer was 25 mM Tris-HCl and 192mM glycine (pH8.1). Slab gels (1.5mm x 12.5cm x 14cm) were run at 20mA per gel for 5 hr or until the Bromophenol Blue tracking dye was 0.5-1.0 cm from the bottom. Gels were stained for protein using Coomassie Brilliant Blue G-250 (0.25% in 50% methanol and 7.5% acetic acid) and were destained by immersion in methanol: H 20 : glacial acetic acid (2: 7:1, vol:vol:vol). SDS-PAGE (Laemmli, 1970) on 10% separating gels and 4% stacking gels was at 120 V per gel for 4-5 hr. Gels were stained for protein using Coomassie Brilliant Blue R-250 (0.1% in 50% methanol and 10% acetic acid) and destained as above.

Elution of ribonuclease activity from native polyacrylamide gels. Duplicate samples were subjected to electrophoresis done on the same native 10% polyacrylamide gel; one of the duplicate lanes was stained for protein and the other was used for determination of activity. The latter were sliced into 1.0cm pieces and placed in 0.8 ml of 50 mM Tris-HC1, pH8.1, 25% ethanol (vol:vol). Slices were then frozen, thawed, hand homogenized, and incubated for 15 min at 37°C. After incubation, mixtures were centrifuged for 5 rain at top speed using a desk top Model CL International Clinical Centrifuge and portions of the supernatant were assayed for activity (10-30 rain; 37°C). Fractions having latent alkaline ribonuclease activity. Latent alkaline ribonuclease fraction and post-ammonium sulfate fraction were prepared as described and designated by Button et al. (1982). Dialyses were against 10raM Tris-HCl (pH 8.1) and 1 mM MgCI 2 buffer for at least 8 hr. Preparation of subcellular fractions. Homogenates were prepared as above and centrifuged at 12,000g to sediment nuclei and mitochondria; supernatants were reserved and pellets resuspended in homogenizing buffer and recentrifuged at 1000 g for 10 rain. These pellets, which contained nuclei, were resuspended in homogenizing buffer. The supernatants, which contained mitochondria, were recentrifuged at 12,000 g for 30 rain. The supernatants from this centrifugation were discarded and the mitochondrial pellets resuspended in homogenizing buffer. The reserved postmitochondrial supernatants were centrifuged (Beckman L5-65) for 2.5hr at 79,000g to pellet microsomes; the supernatants (cytosol) were saved and the microsomal pellets resuspended in homogenizing buffer. Isolation of latent alkaline ribonuclease Homogenization. All manipulations were performed at 4°C unless otherwise stated. Typically 100g of frozen porcine thyroids, stored at -80°C, were ground while still frozen using a commercial meat grinder. The frozen, ground tissue was homogenized in 3 vol cold buffer containing 200 mM NaC1, 50 mM sodium acetate, 5 mM magnesium acetate, pH 6.0. After filtration through cheese cloth, homogenates were centrifuged at 12,000g for 30rain at 4°C. Post-mitochondrial supernatants were filtered through Miracloth and recentrifuged at 100,000g for 2.5 hr to obtain cytosol. Phenolic extraction. Cytosols were made 2% SDS and one-third vol of neutralized 85% phenol (vol:vol) was added as mixtures were stirred. Following additional vigorous stirring for 15 min at room temperature, mixtures were centrifuged at 28,000g for 15rain, also at room temperature, to separate phases. Aqueous phases were removed and re-extracted with equal vols of 85% phenol. The aqueous phases from the second extraction were removed and 3 vols of 95% ethanol added. After storage at - 2 0 ° C overnight, precipitates were collected by centrifugation (28,000 g, 20 rain) and dissolved in 50 mM sodium acetate, 5 mM magnesium acetate, pH 6.0 (approximately one-fourth vol of starting sample). BD-cellulose chromatography. The dissolved material from above was next applied to a BD-cellulose column (2.5 × 15cm) pre-equilibrated in 50mM sodium acetate, 5 m M magnesium acetate, pH6.0. [Phenol:SDS-treated samples were not dialyzed prior to charging because residual phenol (pKa, 10.0) was washed from the column at pH 6.0.] The column was next washed with the same buffer until absorbance was negligible and then eluted with 200 mM NaCI in the same buffer. Fractions of the 0.2 M NaCI eluate containing significant absorbance at 260 nm were combined, dialyzed into 10mM Tris-HC1, l mM MgCI2, pHS.1, condensed on a bed of polyethylene glycol, and re-dialyzed. G-IO0 Sephadex gel chromatography. When necessary, further purification of latent alkaline ribonuclease was achieved by chromatography of 0.2 M samples on a G-100 Sephadex column (1.13 × 57 em) that was pre-equilibrated

