Limited proteolysis of calf thymus terminal deoxynucleotidyl transferase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 202, No. 2, July, pp. 414-419, 1980

Limited MARTIN Department

Proteolysis of Calf Thymus Terminal Deoxynucleotidyl Transferasel

R. DEIBEL,

JR. AND MARY University of Kentucky,

of Biochemistry,

SUE COLEMAN Lexington,

Kentucky

40536

Received January 15, 1980 Controlled, limited proteolysis of homogeneous calf thymus terminal deoxynucleotidyl transferase (EC 2.7.7.31) using immobilized Staphylococcus aweus V-8 proteaae results in a low molecular weight form of the enzyme which possesses unaltered catalytic activity. Analysis of the products of limited proteolysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that only the large subunit, p, is modified from a molecular weight of 30,500 to 25,500. The small subunit, (Y, which has a molecular weight of 9500, is unchanged. A shift in the apparent isoelectric pH of the calf enzyme following proteolysis is observed from PI = 8.2 to 7.8. Both forms of the enzyme are homogeneous in the isoelectric focusing gel system, as determined by coincidence of single protein bands with terminal transferase activity on the gel. The specific activities of cleaved and uncleaved terminal transferase proteins, as well as their thermal stabilities, are comparable. These results suggest that the polypeptide domain involved in terminal transferase enzymatic activity can be probed further bv novel methods involving limited proteolysis without concomitant loss in enzymatic function.

We have successfully used limited proteolytic digestion to cleave a highly sensitive region of homogeneous calf thymus terminal transferase.2 The product is a low molecular weight protein which retains the specific activity observed with the parent enzyme. Terminal transferase activity isolated from a variety of tissues has recently been shown to exist in different size classes. The highest molecular weight form of native terminal transferase which we have isolated from human blasts is 62,000 (1). The molecular weight for the native calf thymus enzyme isolated in our laboratory varies from 40,000 to 44,000, depending on the electrophoretic system utilized (1). While the molecular weight 1 From the Department of Biochemistry, University of Kentucky, Lexington, Ky., 40536. Sup ported by Public Health Service Research Grants CA19492 and CA26391. M. S. Coleman is the recipient of Research Career Development Award K04CA00494. * Abbreviations used: p(dA)c, poly(deoxyadenylic acid) with an averge chain length of 50 residues; terminal transferaae, terminal deoxynucleotidyl transferase; SDS, sodium dodecyl sulfate. 0003~9861/80/080414-06$02.00/O Copyright 0 1980 by Academic Press, Inc. AII rights of reproduction in any form reserved.

414

and subunit structure of terminal transferase differ, the reactions carried out in vitro appear to be identical. The reduction in molecular weight of the calf thymus enzyme but not in catalytic activity which we observe following limited proteolysis, indicates that the catalytic process of nucleotidyl transferase exists in a low molecular weight polypeptide domain. The remainder of the protein mass could be involved solely in the recognition, transport, and/or post-translational modification of terminal transferase for physiological roles which remain to be determined. While enzymatically active low molecular weight fragments of DNA polymerase-I (Escherichia coli) have been observed (2), our procedure is the first report of controlled limited proteolysis of a polymerase protein which has only one major cleavage site with no loss or obvious change in enzymatic function. To probe the active site of terminal transferase, we have devised a technique in which the enzyme, Staphylococcus aureus V-8 protease (3), is immobilized on Sepharose via cyanogen bromide activa-

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OF CALF TERMINAL

tion. The immobilized enzyme retains catalytic activity and is easily separated from the digestion products by centrifugation, so that the products of the reaction can be further characterized in the absence of protease. This report represents an initial study in our laboratory to assign polypeptide domains in terminal transferase to function in wivo. EXPERIMENTAL

