Simplified methods for isolation of ubiquitin from erythrocytes. Generation of ubiquitin polymers

0020-71IX/87 $3.00f 0.w) Copyright Q 1987Pergamon Journals Ltd

fnf, J. Biochem.Vol. 19, No. II, pp. 1055-1061,1987 Printed in Great Britain. All rights reserved

SIMPLIFIED METHODS FOR ISOLATION OF UBIQUITIN FROM ERYTHROCYTES. GENERATION OF UBIQUITIN POLYMERS Max-Planck-Institut

HAROLD F. DEUTSCH* fiir Zellbiologie, 6802 Ladenburg, West Germany (Received 5 January 1987)

Abstract-l. Ubjquitin has been isolated from bovine erythrocytes by procedures in which the hemoglobin was removed by denaturation with either ethanol~hlorofo~ mixtures or by heating. 2. The proteins soluble to the denaturation step were removed by 3% sodium tri~hIoroa~tate (TCA) at pH 2.c2.5 or by S% TCA. 3. Ubiquitin was isolated in relatively high yield from the TCA insoluble fraction by use of single ion-exchange chromatographic and gel permeation steps. 4. Ubiquitin shows relatively little cross-linking upon treatment with glutaraldehyde or with dimethyl suberimidate. Heating of the glutaraldehyde treated material in 4 M guanidine, however, leads to marked aggregation. 5. The polymers of ubiquitin react strongly with antibody in an immunoblot assay.

lNTRODlJCTION Ubiquitin,

a 8565 Dalton protein has been found to

possess a relatively wide range of biological activities. These include an apparent role in the regulation of chromatin structure as related to transcription (Goldknopf er al., 1975; Finley and Varshavsky, 1985; Mueller et al., 1985), its obligatory role in ATPdependent non-lysosomal proteolysis (Ciechanover et al., 1984a,b,c; Finley et al., 1984; Hershko and Ciechanover, 1982; Hershko, 1983), as part of a lymphocyte cell surface receptor (Siegelman et al., 1986; St. John et al., 1986) and a postulated function in heat-shock responses (Ciechanover et al., 1984b, Finley et al., 1984; Bond and Schlesinger, 1985). Ubiquitin also possesses weak, sulfonamide inhibitable carbonic anhydrase and esterase activities (Matsumoto et al., 1984) as well as being able to cleave aromatic phosphate esters at acid pH (Tanigucbi and Matsumoto, 1985). Details of the role of ubiquitin in the ATPdependent breakdown of specific proteins are rapidly emerging. Shanklin et al. (1987) have recently shown that the light mediated conversion of the photoreceptor phytochrome into its far-red light absorbing form increases its proteolytic degradation about lOO-fold due to its rapid conjugation with ubiquitin. Isopeptide linked polyubiquitin complexes when joined to lysozyme increase the rate of proteolytic degradation of lysozyme as compared to monomer linked ubiquitin complexes (Hershko and Heller, 1985). Recent studies by Bachmair et a!. (1986) indicate that the rate of in vitro degradation of proteins undergoing proteolysis over the ubiquitin pathway is a function of their amino-terminal groups. The wide range of studies with ubiquitin require that substantial amounts of this protein be available. *Present address: Department of Physiological Chemistry, University of Wisconsin, Madison, WI 53706, U.S.A.

Two relatively large scale methods for the isolation of ubiquitin from erythrocytes have appeared (Jabusch and Deutsch, 1983; Haas and Wilkinson, 1985). The essential requirement of removing the large amount of hemoglobin has been accomplished in one case (Jabusch and Deutsch, 1983) by denaturation with ethanol-chloroform (CHCl,) by the classical method of Tsuchihashi (1923) and more recently by heating at 90” (Jabusch and Deutsch, 1983). We have reinvestigated the methods to provide a somewhat simpler and rapid method of preparation by which ubiquitin can be isolated from hemolysates to the level of crystallinity by one ion-exchange and one gel permeation step following removal of the hemoglobin. In addition, a study of the ability of ubiquitin to form aggregates by cross-linking procedures has been carried out, EXPERIMENTAL Materials and methcdf The bovine and hog bloods used as the source of erythrocytes were obtained at a local abattoir. Erythrocytes were washed twice with two volumes of 0.15 M NaCl except in several instances where packed cells were used directly. All chemicals employed were of reagent grade. The chromatographic procedures employed DEAE-Sepharose 6B CL (Pharmacia) and Ultrogel AcA-54 (LKB) and fractions were collected with the LKB Ultra-rat 2111 apparatus. Because of the relatively low mol. wt of ubiquitin (856.5) all dialysis experiments were carried out with Spectra/Par 3 membranes with a cut-off of 3500. Ultraviolet abso~tion m~sur~ents utilized a Gilford Model 2600 S~trophotometer and recordings were made with a Hewlett-Packard 7225 A Plotter. High pressure liquid chromatography employed the Waters Associates Model 660 apparatus and a Cl8 Bondapak column. A gradient from 5 to 70% acetonitrile in 0.1% tiifluoroacetic &id was used to elute protein. Electronhoresis was carried out in l&25% SDS-gels. The immunobiot experiments were carried out essentially by the method of Haas and Bright (1985) which utilizes the procedure of Burnette (1981) for the transfer of protein from