291

Stability of latent ribonuclease

A

1

2

3

4

5

6

B

7

800

600

-'~51 kD o

400

200

0 1 2 3 4

5 6 7

8 9 10 11 12 13

Gel segment

Fig. 1. Electrophoretic analysis of PS-alkaline ribonuclease and PS-latent alkaline ribonuclease. (A) SDS-PAGE. Lane 1, standards (from top): myosin, fl-galactosidase, phosphorylase B, bovine serum albumin, ct- and fl-tubulins, ovalbumin, carbonic anhydrase, lysozyme. Lanes 2 and 3, PS-latent alkaline ribonuclease (15 and 30 #g). Lanes 4 and 5, PS-alkaline ribonuclease (1.5 and 3.0 #g). Lanes 6 and 7, G-100 fraction of PS-alkaline ribonuclease, Methods (1.0 and 2.0/zg). (B) Stacked-bar graph showing native PAGE of PS-latent alkaline ribonuclease (a, 9.8 units) and PS-alkaline ribonuclease (b, 7.5 units). Assays were for 10 min at 37°C. and eluted using 200 mM NaC1, 10 mM Tris-HC1, pH 8.1. Absorbance characteristics of several peaks that trailed latent alkaline ribonuclease indicated that they were most likely RNA. RESULTS

Purification by phenolic : SDS extraction and cellular localization of phenol : SDS-resistant alkaline ribonuclease Purification. The purification of alkaline ribonuclease to homogeneity (Fig. 1A, lanes 4-7) using p h e n o l : S D S is described in the Methods section and summarized in Table 1. Although judged pure by protein staining, PS-alkaline ribonuclease contained R N A . Fundamental to purification was that the amount o f alkaline ribonuclease extracted into the

aqueous phase depended on the SDS concentration (Fig. 2).

Cellular localization of PS-alkaline ribonuclease. T o determine if cellular fractions other than cytoplasm contained PS-alkaline ribonuclease, nuclear, mitochondrial, microsomal, and cytosolic (postmicrosomal supernatant) samples were obtained from an homogenate of 100 g tissue (Methods). Portions of each were extracted with p h e n o l : S D S and assayed. Only the cytosolic sample contained PSalkaline rihonuclease activity (1970 units; 288 specific activity). F o r all remaining experiments we used either purified PS-alkaline ribonuclease, the latent alkaline ribonuclease fraction or the post-ammonium sulfate fractions (Methods). Whereas the latent alkaline

Table 1. Isolation of latent alkaline ribonuclease from porcine thyroid by phenol:SDS Absorbance Total Total Specific ratio protein activity activity Fold Sample (260: 280 nm) (mg) (units) (units/rag) purification Post-mitochondrial supernatant 0.91 7603.68 58,624 7.7 0 Cytosol 0.86 6115.20 51,527 8.4 l Phenol: SDS-treated cytosol 1.78 4.37 3299 754.9 98 0.2 M 1.32 0.60 1061 1768.7 229 G-100 1.78 0.06 603 10,055.5 1306 Experimental and procedural details are given in the Methods section. Total activity in the post-mitochondrial supernatant and cytosolic fractions was estimated in the presence and absence of 2 mM pC1-HgPhS assuming 20% inhibition by pCI-HgPhS (Rutherford et al., 1983).

292

BARBARAE. CRtrrE et al. i

100

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Fig. 2. Units of PS-alkaline ribonuclease and total protein recovered from aqueous phases as a function of percentage of SDS used during phenolic extraction. Equal aliquots of a post-ammonium sulfate fraction (Methods) were used (267 units) for phenolic extractions done at the percentages of SDS shown (Q). After phenol:SDS treatment samples were condensed, dialyzed and assayed as described in Methods. Protein was measured (ll) by the method of Bradford (1976). ribonuclease fraction is inactive unless pC1-HgPhS is added to inactivate ribonuclease inhibitor, the postammonium sulfate fraction is fully active (Button et al., 1982). Identification o f the PS-alkaline ribonuclease as latent alkaline ribonuclease Characterization o f PS-alkaline ribonuclease. We compared PS-alkaline ribonuclease with latent alkaline ribonuclease relative to effects of monovalent and divalent salts, pH optimum, mol. wt, and general substrate specificity using procedures described by Button et al. (1982). At ionic strengths greater than 60raM all monovalent salts were inhibitory. PS-latent alkaline ribonuclease was significantly stimulated by 30 mM CsC1 and moderately stimulated by KCI, NH4C1, KI, and KBr. MgC12 and CaCI2 were weakly stimulatory whereas CuCI~ and ZnCI2 were strong inhibitors from 0.1 to 5.0 mM. A single pH optimum of pH 8.0 was obtained in Tris-HC1 when ionic strengths were constant. The mol. wt of PS-alkaline ribonuclease was estimated to be 51,000 + 1500 by G-75 Sephadex, 52,800+1500 by G-100 Sephadex and 50,000 (-t-5000) using denaturing S D S - P A G E (Fig. IB, lanes 6 and 7). Based on its A:60:A2s0 ratio (1.78, Table 1), PS-alkaline ribonuclease preparations contained about 25-30% R N A by weight. PS-alkaline ribonuclease's activity toward porcine liver r R N A and t R N A was limited, suggesting cleavage at single-

