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Use of insoluble heparin for isolation of DNA polymerase enzymes from murine myeloma
ANALYTICAL
BIOCHEMISTRY
87,41 I-417
(1978)
Use of Insoluble Heparin for Isolation of DNA Polymerase Enzymes from Murine Myeloma BARBARA A. BRENNESSEL, Department
of Microbiology 1430 Tulane
D. PAULBUHRER,AND A. ARTHUR GOTTLIEB’ and Immunology. Tulane University School Avenue. NeM, Orleans. Louisiana 70112
of Medicine,
Received June 17, 1977: accepted February 8, 1978 Heparin was found to be a potent inhibitor of a DNA polymerase present in the murine myeloma tumor MOPC-21. By increasing the KCI concentration of the reaction mixture, the inhibition of this enzyme could be completely reversed, suggesting that insoluble heparin might be a useful tool in the isolation of DNA polymerases. When heparin covalently bound to Sepharose was used, some of the DNA polymerases present in MOPC-21 myeloma tumors were separated and partially purified.
In the course of studying the DNA polymerases present in the murine myeloma tumor MOPC-21, it was observed that heparin was a potent inhibitor of several of the DNA polymerase enzymes present in this tumor. In particular, a DNA polymerase with high efficiency on ribopolymeric templates, which we have previously described (l-3), was markedly inhibited by heparin. This enzyme, designated R-l DNA polymerase, appears to share many characteristics with the y DNA polymerase previously described by Weissbach’s laboratory (4) but can be distinguished from the latter by virtue of its larger sedimentation coefficient and distinct isoelectric point (3). Another laboratory has shown that heparin inhibits, as well as dissociates, the polymeric, cytoplasmic DNA polymerase of baby hamster kidney cells (5). The fact that heparin was such a potent inhibitor of these DNA polymerases suggested that selective binding of these enzymes to heparin could be utilized for their isolation. Such a technique has been employed to study the interaction of heparin and lipoprotein lipase (6) and to isolate coagulation factors (7,8) as well as rabbit reticulocyte initiation factors (9). In this report, we show that by using columns of Sepharose conjugated to heparin two DNA polymerases can be isolated from MOPC-21 tumor cells. One of these enzymes was highly active on a “nicked” DNA template, i.e., calf thymus DNA which had been activated as a template-primer by limited deoxyribonuclease treatment. A second ’ Author to whom reprint requests should be sent. 411
0003-2697/78/0872-04 Copyright A,, ri”h,.
11$02.00/O
0 1978 by Academic Press. Inc. ,.F rnrr^A..^.:^:.. ^-. c..- ~-~~
1
412
BRENNESSEL,
BUHRER,
AND
GOTTLIEB
enzyme preferred a ribopolymer template, and this appears to be the R-l DNA polymerase. The results of these studies indicate that the use of heparin-Sepharose provides a simple and rapid method for the separation of DNA polymerase activities from this tumor. MATERIALS
AND METHODS
Reagents. Cyanogen bromide-activated Sepharose 4B and dextran sulfate of approximately 5 x lo5 molecular weight were obtained from Pharmacia Fine Chemicals. [methyl-3H]Deoxythymidine 5’-triphosphate, ([3H]TTP), having a specific activity of greater than 40 Ci/mmol was the product of New England Nuclear Company. Poly rA.oligo(dT),,-,, was purchased from P. L. Biochemicals. Calf thymus DNA (Worthington Biochemical Corp.) was activated as template-primer according to the method of Loeb (10). Heparin, chondroitin sulfate (mixed isomers), and hyaluronic acid were obtained as sodium salts from Sigma Chemical Company. Assays for DNA polymerase activity. DNA polymerase activities were measured by determining the ability of test samples to incorporate [3H]TTP into material precipitable by cold 10% trichloroacetic acid after 1 hr of incubation at 37°C. In assays utilizing 5 pg of activated DNA as template-primer, the reaction mixture (50 ~1) contained the following: 0.05~ Tris, pH 7.8; 5 mM 2-mercaptoethanol; 0.2 mg/ml bovine serum albumin; 40 mM KCl; 4 mM MgCl,; 50 nmol each of dATP, dGTP, and dCTP; 2.5 nmol of TTP; and 4 PCi of [3H]TTP. Assays in which 5 pg of poly rA.