1055

1056

HAROLDF. DEUTSCH

0.15

E c 2 E 0.10 x ?j =l 005

5

65

Fractroo No

Fig. 1. (A) Result for the chromatography of 23ml of bovine erythrocyte (washed) proteins soluble at 90°C on a 3 x 60 cm column DEAE-Sepharose 6B. Full scale absorbance was 0.2 and 9.03 ml fractions were collected. In this and the following chromatograms the arrows indicate the ubiquitin fractions taken. (B) Ubiquitin fractions from experiment A (7.2 ml) applied to a 2 x 75 cm column of AcA-54. Full scale absorbance was 0.2 and 3.82 ml fractions were collected. the SDS-gels to the nitrocellulose and its detection. The antibody employed was the immunoglobulin fraction from the serum of rabbits immunize to ubiquitin by the method reported by Hershko er al. (1982) to give antibody reacting primarily with non-conjugated ubiqnitin. Large scale preparation of ubiquitin Most of the ubiquitin isolations utilized bovine erythrocytes that had been washed twice with 2 vol of isotonic saline at approximately 4°C. The use of unwashed cells appeared to result in larger amounts of hemeprotein products being present in the supernatants to the hemoglobin Table I. Isolation of ubiquitin from erythrocytes

(4

1. 2. 3. 4.

(B)

I. 2. 3.

1.

2. 3.

4. 5. 6. 7.

Removal of hemoglobin with ethanol_CHCl, as in procedure of Jabusch-Deutsch (1983). Na tricbloroacetate added to supematant to a cont. of 3%, temp. 0°C. Add 25% TCA (w/v) to pH 2.0-2.5. Centrifuge off precipitate at 0°C after stirring 30-40 min. Removal of hemoglobin at 90°C as in procedure of Haas-Wilkinson (1985). Ubiquitin precipitated by addition of 100% TCA (w/v) to cont. 5% at 0°C. Precipitate removed at 0°C after stirring 3~min.

Sreps common to. both hemoglobin removal methods TCA precipitate suspended in pH 8.0, 0.5M Tris-HCI and I N NaOH added with rapid stirring to pH 8.0. Suspension dialyzed to equilibrium in cold (2-C) against pH 8.0, 25 mM Tris-HCl. Insoluble material removed by centrifugation near 0°C. Supernatant applied to column of DEAE-Sepharose 6B equilibrated with pH 8.0, 25mM Tri-HCI. Column etuted with linear gradient to pH 8.0, 25 mM Tris-HCl containing 500mM NaCl, amount of eiuting solutions about 4 x column volume. Ubiquitin fractions placed in Spectrapore 3 dialysis tubing and sufficient ammonium sulfate placed outside tubing to effect saturation of solution. Precipitate suspended in small amount of H,O and dialyzed 3O-@min to effect solution. Solution applied to AcA-54 column equilibrated with pH 8.0, IOOmM Tris-HCI ~nt~ning 500 mM NaCI. Elution with same buffer. Ubiquitin fractions pooled, precipitated as in step 4 above. Precipitate suspended in small amount of H,O and dialyzed exhaustively against H,O. Ubiquitin solution held frozen or . lyophilized.