stranded regions. Poly (U) and poly (C) were cleaved, however poly (G), poly (A), single or double-stranded DNA, poly (U):poly (A), poly (C):poly (I), poly (C):poly (G,I), and 2'(Y)-cyclic CMP were not hydrolyzed. All of the characteristics listed above supported the premise that PS-alkaline ribonuclease and the latent alkaline ribonuclease previously purified by Button et al. (1982) were the same. Electrophoresis. A latent alkaline ribonuclease fraction (Methods) was treated with phenol:SDS to yield active PS-latent alkaline ribonuclease free of ribonuclease inhibitor. PS-latent alkaline ribonuclease migrated identically to PS-alkaline ribonuclease on both S D S - P A G E and native PAGE (Fig. 1A, lanes 2 and 3, Fig. 1B). Identical results were obtained in other PAGE experiments using native and denaturing conditions comparing PS-alkaline ribonuclease, PS-latent alkaline ribonuclease and latent alkaline ribonuclease that had not been treated with phenol:SDS. (Data not shown.) Inhibition by ribonuclease inhibitor. Ribonuclease inhibitor provided further evidence that PS-alkaline ribonuclease and latent alkaline ribonuclease represented the same activity (Table 2). Because latent alkaline ribonuclease, PS-latent alkaline ribonuclease, and PS-alkaline ribonuclease all represented the same activity, we will designate that activity as latent alkaline ribonuclease in the remainder of this paper.

Table 2. Effectof ribonucleaseinhibitoron PS-alkalineribonuclease,PS-latentalkalineribonuclease and pancreaticribonucleaseA Units of activity With ribonuclease Per cent Ribonuclease Untreated il,hibitor inhibition PS-alkalineribonuclease 3.00 0.33 89 PS-latent alkalineribonuclease 2.35 0.27 88 Ribonucleas¢A 2.36 0 100 The units of ribonucleasesshown were either preincubatedwith 1.2 units of human placental ribonucleasvinhibitoror buffer prior to assay.

Stability of latent ribonuclease

Factors affecting the distribution of latent alkaline ribonuclease between aqueous and phenolic phases The amount of latent alkaline fibonuclease present in aqueous phases was dependent on the SDS concentration (Fig. 2). Furthermore, considerable activity was recovered (476 units) when phenolic phases that had been saved frozen were combined and re-extracted with buffer (200 mM NaCl; l0 mM Tris-HC1, pH 8.0; 2% SDS). These results, which showed that latent alkaline ribonuclease entering the phenolic phase was not irreversibly denatured, led us to examine other factors that affected its phase distribution. Presence of ribonuclease inhibitor. The yield of latent alkaline ribonuclease prepared by direct phenol: SDS treatment of the latent alkaline ribonuclease fraction was less than that obtained by the procedure described in Methods 0 - 5 units per gram tissue vs 11 units per gram) suggesting the presence of ribonuclease inhibitor during phenolic extraction decreased the amount of recoverable latent alkaline ribonuclease. To test this possibility ribonuclease inhibitor (350 units) was incubated with 762 units of latent alkaline ribonuclease for 10min at room temperature. Following incubation a portion was removed as a control to measure the amount of activity in the complex (89.6%). The remainder was adjusted to 0.2 M NaCI and treated with phenol:SDS. After condensation and dialysis (Methods) only 4.4% of the activity was recovered. However, when ribonuclease inhibitor was absent during phenol: SDS treatment, 36% was recovered. Less activity was re-extractable from phenolic phases if ribonuclease inhibitor was present during extraction than if it was absent (0.9% vs 28%). This suggested that denatured ribonuclease inhibitor remained irreversibly complexed to latent alkaline ribonuclease. Table 3 shows that results using the latent alkaline ribonuclease fraction substantiated those obtained with purified components. Approximately 39% of the latent alkaline ribonuclease in the post ammonium sulfate fraction was recovered following phenol:SDS treatment, whereas less than 1% was recovered by direct phenol: SDS treatment of the latent alkaline ribonuclease fraction. tRNA, pH, NaCl. Thyroid tRNA (2.2 mg) was mixed with 240 units of post ammonium sulfate fraction (molar ratio, tRNA:latent alkaline ribonuclease, 3:1). Two samples were treated identically except buffer was added in place of tRNA. Following 10 min at 25°C, the three samples were adjusted to