(dT),,-,, was used as template-primer were identical except that 200 PM MnCl, was substituted for MgCl, and 4 &i of [3H]TTP was the only nucleoside triphosphate added to the reaction. All polymerase reactions were terminated by the addition of 0.2 ml of saturated sodium pyrophosphate followed by 5 ml of 10% trichloroacetic acid containing 5% saturated sodium pyrophosphate. After remaining at 0 to 4°C for 30 min, the reaction mixtures were filtered and washed as previously described (1). The dried filters were counted in a scintillation fluid containing 0.4% PPO/O.OOS% Bis-MSB in toluene. In these studies, one unit of enzyme activity is defined as the amount of enzyme that catalyzes the incorporation of 1 pmol of TTP per 60 min at 37°C. Preparation of heparin -Sepharose. Heparin was covalently attached to cyanogen bromide-activated Sepharose 4B according to the procedure described by Pharmacia. Unbound heparin was measured by the carbazole reaction (11). Eighty to eighty five percent covalent attachment of the heparin to Sepharose resulted in a final concentration of 3 to 3.5 mg of heparin bound per gram of dry Sepharose. Isolation of DNA polymeruse enzymes. Maintenance of the MOPC-2 1 murine myeloma tumor by serial passage in Balb/c mice and the isolation
ISOLATION
OF
MYELOMA
DNA
POLYMERASE
413
of DNA polymerase enzymes from this tumor have been described (1,2). An enzyme in this tumor which is capable of transcribing ribopolymeric templates has been designated as the R-l polymerase. In order to utilize heparin-Sepharose for isolation of the MOPC-21 polymerases, the tumor homogenate was taken to the 60% saturated(NH&SO, stage of purification noted in reference (1). After resuspension of the crude enzyme in a buffer containing 0.05 M Tris, (pH 7.8), 0.002 M MnCl,, 0.015 M mercaptoethanol, and 30% glycerol, the resultant mixture was dialyzed overnight against this buffer. The resulting sample, obtained from up to 20 g of tumor, was applied to a 0.9 x IO-cm column of heparin-Sepharose. Polymerase enzymes were eluted with a 100 ml linear gradient of 0.0 to 1.0 M KC1 in the dialysis buffer. One-milliliter fractions were collected and lo-p1 aliquots were tested for polymerase activity. Ribopolymer transcribing enzymes could also be absorbed to heparinSepharose in a batchwise procedure. Approximately 0.5 ml of the heparin-Sepharose was added directly to 0.5 ml of the soluble fraction of the MOPC-2l tumor homogenate. After gentle agitation for 30 min at 4°C on a wrist-action shaker, the heparin-Sepharose was recovered by centrifugation and washed once with 0.5 ml of 0.05 M Tris buffer, pH 7.8, containing 0.002 M MnCl,, 0.015 M 2-mercaptoethanol, and 30% glycerol (TMMG buffer). The heparin-Sepharose was then eluted successively with 0.5 ml of 0.3 M KCI, 0.5 M KC], and 1.0 M KC1 in TMMG buffer. Ten-microliter aliquots of the eluted fractions were assayed for polymerase activity. RESULTS
Effect of Glycosaminoglycans
AND DiSCUSSION
on Polymerase
Enzymes
Heparin was found to be a potent inhibitor of the ribopolymertranscribing (R-l) DNA polymerase obtained from MOPC-21 tumor cells (Fig. 1B). The concentration of heparin which caused 50% inhibition of one unit of this enzyme was 200 pg/ml. The related, naturally occurring glycosaminoglycans, chondroitin sulfate (Fig. IC) and hyaluronic acid (Fig. IA) were without significant and consistent inhibitory effects on the enzyme. The polyanion dextran sulfate (Fig. lD), however, was a more potent enzyme inhibitor than heparin, since greater than 75% inhibition of enzyme activity resulted with concentrations as low as 2 pg/ml. In experiments designed to explore the mechanism of heparin inhibition, it was observed that increasing concentrations of Mn2+ had no effect on inhibition, indicating that heparin was not acting simply as a chelator of divalent cations. Kinetic analyses utilizing Lineweaver-Burk plots revealed that the inhibitory effect of heparin on R-l polymerase was noncompetitive with regard to the binding of poly rA.(dT),,-,, (our
414
BRENNESSEL,
BUHRER, AND GOTTLIEB
100
100
0’ 75
75
E 50 z o 25
50
Y L 5
25
75
75
$2 50
53
25
25 I
2
3456
GLYCOSAMINOGLYCAN
(-log
I
2
3456
CONCENTRATION
,uq/ml
I
FIG, 1. The effect of glycosaminoglycans on the myeloma ribopolymer-transcribing DNA Polymerase: (A) hyaluronic acid; (B) heparin; (C) chondroitin sulfate: (D) dextran sulfate. Samples of glycosaminoglycans were preincubated with 1 unit of enzyme in a total volume of 20 ~1 for 30 min at room temperature and, 30 ~1 of the remaining assay mixture was added. After a 60-min incubation at 37”C, radioactivity incorporated into trichloroacetic acid precipitates was determined as described in the Materials and Methods section.