PROCEDURES

Materials. Radioactive deoxynucleoside 5’-triphosphates were purchased from New England Nuclear Corporation. Unlabeled deoxynucleoside 5’-triphosphates were from Sigma Chemical Company, and were further purified (5). Acrylamide, bisacrylamide, Bio-Lytes (pH 3-10 and pH 8-10) were from BioRad Laboratories, Oligo(dT)-cellulose (Type 7) was purchased from P-L Biochemicals, Inc. S. aureus V-8 protease was obtained from Miles Laboratories (500 units/mg protein at pH 7.8 with casein as substrate). Cyanogen bromide-activated Sepharose was obtained through Sigma Chemical Company. Enzyme assays. The assay of calf thymus terminal transferase has been described (6). The assay mixture in a final volume of 125 ~1 contained 0.2~ potassium cacodylate, pH 7.5, 1 mM 2-mercaptoethanol, 0.010 mM p(dA),, and 1 mM [3H]dATP (100 cpm/pmol) with 8 mM MgCl,, or 1 mM L3H]dGTP (100 cpm/pmol) with 0.5 mM MnCl,. The reactions were incubated at 35°C for varying time periods, and terminated by application of 25-~1 aliquots onto glass fiber papers (GF/C, Whatman) as previously described (7). One unit of enzyme activity is defined as 1 nmol of radioactive deoxynucleotide incorporated per hour, and specific activity is expressed as units of activity per milligram of protein. Enzyme protein was measured by the method of Lowry et al. (8) using bovine serum albumin as the standard protein. Preparation of homogeneous calf thymus terminal trunsferase. Terminal transferase was purified from calf thymus tissue by standard chromatographic techniques as shown in Table I. Several modifications, including carboxymethyl-Sephadex and oligo(dT)-cellulose chromatography were introduced into the procedure devised by Bollum (4) to avoid the necessity of using the low pH incubation step. The column steps which are novel to this procedure have been described in detail elsewhere (1, 9). The purified protein was determined to be at least 99% pure by the following criteria: (a) the specific activity of the enzyme was 111,205 units/mg, (b) only the p and (Y subunits were observed by SDS-polyacrylamide gel electrophoresis, (c) immunoprecipitin bands are obtained on Ouchterlony plates against rabbit

415

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anti-calf antisera (data not shown), and (d) a single protein band coincident with terminal transferase activity was noted on isoelectric focusing polyacrylamide gels (see Fig. 2). Preparation of immobilized S. aureus V-8 protease. S. aureus V-8 protease protein (2 mg) was coupled to Sepharose 4B (5 ml swollen bed volume) by the method of cyanogen bromide activation as described previously (10). Protease protein was mixed with commercially available CNBr-Sepharose (Sigma Chemical Co.) following preliminary washing and swelling of the gel with 1 mM HCl followed by incubation of the gel in 10 ml of 0.2 M potassium phosphate, pH 8.0. The protein-ligand was subsequently added in 5 ml of the same buffer, and the resulting suspension was mixed by gentle rotation at 4°C for 24 h. Following the coupling step, the gel was exhaustively washed with 0.2M potassium phosphate, pH 8.0, and finally was incubated in 10 ml of 0.1 M ethanolamine, pH 7.0, for 24 h at 4°C to eliminate remaining reactive sites on the gel. The gel was then washed repeatedly with 0.2~ potassium phosphate, pH 8.0, 0.2~ sodium acetate, pH 5.0, and 0.2~ potassium phosphate, pH 10.0. The protease-gel was subsequently stored in the 0.2~ potassium phosphate buffer, pH 8.0, containing 50% glycerol, and stored at -20°C until required. The yield of coupled protein was estimated to be 5-10% of the total A,,, units applied, and the protease-gel was determined to be enzymatically active by analysis of the hydrolysis of 1 mg/ml solutions of azoalbumin as previously described (11). The immobilized V-8 protease has been stored at 4°C for 6 months and still retains at least 50% of the activity originally detected by hydrolysis of solutions of azoalbumin. Controlled, limited terminal transferuse.

proteolysis

of

calf

thymus

Purified calf thymus terminal transferase was quantitatively cleaved into a lower molecular weight protein (maintaining the specific activity of the parent enzyme) by incubation with immobilized V-8 protease (prepared as described above). For this procedure, 147 pg of homogeneous calf terminal transferase was incubated with gentle rotation at 4°C for 3 days in the presence of 5 ml (bed volume) of immobilized V-8 protease, in a buffer (1 ml) consisting of 25 mM potassium phosphate, pH 7.2, containing 1 mM 2-mercaptoethanol. The suspension was subsequently centrifuged and the supernatant removed. The gel was further washed with 0.5 ml of 50 mM potassium phosphate, pH 7.2, containing 1 mM 2-mercaptoethanol, and the washings were combined with the original supernatant. This preparation was further concentrated by ultrafiltration (PM-10 membrane, Amicon Corp.) to a final volume of 0.25 ml. To account for all products of proteolysis using this method, direct aliquots of the reaction supernatant were analyzed directly by SDS-

416

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AND COLEMAN TABLE

I

PURIFICATION OFCALFTHYMUSTERMINALDEOXYNUCLEOTIDYLTRANSFERASE

Fraction

Volume (ml)

Protein (mg)

Specific activity” (units/mg)

Crude extract Phosphocelhdose I DEAE-cellulose CM Sephadex I Hydroxylapatite I Sephadex C-100 Hydroxylapatite II CM Sephadex II Oligo(dT)cellulose