293

0.2 M NaCI and a fourth sample was prepared in 0.2 M sodium acetate pH 6.0 (not preincubated). All four samples were then treated with phenol:SDS as usual. The aqueous phases from the tRNA sample, the pH6.0 sample, one pH8.1 sample, and the aqueous phase plus interphase from the other pH 8.1 sample were condensed, dialyzed and assayed (Methods). Less latent alkaline ribonuclease was in the aqueous phase at pH 6.0 (34 units) than at pH 8.1 (55 units), addition of tRNA prior to phenol:SDS extraction decreased the amount of activity recovered (18 units), and significant activity was in the interphase (96 units). The phenolic phases from the combined samples (Methods) contained 480 units, 71% of the initial activity. Control experiments using ribonuclease .4. We examined effects of phenol:SDS on ribonuclease A because pancreatic ribonuclease A has previously been shown to be resistant to phenol (Kickhofen and Burger, 1962). First, 5.3 x 107 units of ribonuclease A supplemented with 180 mg of bovine serum albumin were extracted with phenol using 0.1% SDS. No activity was recovered from the aqueous phase. Second, 2.1 x 107 units of ribonuclease A were mixed with 60 mg bovine serum albumin and then extracted with phenol using 2% SDS. A negligible amount of activity (1.4 x 10-4%) was detected in the aqueous phase. Finally tRNA (1.7 mg), bovine serum albumin (600 mg), and ribonuclease A (3.2 x 103 units) were combined in a vol of 30 ml buffer (approximately the same amounts of activity, protein, and RNA as present in an equal vol of cytosol) and again extracted with phenol using 2% SDS. No activity was recovered from the aqueous phase.

Stability of latent alkaline ribonuclease Latent alkaline ribonuclease was unusually stable to phenol:SDS, a treatment that would denature most proteins. Therefore, we tested other procedures usually effective in protein denaturation. Time in phenol. A portion of post ammonium sulfate fraction was made 0.2 M NaCI, treated with phenol: SDS, and the mixture stirred. Equal portions were removed at 15, 30, 45, and 6 0 imn and centrifuged to separate the aqueous and phenolic phases, and each condensed, dialyzed, and assayed (Methods). There was no correlation between the amount of latent alkaline ribonuclease recovered from the aqueous phases and the time of exposure to

Table 3. Recovery of latent alkaline ribonuclease following various treatments of latent alkaline ribonuclease fraction Sample or treatment Latent alkaline ribonuclease fraction +pCI-HgPhS -pCI-HgPhS Latent alkaline ribonuclease fraction (treated with phenol: SDS) Latent alkaline ribonuclease fraction (stirred 30 min before phenol : SDS) Post-ammonium sulfate fraction Post-ammonium sulfate fraction (treated with phenol: SDS)

Total units

Percentage of original

8035 473 72

100.0 5.9 0.9

151

1.9

2162 840

26.9 10.5

Data are expressed in units of activity normalized to that obtained from the original 50 g of tissue. The total activity of the latent alkaline ribonuclease fraction was estimated using pCI-HgPhS, the only sample that showed an increase in its presence.

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BARBARAE. CRUTE et al.

phenol : SDS. Furthermore, the sum of the activities recovered from aqueous and phenolic phases showed that complete activity was recovered for each time point. Urea and guanidinium:HCl. Exposure of post ammonium sulfate fraction at 25 ° to either 7 M urea or 3 M guanidinium: HC1 for 30, 60, and 120 min had no effect on latent alkaline ribonuclease. Samples were dialyzed for 18 hr prior to assay. Chloroform :isoamyl alcohol, chloroform :isoamyl alcohol:phenol. A portion of post ammonium sulfate fraction was made 0.2 M NaCI and 2.0% SDS and then mixed with an equal vol of chloroform:isoamyl alcohol (24:1; vol:vol) for 20 min at room temperature. Following centrifugation, the aqueous phase was condensed, dialyzed and assayed (Methods). There was no loss of activity under these conditions; all activity (620 units) remained in the aqueous phase. When an identical portion of post ammonium sulfate fraction was mixed with equal vols of chloroform/isoamyl alcohol and phenol, there was less activity recovered than from the control that was treated by phenol:SDS (5.9 units vs 117 units). Analysis of the organic phases revealed that the enzyme was relatively stable to the mixed solvent (51% of the total activity treated was recovered) but was relatively more soluble in the mixed organic phase (318 of the total 324 units went into this phase) than in either the phenolic phase (483 units; 78%) or chloroform/isoamyl alcoholic phases (7.8 units; 1%) alone. Heat and ionic strength. Figure 3A shows the heat stability of latent alkaline ribonuclease. The ribonuclease A control had 40 times more activity than the latent alkaline ribonuclease sample and its protein concentration was normalized to that of the latent alkaline ribonuclease sample by the addition of bovine serum albumin. Ribonuclease A was stable to 70°C, but was rapidly denatured at higher temperatures. There was no loss of latent alkaline ribonuclease activity at any temperature tested. The effect of combined heat and ionic strength on latent alkaline ribonuclease activity was also examined. As shown in Fig. 3B and C, latent alkaline ribonuclease was essentially unaffected by the presence or absence of 100 mM NaC1 during preincubation; more than 95% of the activity remained in both cases, whereas ribonuclease A was rapidly inactivated (t t/2 minus NaCI, 10 min; plus NaC1, 3 min). Ethanol. Latent alkaline ribonuclease was routinely concentrated by taking advantage of its insolubility in 75% ethanol. Therefore, we examined the effects of ethanol during incubations and found it had essentially no effect on activity (3 units per assay preincubated 5 min with varied amounts of ethanol) at all concentrations tested (4.5, 9, 18.8, 30, 37.5% by vol in preincubations; 3, 6, 12, 20, and 25% in the incubations). SDS. SDS was an effective inhibitor of latent alkaline ribonuclease at incubation concentrations greater than 0.013%; 91% inhibition at 0.05%, 96% inhibition at 0.5% SDS. The fact that activity was routinely recovered from the aqueous phases of the phenol extractions done in the presence of 2% SDS indicated that inhibition by SDS was easily reversible.