unpublished observations). Moreover, the inhibitory effects of heparin on the R-l polymerase could be completely reversed by increasing the KC1 concentration of the enzyme assay reaction mixture from 50 mM to 150 mM (Table 1). Fully active enzyme generated by this procedure could once again be inhibited by heparin if the KC1 concentration was reduced to 50 mM. The above observations suggested that the interaction of heparin with the R-l DNA polymerase was ionic in nature. Furthermore, the fact that fully active enzyme could be recovered by increasing the KC1 concentration of the reaction mixture indicated that heparin could, in principle, be useful in the isolation of the R-l polymerase, and perhaps other DNA polymerases as well. Isolation of Polymerases by Use of Heparin depharose
Columns of heparin-Sepharose were utilized for the isolation of DNA polymerase activities from MOPC-21 tumor cells. As shown in Fig. 2, the ribopolymer-transcribing activity of the MOPC-21 tumor eluted at 0.4 to 0.45 M KU. MOPC-21 DNA polymerase activity, which utilized activated DNA as template-primer, eluted from heparin-Sepharose at 0.28 to 0.32 M KCl. This activity most likely represents the DNA-directed polymerase activities which can be further separated on DEAE-cellulose (1,2). A summary of the results of the heparin-Sepharose isolation procedure for each of the MOPC-21 tumor enzymes studied appears in Table 2.
ISOLATION
OF MYELOMA TABLE
REVERSIBILITY
OF HEPARIN
DNA POLYMERASE
415
I INHIBITION
BY
KCI”
Ratio of counts per minute for heparin-treated and control samples
KC1 concentration (I-m) 50” 150’ 50”
82.4 104.0 60.2
(1Assays for the R-l polymerase were performed in duplicate as described in Materials and Methods. After 20 min of incubation with 50 mM KCI, aliquots were removed from the reaction mixture, and KC1 concentration was increased to 150 mM. After an additional 20 min of incubation. aliquots were again removed, and KCI concentration was reduced to 50 mM by dilution. Aliquots were removed after an additional 20 min of incubation. Concentrations of all other reagents in the enzyme assay were kept constant. Net incorporation of [“H]TTP was calculated for each 20 min of incubation, and results were compared to a parallel incubation which did not contain heparin. The final concentration of heparin in the assay was 200 pgiml. b First 20-min incubation period. (’ Second 20-min incubation period. ‘r Third 20-min incubation period.
For the ribopolymer-transcribing (R-l) activity of MOPC-21 tumors, this procedure represents a considerable improvement over previous methods used to prepare the R-l enzyme, with respect to yield, extent of purification, and ease of isolation.
I 0
00 20 40 60 80 100 120 140 160 180 200 220 240 ELUTION
VOLUME
FIG. 2. Elution of DNA polymerase enzymes from heparin-Sepharose. DNA polymerase enzymes were applied to and eluted from columns of heparin-Sepharose as described in the Materials and Methods section. Ten-microliter aliquots of the fractions were assayed for enzyme activity with (0) “nicked” DNA and with (0) poly rA.oligo dT as template-primers, as described in the Materials and Methods section. The KCI gradient is indicated.