-!60 Fractm

No

Fig. 2. (A) Result for the chromatography of 72 ml of porcine erythrocyte (unwashed) proteins soluble at 90°C on a 3 x 64 cm column of DEAE-Sepharose 6B. Full scale absorbance was 0.2 and 8.35 ml fractions were collected. (3) Ubiquitin fractions from experiment shown in A (24ml) applied to a 2 x 74 cm column of AcA-54. Full scale absorbance was 0.2 and 3.89 ml fractions were collected. denaturation step that bound irreversibly to DEAESepharose 6B. The levels of hemoglobin in the frozen and thawed hemolysates was determined by dry weight analyses or refractometrically and were in the range of 28-30%. Removal of this protein from the lysates was accomplished with ethanoLCHC1, as previously described (Jabusch and Deutsch, 1983) or by heating at 90°C as in the procedure of Haas and Wilkinson (1985). Sodium trichloroacetate (TCA) was added to the supernatant to the ethanol-CHCl, to a level of 3%, the temperature being maintained at O”C, and 25% aqueous TCA (w/v) was then added until the pH was between 2.0 and 2.5. In the case of the filtrate to the heated hemolysate, 100% TCA (w/v) was added to a cont. of 5% as in the method of Haas and Wilkinson (1985). This results in a pH below 1. Following stirring for 3&60 min at near 0°C the precipitates formed were removed by centrifugation at this temperature. The remaining steps were identical for the two procedures and are given in the fractionation steps outlined in Table I. Typical results for the chromatography on DEAESepharose 6B and subsequent gel filtration on AcA-54 columns of the TCA precipitate of ubiquitin from material prepared by the ethanolCHC1, precipitation and by the heat denaturation of hemolysates are shown in Figs I-3.

Fraction No

Fig. 3. (A) Resuit for the chromatography of 98ml of bovine erythrocytes (unwashed) proteins soluble at 90°C on a 3 x 65 cm column of DEAE-Sepharose 6B. Full scale absorbance was 0.2 and 7.98 ml fractions were collected. (B) Ubiquitin fractions from experiment shown in A (98 ml) applied to a 2 x 75 cm column of AcA-54. Full scale absorption was 0.5 and 3.88 ml fractions were collected.

Fig. 4. (A) Results of SDS-ge1 (l&25%) ekctrophoresis of unmodi~ed ubiquitin (lanes 1, 2 and S), of dimethyl suberimidate (lane 4) and glutaraIdeh~de (lane 3) treated protein. The corresponding immunoblots are shown in (B).

Fig. 6. (A) Results of SDS-ge1 (lO-25%) ekctrophoresis of ubiquitin polymers as separated on an AcA-54 column (see Fig. 5). The corresponding immunoblots are shown in (B).

1057

Preparation of ubiquitin The left-hand figures labelled A are the results for the DEAE-Sepharose chromatography, the accompanying right-hand ones labelled B are the AcA-54 gel permeation results of the fractions pooled in the experiments in A. Figure 1 is a typical experiment using I560 ml of hemolysate from washed bovine erythrocytes containing 30% Hb. The yield of ubiquitin based on a value of A $$iaa = 1.6 was 173 pg per g of hemoglobin. Similar results were obtained for experiments in which the hemoglobin was removed by the ethanolCHC1, method except that lower yields were obtained (see Table 2). The NaTCA precipitates of this method contained less hemeprotein products than the heat denatured ones and smaller amounts of dark-brown material which could not be eluted with 1M NaCl were bound to the DEAE-Sepharose. This upper portion of the DEAESepharose columns was usually discarded after several experiments had been run on a given column. Thus the ethanoKHC1, experiments resulted in the loss of smaller amounts of DEAE-Sepharose. The results for experiments using unwashed porcine and bovine erythrocytes in which the hemoglobin was removed by the heat denaturation method are shown in Figs 2 and 3, respectively. In the case of the unwashed bovine erythrocyte hemolysate the ubiquitin fraction did not separate well from the major protein fractions on the DEAE-Sepharose column because 98 ml of protein was applied to a 460 ml column. In addition, the material pooled had a green-brown color. Upon chromatography of this fraction on AcA-54 the material eluting before the ubiquitin fraction was bright green. A spectroscopic scan of this material from 220 to 700 nm in the presence and absence of H,O, indicated that it was most likely a mixture of eosinophil and myeloperoxidase (Olson and Little, 1983) derived from the white blood cells in the unwashed erythrocytes. The elution of the ubiquitin fraction from the DEAESepharose 6B began at I .05-l .07 column volumes and from the AcA-54 columns at 0.75 column volumes. Various determinations of the 280 nm absorbtivities of the purified ubiquitin fractions gave A:z;irnm values near 1.6. This is in agreement with the results of Haas and Wilkinson (1985). The purified ubiquitin gave a single peak on SDS-gel electrophoresis and a single component on high pressure liquid chromatography. It is interesting that upon dialysis against H,O of the ammonium sulfate precipitated ubiquitin fractions from both the DEAE-Sepharose 6B and AcA-54 columns, the material existed as a mass of birefringent micro-crystals prior to solution of the protein, The yields of ubiquitin experienced in a series of fractionation experiments employing heat denaturation to remove the hemoglobin gave somewhat variable results but on the average about 60% better yields than by the ethanol-CHCl, method. This would appear to be due to the ability to remove the occluded ubiquitin in the massive precipitate of heat denatured hemoglobin by washing with hot H,O. The nature of the ethanolCHC1, precipitate of Hb does not readily permit such an extraction step. The use of unwashed hog erythrocytes in a single experiment gave a yield of 204 I.cg per g of hemoglobin while the unwashed bovine cells yielded only 73 pg. It is not known whether the concentration of ubiquitin in the porcine erythrocytes is significantly higher than in the bovine cells. Only

1059

a single experiment was performed on the porcine source cells. The results indicate that erythrocytes from both of these readily available animals may be employed to generate substantial amounts of ubiquitin.