8,300 1,100 1,475 220 350 101 99 6.8 2.1

2.6 x lo5 5.2 x lo3 5.9 x 103 737 528 122 31 11 2.1

9.3 312 242 1,397 1,974 10,000 14,866 68,630 111,205

Total activity (units) 2.5 1.6 1.4 1.0 1.0 1.2 0.5 0.8 0.25

x x x x x x x x x

Yield 6)

lo6 lo8 106 106 106 10s 10s lo8 10s

100 65 57 41 41 48 19 30 10

Purification factor 1 33 26 150 212 1,075 1,598 7,379 11,957

(1Represents polymerization of MgdATP onto a synthetic initiator, p(dA)w. One unit of enzyme activity is defined as 1 nmol of radioactive deoxynucleotide incorporated per hour at 35°C. gel electrophoresis, avoiding the ultrafiltration step. A summary of the results of this procedure are shown in Table II. No protease activity is detected in the supernatant of the protease-gel, and in no instances have we ever detected protein bands of the protease on SDS-gels in control experiments when no substrate (terminal transferase) has been added. Analytical isoelectric focusing. Terminal transferase (native form and proteolytically cleaved form) was analyzed by isoelectric focusing using a vertical phase slab gel apparatus (Bio-Rad Model 220). The gels were polymerized to a thickness of 1.5 mm. Polymerization was conducted using 5% acrylamide with ammonium persulfate catalysis (0.75%) at a temperature of 4°C. The gel was allowed to remain at 4°C for an additional 3 h to insure completeness of polymerization. Samples of terminal transferase protein were applied to individual wells in a total volume of 0.025 ml (50% glycerol and 1 mM 2-mercaptoethanol). Protein markers of known isoelectric pH were applied under comparable conditions to separate wells. An ampholyte system consisting of 2% (w/v) pH 3-10 and 0.5% (w/v) pH 8- 10 was determined previously to provide maximum results for this enzyme (1). The anodic and cathodic tank solutions were 0.1 M phosphoric acid and 0.15 M ethanolamine, respectively. Samples were applied to the basic pH end of the ampholyte pH gradient. Electrofocusing was performed using an Ortec pulsed, constant power supply for a total of 8 h, using 200 V, 300 pulses/s (pulse rate), and 1.0 mfd (discharge capacitance). After 4 h, no further changes in current could be detected. Current readings of 2 mA per sample were observed in the initial stages of electrofocusing, and a final plateau current of 0.5 mA per sample was determined. Duplicate tracks of

the gel were either stained for protein or analyzed for terminal transferase activity. In the former method, the gel was stained for 12 h in 0.2% (w/v) Coomassie brilliant blue R-250 (50% methanol and 10% acetic acid), and destained in methanol-acetic acid alone. For the assay of terminal transferase activity, tracks of the gel were cut into 2-mm sections, and incubated for 12 h in 50 ~1 of 50 mM potassium phosphate, pH 7.2, containing 1 mM 2-mercaptoethanol. Volumes of 10 ~1 were removed and assayed for terminal transferase activity as described above, except that the reaction was conducted over a 12-h period in a total assay volume of 50 ~1. Aliquots of 25 ~1 were applied to glass fiber papers and TABLE

II

LIMITEDPROTEOLYSISOFCALF THYMUS TERMINAL DEOXYNUCIJWTIDYLTRANSFERASE Specific activity” (units/mg)

Protein (Yield)

Activity (Yield)

Native

147 I% ( lOO%)b

16,347 units (lOO%)b

111,205

Cleaved

65 cLg (44%)

6,831 units (42%)

105,096

Fraction

a Specific activity was determined by the polymerization of M$+dATP onto a synthetic initiator, p(dA)E. One unit of enzyme activity is defined as 1 nmol of radioactive dAMP incorporated per hour at 35°C. b Represents total protein and activity utilized in this preparative limited proteolysis experiment (immobihzed V-8 protease).