416
BRENNESSEL,
BUHRER, TABLE
PURIFICATION
OF POLYMERASE
AND GOTTLIEB 2
ENZYMES
WITH HEPARIN-SEPHAROSE
Stage of purification
Total units of enzyme activity (X 10-S)
Specific activity
Purification (-fold)
Recovery (%I
Ribopolymer-directed polymerase Crude homogenate Soluble fraction of homogenate 0.4 M (NH&SO, eluate from DEAE Dialyzed 0.4 M (NH&SO4 eluate Heparin-Sepharose peak
114 74 48 8.1 8.2
8.7 21 59 25 2,680
-
100 65 43 7.1 7.2
“Nicked” DNA-directed polymerase Crude homogenate Soluble fraction of homogenate 0.4 M (NH&SO, eluate from DEAE Dialyzed 0.4 M (NH&SO, eluate Heparin-Sepharose peak
396 I8 30 75 59
31 5.1 36 237 29,800
2.4 6.7 2.9 307
-
100 4.5 7.5 19 14
1.2 7.8 980
To effect separation of DNA polymerases which were highly active on “nicked” DNA and the ribopolymer-directed enzyme, it was also possible to add heparin-Sepharose directly to the soluble fraction of the tumor homogenate (Table 3). By this procedure, it was found that 85% of the protein in the homogenate did not bind to the heparin-Sepharose. Although 80% of the polymerase activity on “nicked” DNA was present in the nonbinding fraction, virtually all of the ribopolymer-transscribing activity bound to the heparin-Sepharose and could be subsequently eluted with increasing concentrations of KCl. Over 50% of the activity of the ribopolymer-directed enzyme was thus recovered in the combined 0.5 M and 1.O M KC1 eluates, and a partial purification of this enzyme was achieved at a very early stage of the isolation procedure. TABLE BATCHWISE
SEPARATION
OF
3
MOPC-21 DNA POLYMERASES
WITH
HEPARIN-SEPHAROSE
Enzyme activity (% recovery) Fraction Nonbinding Wash 0.3 M KCI eluate 0.5 M KCI eluate 1.O M KCI eluate
“Nicked” 80.6 1.7 16.0 12.2 5.1
DNA
Poly rA.(dT),,-,,
Protein” (% recovery)
2.7 0.0 1.5 27.3 26.7
4 Protein was determined by use of fluorescamine (Fluram, Roche Diagnostics).
84.6 23.3 16.4 9.5 4.7
ISOLATION
OF MYELOMA
DNA POLYMERASE
417
The rapidity of isolation and separation of these DNA polymerase enzymes on heparin-Sepharose should simplify the study of other eukaryotic polymerases. Moreover, heparin-Sepharose may be useful in determining the subunit structure and mechanism of action of eukaryotic DNA polymerases, particularly since the inhibition of at least one cellular DNA polymerase enzyme by heparin can be correlated with a dissociation of the polymeric form of the enzyme into a smaller active species (5). The comparison of the size of the heparin-Sepharose-isolated enzymes to their size when isolated by conventional methods may help to determine the structure and function of these important cellular enzymes. ACKNOWLEDGMENT Supported in part by a grant from the National Cancer Institute (CA-19164) and grants from the Edward G. Schleider Foundation and Metzger-Price Fund. We are greatly indebted to Ms. J. Chang for expert technical assistance in the execution of these studies.
REFERENCES 1. Persico, F. J., Nicholson, D. E.. and Gottlieb, A. A. (1973) Cancer Res. 33, 1210-1216. 2. Persico, F. J., and Gottlieb. A. A. (1972) Nature New Biol. 239, 173-176. 3. Gottlieb, A. A., Smith, A. H., Plescia, 0. J., Nicholson, D. E., Bowers, S., Pankuch, E., and Berokoben, D. (1975) in Fundamental Aspects of Neoplasia (Gottlieb, A. A., Plesica, 0. J., and Bishop, D. H. L.. eds.), pp. 269-277, SpringerVerlag, New York. 4. Weissbach. A. (1975) Cell 5, 101-108. 5. Lazarus, L. H., and Kitron, N. (1974) Arch. Biochem. Biophys. 164, 414-419. 6. Iverius, P. H., Lindahl, U., Egelrud, T., and Olivecrona. T. (1972) J. Biol. Chem. 247, 6610-6616. 7. Gentry, P. W.. and Alexander. B. (1973) Biochem. Biophys. Res. Commun. 50, 500-509. 8. Rosenberg, R. D., and Damus, P. S. (1973) J. Biol. Chem. 248, 6490-6505. 9. Waldman, A. A., Marx, G., and Goldstein, J. (1975) Proc. Nut. Acad. Sci. USA 72, 2352-2356. 10. Loeb, L. A. (1969) J. Biol. Chem. 244, 1672-1681. 11. Galambos, J. T. (1967)AnaI. Biochem. 19, 119-132.