Since ubiquitin appears to be synthesized as a multiunit protein in various organisms (Bond and Schlesinger, 1985; Ozkaynak a! al., 1984; Dworkin-Rastl et al., 1984) it was of interest to form some artificial polymers of ubiquitin as a preliminary to searching for the presence of naturally occurring ones. Two methods of cross-linking of ubiquitin were attempted. One of these empioyed dimethyl suberimidate (Davies and Stark, 1970), the other ~utaraldehyde. Dimethyl suberimidate (Pierce) in a 10 fold excess over ubiquitin (5 x 10m6mol) in pH 8.7,0.2 M triethanolamine buffer containing 4 M guanidine was allowed to react for 4 hr at room temperature when the reaction was terminated by adding an excess of ammonium acetate. When this material was subjected to filtration over an AcA-54 column only a small amount, i.e. less than 10% of the protein was present as an aggregate. SDS-acryiamide gel electrophoresis of this material also showed the presence of only a small amount of what appeared to be a ubiquitin dimer. This minor component, however, appeared to react more strongly with antibody in an immunoblotting experiment than the major monomer component (see Fig. 4B). The antibody employed was to a complex of ubiquitin with human serum albumin that was subsequently adsorbed with albumin. According to Hershko et at. (1982) this procedure results in antibody that is primarily directed against the non-conjugated form of ubiquitin. Ubiquitin (1.2 x 10-6mol) in pH 8.7, 0.2 M triethanolamine buffer was also reacted for 5 hr at room temperature with glutaratdehyde in amounts ranging from 0.5 to 16 times the molar levels of ubiquitin. All of these samples showed extensive aggregation when assayed by SDS-gel electrophoresis. The result for a 5: 1 molar ratio glutaraldehyde:ubiquitin reaction mixture is included in Fig. 4. It is apparent that a marked formation of polymers has occurred. However, even at the higher molar ratios of glutaraldehyde, only small amounts, i.e. less than 10% of dimerized protein was noted when the reaction mixtures were subjected to gel permeation chromatography on AcA54 columns. This result suggested that polymerization of the glutaraldehyde derivatized material occurred only after heating of the sample with SDS at 100°C prior to electrophoresis. Another sample of ubiquitin (1.2 x 10s6 mol) was treated with the same amount of glutaraldehyde for 4 hr at room temperature. The mixture was then extensively dialyzed,

E 0.6

1 1 12131 I

8 c 0.3 B $ 0.2 9 0.1

Table 2. Amounts of ubiquitin recovered from

bovine erythrocytes

pg ubiquitinig Hb Hemoglobin WS?lOVt4

Number ~~ experiments Range

Heating Ethanol-CHCl,

6 4

Heating* Heating* (porcine)

I I

*Unwashed erythrocytes employed.

92-173 57-90

~~

5

10 15 20 Fraction No

25

Average 130 76 13 206

Fig. 5. Results of the chromatography of IO mg of ubiq~tin treated with an equi-molar level of glutaraldehyde and denatured in 4 M guanidine-HCl at 100°C. The protein in 1.5 ml was applied to a I x 45 cm column of AcA-54. Full scale absorbance was 0.1 and 1.84 ml fractions were collected. The material pooled is indicated by the vertical bars.

1060

HAROLDF. DEUTXH

lyophilized and taken up in 1.0 ml of pH 8.7, 0.2 M triethanolamine buffer containing 4 M guanidine-HCl. After heating for 2min at 100°C the sample was applied to an AcA-54 column to give the result shown in Fig. 5. The fractions separated were pooled as indicated, dialyzed against distilled water and lyophilized. A recovery of 83% of the protein applied to the column was obtained, The fractions separated on the AcA-54 column were subjected to SDS-gel electrophoresis and immunoblotting to give the result shown in Fig. 6. It appears that fraction 4 is largely ubiquitin monomer, 3 is largely the dimer and that the main component of fraction 2 migrates in an area that indicates it is a trimer. The first fraction eluting from the AcA-54 column contains all of the above noted components plus higher molecular weight ones. The presence of what appears to be monomer, dimer and trimer units in fraction 1 from the AcA-54 column (see Fig. 5) suggests that the glutaraldehyde induced higher mol. wt polymers of ubiquitin separated on the AcA-54 column may be able to slowly dissociate. Nevertheless, it is clear that the initial glutaraldehyde addition derivatives of ubiquitin remain as monomers that are able to undergo extensive polymerization upon heating. The results of Fig. 6B show that even when the polymers are present at low levels they react strongly with the antibody employed. DISCUSSION