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Lactate dahydroganose Carbonic onhydmse

46 -

4.2.

a

341

02

04

RELATIVE

0.6

\

0.8

IO

MIGRATION,

Rm

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of homogeneous calf thymus terminal deoxynucleotidyl transferase and the products of limited proteolysis with immobilized S. aureus V-8 protease. Conditions for gel electrophoresis were described in Experimental Procedures. Markers for molecular weight standardization were as follows: ovalbumin (45,000), lactate dehydrogenase (SS,OOO), carbonic anhydrase (30,000), a-chymotrypsinogen-A (ZS,OOO),and cytochrome c (12,000). Calf thymus terminal transferase protein (5 pg) and the product obtained by limited proteolysis (5 I.cg) are shown in positions B and A, respectively.

analyzed by liquid scintillation counting as described above. The pH gradient generated by electrofocusing was determined by incubation of 2-mm sections of a control gel track in 0.5 ml of water (CO,-free) for 2 h at room temperature. The pH of each section was determined by the use of a microelectrode (Markson). Sodium

dodecyl

sulfate

-polyacrylamide

gel electro-

phoresti. SDS-gel electrophoresis was conducted in 12% polyacrylamide gels according to the method of Weber and Osborn (12), using a slab gel apparatus (Model 4214 cell and Model 4200 tank) manufactured by Ortec, Inc. Protein samples, containing 10 mM 2-mercaptoethanol, 25% glycerol, 1% SDS, 0.02% bromphenol blue, and the sodium phosphate buffer system described by Weber and Osborn (12), were immersed in a boiling water bath for 3 to 5 min. Electrophoresis was performed using a constant, pulsed power supply (Ortec, Inc., Model 4100) with initial settings of 300 pps (pulse rate), 1.0 mfd

TRANSFERASE

417

(discharge capacitance), and 5 to lO-mA current per sample. A set of protein standards was run simultaneously in separate wells. Gels were stained by incubation for 12 h in a solution of 0.2% Coomassie brilliant blue R-250 in 50% methanol and 10% acetic acid, and destained in a solution containing 50% methanol and 10% acetic acid. Analysis of oligopeptide fragments (with molecular weights less than 10,000) resulting from limited proteolysis was made by SDS-urea polyacrylamide gel electrophoresis according to a system similar to that described by Swank and Munkres (13). Gels were polymerized in a Bio-Rad slab gel apparatus (Model 220) having a thickness of 1.5 mm. Reagents and their concentrations in the polymerized gel were as follows: acrylamide (acrylamide:bisacrylamide ratio of 3’7.5:1.0), 17.5%; Trisphosphate buffer, pH 6.8 (0.2 M phosphoric acid titrated to pH 6.8 with Tris base); sodium dodecyl sulfate, 1%; and urea, 4 M. Samples were prepared for electrophoresis in a manner similar to that described above, and denaturation was conducted in the presence of all reagents in the gel system, except acrylamide. Electrophoresis was performed using an Ortec pulsed constant power supply with settings as described for the Weber and Osborn system. RESULTS

AND DISCUSSION

Terminal deoxynucleotidyl transferase from calf thymus tissue is a protein comprised of two nonidentical subunits of molecular weight 30,500 and 9500, respectively. The purified enzyme utilized here was obtained by a modification of the procedure reported by Chang and Bollum (4) as shown in Table I. A total purification factor of 11,957 was achieved with an overall yield of 10%. A specific activity of 111,205 units/mg was determined for the homogeneous enzyme, which is comparable to the earlier reported value (4). The procedure we devised results in homogeneous enzyme without prolonged incubation of the protein at low pH. The calf thymus terminal transferase protein was selectively cleaved by controlled incubation in the presence of V-8 protease immobilized on Sepharose. The results of this preparative procedure are summarized in Table II. Terminal transferase protein was analyzed by SDS-polyacrylamide gel electrophoresis following limited proteolysis to determine the extent of the modification. As shown in Fig. 1, a reduction in the molecular weight is noted for only one sub-

418

DEIBEL

AND COLEMAN

unit of the protein. A subunit molecular weight for p’ of 25,500 was determined, which represents a loss of approximately 5000 in molecular weight compared to unmodified terminal transferase subunit /3. The LY subunit was unaffected by the limited proteolysis, and was shown to maintain a subunit molecular weight of 9500, Attempts to identify one or more oligopeptides having molecular weights less than 10,000 by SDS-gel electrophoresis according to the method of Swank and Munkres (13) were unsuccessful following limited proteolysis with immobilized V-8 protease. However, following proteolysis with free V-8 protease under comparable conditions, we were able to identify one major oligopeptide having a molecular weight of approximately 5000 (data not shown). Why the peptide is not observed after treatment with the immobilized protease is not understood. Since the primary specificity of the S. aureus V-8 protease is for peptide bonds containing the -COOH terminus of glutamic acid (3, 14) we are inclined to believe that only one major and sensitive glutamic acid site exists on the native protein under the conditions utilized. Determination of the amino acid composition of homogeneous calf thymus terminal transferase (4) indicated that the protein is comprised of a large population of either glutamic acid or glutamine residues. Bollum (4) originally suggested that the majority of these residues were glutamine, since the isoelectric pH of the protein is rather basic (PI = 8.2). Our results are in agreement, since glutamine residues cannot serve as substrates for the V-8 protease. Biochemical properties of the native and cleaved enzymes were analyzed by isoelectric focusing studies and thermal denaturation at 45°C. The results from isoelectric focusing experiments are shown in Fig. 2. Enzymatic activity is coincident with a single protein band for both cleaved and uncleaved proteins, although a change in isoelectric pH is evident. The original protein has an isoelectric pH equal to 8.2, while that for the modified protein is 7.8. Thermal stabilities of the two terminal transferase enzymes were analyzed by incubation for variable timed intervals at