BIOCHEMISTRY
87,41 I-417
(1978)
Use of Insoluble Heparin for Isolation of DNA Polymerase Enzymes from Murine Myeloma BARBARA A. BRENNESSEL, Department
of Microbiology 1430 Tulane
D. PAULBUHRER,AND A. ARTHUR GOTTLIEB’ and Immunology. Tulane University School Avenue. NeM, Orleans. Louisiana 70112
of Medicine,
Received June 17, 1977: accepted February 8, 1978 Heparin was found to be a potent inhibitor of a DNA polymerase present in the murine myeloma tumor MOPC-21. By increasing the KCI concentration of the reaction mixture, the inhibition of this enzyme could be completely reversed, suggesting that insoluble heparin might be a useful tool in the isolation of DNA polymerases. When heparin covalently bound to Sepharose was used, some of the DNA polymerases present in MOPC-21 myeloma tumors were separated and partially purified.
In the course of studying the DNA polymerases present in the murine myeloma tumor MOPC-21, it was observed that heparin was a potent inhibitor of several of the DNA polymerase enzymes present in this tumor. In particular, a DNA polymerase with high efficiency on ribopolymeric templates, which we have previously described (l-3), was markedly inhibited by heparin. This enzyme, designated R-l DNA polymerase, appears to share many characteristics with the y DNA polymerase previously described by Weissbach’s laboratory (4) but can be distinguished from the latter by virtue of its larger sedimentation coefficient and distinct isoelectric point (3). Another laboratory has shown that heparin inhibits, as well as dissociates, the polymeric, cytoplasmic DNA polymerase of baby hamster kidney cells (5). The fact that heparin was such a potent inhibitor of these DNA polymerases suggested that selective binding of these enzymes to heparin could be utilized for their isolation. Such a technique has been employed to study the interaction of heparin and lipoprotein lipase (6) and to isolate coagulation factors (7,8) as well as rabbit reticulocyte initiation factors (9). In this report, we show that by using columns of Sepharose conjugated to heparin two DNA polymerases can be isolated from MOPC-21 tumor cells. One of these enzymes was highly active on a “nicked” DNA template, i.e., calf thymus DNA which had been activated as a template-primer by limited deoxyribonuclease treatment. A second ’ Author to whom reprint requests should be sent. 411
0003-2697/78/0872-04 Copyright A,, ri”h,.