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Fig. 3. Effect of temperature on latent alkaline ribonuclease (O) and ribonuclease A (11). (A) General thermal denaturation of pancreatic ribonuclease A and latent alkaline ribonuclease. Samples of latent alkaline ribonuclease (26 units/ml, 0.35mg/ml protein) and ribonuclease A (1062units/ml, 0.35 mg/ml BSA) were treated for 10 min at the temperatures indicated and then placed on ice until assayed. (B) and (C) Effect of NaC1 on the stability of ribonuclease A and latent alkaline ribonuclease at 80°C. Duplicate samples of latent alkaline ribonuclease (51 units/ml, 0.71 mg/ml protein) and ribonuclease A (43 units/ml, 0.71 mg/ml BSA) without (B), or with (C), 100 mM NaC1 were incubated at 80°C. At indicated times, 25/xl portions were removed and placed on ice prior to assays done at a constant ionic strength of 67 mM NaCI. Data are given as per cent activity relative to controls also incubated at 23°C.

Proteinase K. Proteinase K catalyzes the hydrolysis of many proteins, most efficiently in the presence of up to 0.5% SDS (Ebeling et al., 1974), and it is typically used to remove contaminating nucleases from D N A or RNA preparations. The effect of proteinase K on latent alkaline ribonuclease and ribonuclease A is shown in Table 4. In the absence of SDS, latent alkaline ribonuclease was much more resistant than ribonuclease A to proteinase K. However, because latent alkaline ribonuclease and ribonuclease A were strongly inhibited by SDS, assays done in the presence of SDS were inaccurate.

295

Stability of latent ribonuclease Table 4. Effectof proteinaseK on latent alkalineribonucleas¢and pancreaticribonuclease A in the presence and absence o f S D S Latent alkaline ribonuclease

Ribonucleas¢ A

ProteinaseK

Initial

After 24 hr

Initial

After 24 hr

-

-

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+

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0.4

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0.1 0

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+ + 0 0 0 0 Data are expressedas units of activityremainingafter treatment relativeto controls. Samples were preincubated plus or minus I unit/ml of proteinase K, with and withoutthe inclusionof 0.05% SDS.Extraprotein(bovineserumalbumin, 17#g/ml) was added to the ribonucleaseA controls so that they had the same amount of protein as the latent alkalineribonucleasesample.To compensatefor inhibitionby SDS, sampleswere dialyzedafter the initial assay and re-assayed24 hr later (text).

Therefore, all samples were exhaustively dialyzed and then re-assayed 24hr after initial exposure to proteinase K. DISCUSSION Initially, 0.1% SDS was used to purify latent alkaline ribonuclease using phenol. Experience, popular wisdom, and results obtained by others (Parish, 1972), suggested that higher concentrations of SDS are more effective in protein removal than lower concentrations. Therefore, we anticipated that an experiment based on the widespread use of higher concentrations of SDS (1-2%) in preparation of m R N A (e.g. Cashmore, 1982) would eliminate the latent alkaline ribonuclease. Surprisingly, the amount extracted at higher SDS concentrations was increased. These results led to isolation of latent alkaline fibonuclease but raised questions about the chemical basis of latent alkaline ribonuclease's partitioning into the aqueous phase in the presence of SDS. It is likely that latent alkaline ribonuclease interacts directly with SDS below the critical miceller concentration of SDS (approximately 0.1% at 25 mM ionic strength; Helenius et al., 1979) because SDS, at low concentrations (0.013%), was a potent inhibitor. However, inhibition was still incomplete at 0.5% SDS, well above the critical miceller concentration of 0.1%, providing evidence that latent alkaline ribonuclease does not form a mixed micelle with the SDS. Because of the probability of charge repulsion between SDS and RNA, inhibition of latent alkaline ribonuclease by SDS is less likely due to secondary effects resulting from interactions between SDS and substrate than SDS and latent alkaline ribonuclease. At the salt concentration used during phenolic extraction (200 mM NaC1) the pronounced increase in the amount of latent alkaline ribonuclease entering the aqueous phase occurred well after the critical miceller concentration of SDS at this salt concentration was exceeded (0.026%). However, in the presence of phenol the critical miceller concentration of SDS in the mixture is most likely altered because of SDS's solubility in phenol. Therefore, the occurrence of latent alkaline ribonuclease in aqueous phases can be interpreted as the result of competition between SDS and latent alkaline ribonuclease for phenol. At low SDS concentrations the phenolic phase is unsaturated with SDS whereas at higher concentrations it is. Thus, latent