_

6001

I

AB

7 PH 5

IO

20

GEL

SLICE

30

40

NUMBER

FIG. 2. Analytical isoelectric focusing of calf thymus terminal transferase and the cleaved product obtained by limited proteolytic digestion with immobilized V-8 protease. The preparation of the gel and the electrofocusing experiment are described in Experimental Procedures. (A) Calf thymus terminal transferase (4.9 pg) and (B) proteolytically cleaved calf thymus terminal transferase (1.2 pg).

45”C, followed by the standard assay as described under Experimental Procedures. The modified protein showed an identical denaturation profile to that of the control, original enzyme protein (data not shown). These results indicate that the protein subjected to limited proteolysis by immobilized V-8 protease maintains the stability inherent in the parent enzyme, despite the loss of a sizable fragment of terminal transferase protein. Our results using limited proteolysis as a novel tool for investigating the structural composition of calf thymus terminal transferase provide convincing evidence that the polypeptide domain responsible for enzymatic function is maintained in a low molecular weight protein. The subunit molecular weight we have obtained following limited proteolysis with S. aureua V-8 protease .is surprisingly comparable to values previously reported in the literature for this protein (4). It is conceivable that our purification scheme eliminates one or more proteolytic cleavages of terminal

LIMITED

PROTEOLYSIS

OF CALF

transferase during standard isolation procedures. However, the identity or substrate specificity of such putative proteolytic enzymes in calf thymus tissue extracts is not currently known, and the use of S. aureus V-8 protease is probably not representative of in wivo metabolic pathways. Our results do imply that proteolytic degradation of terminal transferase may occur. Whether our limited proteolysis experiment simulates in wivo processing of the protein, or proteolysis during routine enzyme purification is unknown. However, this report represents initial attempts to characterize calf thymus terminal transferase by novel procedures which eventually could lead to a better understanding of both protein processing and the structure of the catalytic domain of the enzyme. ACKNOWLEDGMENTS We wish to thank Dr. Judith Lesnaw for her valuable discussions of the experiments, and Ms. Karen Acree for her expert technical help in the purification of the enzyme.

M. S. (1979)

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2. YOSHIDA, S., AND CAVALIERI, L. F. Proc. Nut. Acad. Ski. USA 68, 200-204.

(1971)

G. R., BOILY, Y., AND

3. DRAPEAU, (1972) J. Biol. 4. CHANG, Biol.

Chem.

HOUMARD, 247, 6720-6’726.

L. M. S., AND BOLLUM, Chem. 246, 909-916.

J.

F. J. (1971)

J.

5. CHANG, L. M. S., AND BOLLUM, F, J. (1970) Proc. Nat. Acad. Sci. USA 65, 1041-1048. 6. COLEMAN, M. S. (1977) Arch. Biochem. Biophys. 182, 525-532. 7. BOLLUM, F. J. (1966) in Procedures in Nucleic Acid Research (Cantoni, G., and Davies, D., eds.), p. 577, Harper & Row, New York. N. J., FARR, A. L., 8. LOWRY, 0. H., ROSEBROUGH, AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 9. OKAMURA, S., CRANE, F., MESSNER, H. A., AND MAK, T. W. (1978) J. Biol. Chem. 253, 37653767. 10. CUATRECASAS, P., AND ANFINSEN, C. B. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 345-385, Academic Press, New York. 11. TOMARELLI, R. M., CHARNEY, J., AND HARDING, M. L. (1949) J. Lab. Clin. Med. 34, 428-433. 12. WEBER, Chem. 13. SWANK, Anal.

REFERENCES 1. DEIBEL, M. R., JR., AND COLEMAN, J. Biol. Chem. 254, 8634-8640.

TERMINAL

K., AND OSBORN, 244, 4406-4412. R. T., Biochem.

M.

AND MUNKRES, 39, 462-477.

(1969) K.

J. D.

14. HOUMARD, J., AND DRAPEAU, G. R. (1972) Nat. Acad. Ski. USA 69, 3506-3509.

Biol. (1971) PTOC.