The two large scale methods for the preparation of ubiquitin that have appeared in the literature (Jab-

usch and Deutsch, 1983; Haas and Wilkinson, 1985) have been modified. The use of ethanolKHC1, to denature hemoglobin usually requires that the organic solvents be removed by dialysis or evaporation following removal of the massive precipitate of Hb. The present method uses NaTCA at pH 2.0-2.5 to precipitate the ubiquitin present in this solution. This appears to result in the denaturation of such erythrocyte enzymes as carbonic anhydrase, catalase, superoxide dismutase, etc. If the separation of any or all of these entities along with ubiquitin is desired, the organic solvents can be removed by dialysis as was carried out in our earlier procedure (Jabusch and Deutsch, 1983). The dialysis procedure, however, is time consuming and requires use of relatively expensive Spectra/Par 3 membranes. The use of NaTCA precipitation results in a somewhat lower yield of ubiquitin than when the heat denaturation method of Haas and Wilkinson (1985) is employed. We have modified the latter workers method to include only 2 chromatographic steps. The method as applied to bovine and porcine erythrocytes appears to give higher yields of ubiquitin than Haas and Wilkinson (1985) obtained using outdated human blood. The ethanol-CHCl, method gives somewhat cleaner supernatants to chromatograph following removal of hemoglobin, which also results in an eventual saving of some of the DEAE-Sepharose which appears to bind irreversibly to some denatured hemeprotein or heme fraction. The method presented is relatively simple and amounts of pure ubiquitin in the 1IO-150 mg range can be readily isolated from 1500 to 2000 ml of packed erythrocytes. The formation of ubiquitin polymers has been explored. Glutaraldehyde addition products of ubiquitin undergo extensive aggregation when heated in the presence of guanidineHC1. Little polymer formation is experienced when ubiquitin is reacted with

dimethyl suberdiimidate. However, in this case the possibility of an amidination addition product to polymerize upon heating of the protein was not explored. The result obtained with glutaraldehyde indicates that this substance is able to form a complex with ubiquitin but due to steric hindrances cannot cross-link with other molecules. However, when subjected to denaturing conditions these ubiquitin derivatives can now crossreact with other such molecules to form polymers. The marked reactivity of the artificial polymers of ubiquitin with antibody is of interest. These polymers form by the cross-linking of the amino groups of lysine. Antibody that was predominantly to the free form of ubiquitin as was employed in the present study has been reported to react with an epitope requiring a carboxyl terminal diglycine sequence. Removal of these residues leads to a marked diminution of the antigenicity of ubiquitin (Haas et al., 1985; Vierstra et al., 1985). A search for the presence of naturally occurring ubiquitin polymers using an antibody reaction may pose problems since these complexes would be “head-to-tail” bonded through the carboxyl-terminal glycine residue with the amino terminal methionine. Another type of complex are polyubiquitins formed through a C-terminal glycine of ubiquitin with a lysine residue of another such molecule as noted in the ATP dependent degradation of proteins (Hershko and Heller, 1985). It is not known whether such a complex would diminish the reactivity of the antibody requiring a C-terminal diglycine sequence. However, various complexes of ubiquitin in which its C-terminal glycine is bound to the t-amino group of the lysines of various proteins appears to undergo good reactions with antibody to ubiquitin (Haas and Bright, 1985). The ability to immunologically distinguish the latter complexes from naturally occurring head-to-tail polymers of ubiquitin might permit one to determine the level of the latter material and to eventually explore if they play a significant biologic role. Acknowledgements-The author gratefully acknowledges the award of a visiting professorship from the Alexander von Humboldt Foundation as well as support from the University of Wisconsin Graduate School. Thanks are due to Annemarie Scherbarth for performing the gel electrophoresis and immunoblot analyses and to Dr S. Kuhn for the high pressure liquid chromatographic experiments. It is a pleasure to acknowledge the support of Professor Peter Traub in making available the facilities of his laboratory and for discussions relating to this study. REFERENCES

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