alkaline ribonuclcase's interactions with the more polar aqueous medium are favored. The fact that latent alkaline ribonuclease enters the aqueous phases when mammalian cytosols are extracted with phenol:SDS raises questions about preparation of mRNA. It would seem prudent to consider the possibility of contamination by latent alkaline dbonuclease if mRNA's from mammalian tissue are being studied that were prepared using this or similar techniques (e.g. phenol: SDS :chloroform). Although alkaline ribonuclease's occur in various cellular fractions (membrane: Aronson and Yannarell, 1975; nuclei: Niedergang et al., 1974; nucleoli: Sierakowska and Shugar, 1977; microsomes: Bachman et al., 1983) the only cellular fraction that contained phenol:SDS resistant latent alkaline ribonuclease was the cytosol, the same fraction in which Roth (1967) originally reported latent alkaline ribonuclease. The phenol: SDS: BD-cellulose method of purification of alkaline ribonuclease obliged us to determine: first, if the activity we isolated was the same as the latent alkaline ribonuclease purified by Button et al. (1982); and second, if it was affected by phenol:SDS. Based on characterization, behavior on native and denaturing PAGE, and inhibition by ribonuclease inhibitor, we conclude that the activity is latent alkaline ribonuclease and that its properties were unchanged by phenol:SDS. Latent alkaline ribonuclease obtained by Button et al. (1982) using conventional protein isolation techniques and latent alkaline ribonuclease isolated using phenol:SDS both yielded diffuse, proteinstained bands on SDS-PAGE. At least two possibilities could account for these observations. First, purified latent alkaline ribonuclease contains RNA. This RNA, which could be a contaminant, an associated degradation product, or necessary for activity, appeared as a smeared band on gels stained for R N A and migrated faster than latent alkaline ribonuclease but slower than tRNA. Therefore, the diffuse nature of the latent alkaline ribonuclease band could be the result of a slow dissociation of RNA from protein during electrophoresis. Second, some glycoproteins present similar diffuse bands. However, PAGE gels stained with periodic acid-Schiff reagent were negative and the likelihood of a cytosolic glycoprotein is minimal Of most interest relative to latent alkaline ribonuclease's characterization were its mol. wt and general specificity. The average mol. wt of latent alkaline

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ribonuclease obtained by gel filtration and SDS-PAGE was 51,000, a value essentially the same as previously reported for thyroid latent alkaline ribonuclease (Button et al., 1982; Rutherford et al., 1983; Turner, 1985). Latent alkaline ribonuclease catalyzed hydrolyses of tRNA and rRNA were incomplete after 2 hr; the majority of products were large enough to be excluded from G-50 Sephadex. Various double-stranded synthetic RNAs were unaffected by latent alkaline ribonuclease indicating single-strand specificity. Latent alkaline ribonucleases from bovine brain (Okazaki et aL, 1975) and chicken liver (Levy and Karpetsky, 1980) differ sufficiently from thyroid latent alkaline ribonuclease in mol. wt and other criteria to conclude they represent different activities. Factors affecting the distribution of latent alkaline ribonuclease between the aqueous and phenolic phases were of obvious importance to maximize yield and led to some interesting observations. Use of purified latent alkaline ribonuclease and ribonuclease inhibitor and the latent alkaline ribonuclease fraction revealed that the presence of ribonuclease inhibitor during phenol: SDS extraction decreased the amount of latent alkaline ribonuclease that could be recovered. Back-extraction of latent alkaline ribonuclease fraction with ammonium sulfate showed that the yield of latent alkaline ribonuclease from the postammonium sulfate fraction was greater than the amount of free latent alkaline ribonuclease originally present in the latent alkaline ribonuclease fraction. Thus, latent alkaline ribonuclease complexed with ribonuclease inhibitor was irreversibly lost when treated with phenol:SDS. These results agree with studies using porcine liver from which we could only obtain latent alkaline ribonuclease if ribonuclease inhibitor had been previously removed (A. W. Specht and F. J. Kull, unpublished). Therefore, the observation that less latent alkaline ribonuclease was obtained by phenol:SDS treatment of the latent alkaline ribonuclease fraction than from phenol: SDS-treated cytosol can be explained by the fact that the latter method destabilized ribonuclease inhibitor (i.e. buffers did not contain 2-mercaptoethanol and did contain NaCI). Additional experiments examining phase partitioning of latent alkaline ribonuclease can be summarized as follows: in the absence of ribonuclease inhibitor and in the presence of endogenous RNA, the distribution of latent alkaline ribonuclease between the aqueous and phenolic phases depended on the relative amount of RNA; the less RNA, the more latent alkaline ribonuclease that partitioned into the phenolic phase. When exogenous RNA in the form of tRNA was added prior to phenol:SDS treatment fewer units of activity were recovered than in its absence, possibly suggesting that the endogenous RNA associated with latent alkaline ribonuclease is other than a degradation product of tRNA. However, because some tRNA or its degradation products most likely carried through the phenolic procedure, the apparent units of latent alkaline ribonuclease recovered probably reflected competition between the unlabelled and labelled substrates and/or product inhibition. Distribution also depended on pH; less activity

was found in the aqueous phase at pH 6.0 than at 8.1. Similar to latent alkaline ribonuclease, ribonuclease A is stable to treatment with phenol and can be recovered from the phenolic phase (Kickhofen and Burger, 1962). However, these studies were done in the absence of SDS. We found that in contrast to latent alkaline ribonuclease, ribonuclease A quantitatively entered the phenolic phase whether or not SDS was present. Fully active latent alkaline ribonuclease was recovered from frozen and re-thawed phenolic phases indicating that little irreversible denaturation had occurred. Activity could also be recovered from phenolic phases by adding ethanol to about 75% concentration, storage at - 2 0 ° C for a few hours, centrifugation, dissolving precipitates in buffer and then dialysis. Routine dialysis alone did not remove SDS from phenolic phases resulting in underestimation of latent alkaline ribonuclease's recovery due to inhibition. Activity could not be recovered from the phenolic phases when phenolic treatment was done in the presence of ribonuclease inhibitor. Complete recovery of latent alkaline ribonuclease was also achieved when either 7 M urea or 3 M guanidinium:HC1 were removed by dialysis. Either these agents did not denature latent alkaline ribonuclease or it readily renatured during dialysis. Latent alkaline ribonuclease was neither affected by heating to 100°C for 10 min nor destabilized by heating to 80°C in the presence of 100raM NaC1. Because latent alkaline ribonuclease is almost certainly the same ribonuclease studied by Rutherford et al. (1983), it is also stable to mineral acids. In contrast, ribonuclease A was inactivated by heat at around 80°C and its activity was more rapidly lost in the presence of 100 mM NaC1. The effects of ethanol on alkaline ribonucleases have not been previously reported. Ethanol is widely used as a non-denaturing precipitant of nucleic acids and some enzymes, and it and glycol analogs (ethylene, propylene) have been found to stimulate some enzymes that interact with tRNA (Ritter et al., 1970; Kull and Jacobson, 1971). Latent alkaline ribonuclease's activity in the presence of ethanol made it possible to extract activity from native PAGE gels. Nearly complete inhibition of latent alkaline ribonuclease was seen at 0.05% SDS (about 2 mM), but inhibition was readily reversible by ethanolic precipitation and dialysis. The strong inhibition of latent alkaline ribonuclease by SDS requires a certainty that all traces of SDS are eliminated from incubations; a task easier said than done. Fortunately, SDS is soluble in phenol and both phenol and SDS are soluble in ethanol. In contrast to ribonuclease A, which was destroyed by proteinase K within 10 rain, significant amounts of latent alkaline ribonuclease remained even after a 24 hr exposure. This resistance to proteolysis, and others of its rather remarkable properties, could be due to latent alkaline ribonuclease's association with RNA. Based on direct detection of RNA in latent alkaline ribonuclease preparations, A2s0:A260ratios of 0.56, and a mol. wt of 51,000, each molecule of latent alkaline ribonuclease could be associated with 21,900 Da of RNA.

Stability of latent ribonuclease Acknowledgements--This research was supported by S&E funds from OCC of SUNY-Binghamton to F.J.K.

REFERENCES

Aoki Y. and Natori S. (1981) Activation of latent ribonuclease in fat-body of fleshfly (Sarcophaga peregina) larvae on pupation. Biochem. J. 196, 699-703. Aronson N. N. Jr and Yannarell A. (1975) Effects of membrane ribonuclease and Ynucleotidase on the digestion of polyuridylic acid by rat liver plasma membrane. Biochim. biophys. Acta 413, 135-142. Bachman M., Zahn R. K. and Muller W. E. G. (1983) Purification and properties of a novel pyrimidine-specific endoribonuclease termed endoribonuclease VII from calf thymus that is modulated by polyadenylate. J. biol. Chem. 258, 7033-7040. Bartholeyns J. and Baudhuin P. (1977) Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases. Biochem. J. 163, 675-683. Blobel G. and Potter V. R. (1966) Relation of ribonuclease inhibitor to the isolation of polysomes from rat liver. Proc. natn. Acad. Sci. USA 55, 1283-1288. Bradford M. M. (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Button E. E., Guggenheimer R. and Kull F. J. (1982) Purification of cytosolic, latent endoribonuclease from porcine thyroid. Archs Biochem. Biophys. 219, 249-260. Cashmore A. R. (1982) In Methods in Chloroplast Molecular Biology (Edited by Edelman M., Hallick R. B. and N.-H. Chua), pp. 387-392. Elsevier Biomedical Press, New York. Davis B. J. (1964) Disc electrophoresis-II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. Davis D. E., Karpetzky T. P. and Levy C. C. (1980) The ribonucleases of bovine skeletal muscle. Biochem. J. 189, 263-275. Ebeling W., Hennrich N., Klockow M., Metz H., Orth H. D. and Lang H. (1974) Proteinase K from Tritirachium album Limber. Eur. J. Biochem. 47, 91-97. Goldman R. E., Lerea K. M. and KUll F. J. (1981) A procedure for the rapid preparation of ~4C-aminoacyltRNA and its use in the assay of Class I ribonuclease and ribonuclease inhibitor. Prep. Biochem. I1, 49-67. Helenius A., McCaslin D. R., Fries E. and Tanford C. (1979) Properties of detergents. Meth. Enzymol. 56, 734-739. Kickhofen G. and Burger M. (1962) Das verhalten von Proteinen in Phenol I. Ribonuclease. Biochim. biophys. Acta 65, 190-199. Kraft N. and Shortman K. (1970) A suggested control function for the animal tissue ribonuclease-ribonuclease inhibitor system, based on studies of isolated and phytohaemagglutinin-transformed lymphocytes. Biochim. biophys. Acta 217, 164-175.

297

Kull F. J. and Jacobson K. B. (1971) Cytoplasmic phenylalanyl tRNA synthetase from Neurospora crassa: purification and properties when reacting with heterologous tRNA. Meth. Enzymol. 20C, 220-232. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Levy C. C. (1975) Roles of RNases in cellular regulatory mechanisms. Life Sci. 17, 311-316. Levy C. C. and Karpetsky T. P. (1980) The purification and properties of chicken liver RNase. J. biol. Chem. 255, 2153-2159. Niedergang C., Okasaki H., Ittel M. E., Munoz D., Petek F. and Mandel P. (1974) Ribonuclease of beef brain nuclei. Purification and characterization of an alkaline RNase. Biochim. biophys. Acta 358, 91-104. Okazaki H., Ittel M. E., Niedergang C. and Mandel, P. (1975) Purification of an alkaline ribonuclease from soluble fraction of beef brain. Biochim. biophys. Acta 391, 84-95. Parish J. H. (1972) In Principles and Practice of Experiments with Nucleic Acids, pp. 107-146. John Wiley, New York. Ritter P. O., KUll F. J. and Jacobson K. B. (1970) Aminoacylation of Escherichia coli valine transfer ribonucleic acid by Neurospora crassa phenylalanyl transfer ribonucleic acid synthase in Tris(hydroxymethyl) aminomethane hydrochloric acid and potassium cacodylate buffers. J.biol. Chem. 245, 2114-2120. Roth J. S. (1956) Ribonuclease V. Studies on the properties and distribution of ribonuclease inhibitor in the rat. Biochim. biophys. Acta 21, 34-43. Roth J. S. (1958) Ribonuclease. Studies on the inactive ribonuclease in the supernatant fraction of rat liver. J. biol. Chem. 231, 1097-1105. Roth J. S. (1967) Some observations on the assay and properties of ribonucleases in normal and tumor tissues. Meth. Cancer Res. 3, 153-242. Rutherford K. D., Button E. E. and Kull F. J. (1983) Porcine thyroid cytosolic, latent, alkaline ribonuclease: does an acidification step during purification alter the enzyme's properties? Comp. Biochem. Physiol. 75B, 545-552. Shortman K. (1962) Studies of cellular inhibitors of ribonuclease. III. The levels of ribonuclease and ribonuclease inhibitor during the regeneration of rat liver. Biochim. biophys. Acta 61, 50-55. Sierakowska H. and Shugar D. (1977) Mammalian nucleolytic enzymes. Prog. nucL Acid Res, molec. Biol. 20, 59-130. Turner P. M. (1985) The interaction of porcine thyroid ribonuclease inhibitor with pancreatic ribonuclease A and porcine thyroid alkaline ribonuclease. PhD thesis, University Center at Binghamton, New York. Turner P. M., Lerea K. M. and Kull F. J. (1983) The ribonuclease inhibitors from porcine thyroid and liver are slow, tight-binding inhibitors of bovine pancreatic ribonuclease A. Biochem. biophys. Res. Commun. 114, 1154-1160. Waters L. C. and NoveUi G. D. (1971) Analytical reversedphase chromatography of Escherichia coli aminoacyl-tRNAs. Meth. Enzymol. 20, 39-44.