11$02.00/O
0 1978 by Academic Press. Inc. ,.F rnrr^A..^.:^:.. ^-. c..- ~-~~
1
412
BRENNESSEL,
BUHRER,
AND
GOTTLIEB
enzyme preferred a ribopolymer template, and this appears to be the R-l DNA polymerase. The results of these studies indicate that the use of heparin-Sepharose provides a simple and rapid method for the separation of DNA polymerase activities from this tumor. MATERIALS
AND METHODS
Reagents. Cyanogen bromide-activated Sepharose 4B and dextran sulfate of approximately 5 x lo5 molecular weight were obtained from Pharmacia Fine Chemicals. [methyl-3H]Deoxythymidine 5’-triphosphate, ([3H]TTP), having a specific activity of greater than 40 Ci/mmol was the product of New England Nuclear Company. Poly rA.oligo(dT),,-,, was purchased from P. L. Biochemicals. Calf thymus DNA (Worthington Biochemical Corp.) was activated as template-primer according to the method of Loeb (10). Heparin, chondroitin sulfate (mixed isomers), and hyaluronic acid were obtained as sodium salts from Sigma Chemical Company. Assays for DNA polymerase activity. DNA polymerase activities were measured by determining the ability of test samples to incorporate [3H]TTP into material precipitable by cold 10% trichloroacetic acid after 1 hr of incubation at 37°C. In assays utilizing 5 pg of activated DNA as template-primer, the reaction mixture (50 ~1) contained the following: 0.05~ Tris, pH 7.8; 5 mM 2-mercaptoethanol; 0.2 mg/ml bovine serum albumin; 40 mM KCl; 4 mM MgCl,; 50 nmol each of dATP, dGTP, and dCTP; 2.5 nmol of TTP; and 4 PCi of [3H]TTP. Assays in which 5 pg of poly rA.(dT),,-,, was used as template-primer were identical except that 200 PM MnCl, was substituted for MgCl, and 4 &i of [3H]TTP was the only nucleoside triphosphate added to the reaction. All polymerase reactions were terminated by the addition of 0.2 ml of saturated sodium pyrophosphate followed by 5 ml of 10% trichloroacetic acid containing 5% saturated sodium pyrophosphate. After remaining at 0 to 4°C for 30 min, the reaction mixtures were filtered and washed as previously described (1). The dried filters were counted in a scintillation fluid containing 0.4% PPO/O.OOS% Bis-MSB in toluene. In these studies, one unit of enzyme activity is defined as the amount of enzyme that catalyzes the incorporation of 1 pmol of TTP per 60 min at 37°C. Preparation of heparin -Sepharose. Heparin was covalently attached to cyanogen bromide-activated Sepharose 4B according to the procedure described by Pharmacia. Unbound heparin was measured by the carbazole reaction (11). Eighty to eighty five percent covalent attachment of the heparin to Sepharose resulted in a final concentration of 3 to 3.5 mg of heparin bound per gram of dry Sepharose. Isolation of DNA polymeruse enzymes. Maintenance of the MOPC-2 1 murine myeloma tumor by serial passage in Balb/c mice and the isolation
ISOLATION
OF
MYELOMA
DNA
POLYMERASE
413
of DNA polymerase enzymes from this tumor have been described (1,2). An enzyme in this tumor which is capable of transcribing ribopolymeric templates has been designated as the R-l polymerase. In order to utilize heparin-Sepharose for isolation of the MOPC-21 polymerases, the tumor homogenate was taken to the 60% saturated(NH&SO, stage of purification noted in reference (1). After resuspension of the crude enzyme in a buffer containing 0.05 M Tris, (pH 7.8), 0.002 M MnCl,, 0.015 M mercaptoethanol, and 30% glycerol, the resultant mixture was dialyzed overnight against this buffer. The resulting sample, obtained from up to 20 g of tumor, was applied to a 0.9 x IO-cm column of heparin-Sepharose. Polymerase enzymes were eluted with a 100 ml linear gradient of 0.0 to 1.0 M KC1 in the dialysis buffer. One-milliliter fractions were collected and lo-p1 aliquots were tested for polymerase activity. Ribopolymer transcribing enzymes could also be absorbed to heparinSepharose in a batchwise procedure. Approximately 0.5 ml of the heparin-Sepharose was added directly to 0.5 ml of the soluble fraction of the MOPC-2l tumor homogenate. After gentle agitation for 30 min at 4°C on a wrist-action shaker, the heparin-Sepharose was recovered by centrifugation and washed once with 0.5 ml of 0.05 M Tris buffer, pH 7.8, containing 0.002 M MnCl,, 0.015 M 2-mercaptoethanol, and 30% glycerol (TMMG buffer). The heparin-Sepharose was then eluted successively with 0.5 ml of 0.3 M KCI, 0.5 M KC], and 1.0 M KC1 in TMMG buffer. Ten-microliter aliquots of the eluted fractions were assayed for polymerase activity. RESULTS
Effect of Glycosaminoglycans
AND DiSCUSSION
on Polymerase
Enzymes
Heparin was found to be a potent inhibitor of the ribopolymertranscribing (R-l) DNA polymerase obtained from MOPC-21 tumor cells (Fig. 1B). The concentration of heparin which caused 50% inhibition of one unit of this enzyme was 200 pg/ml. The related, naturally occurring glycosaminoglycans, chondroitin sulfate (Fig. IC) and hyaluronic acid (Fig. IA) were without significant and consistent inhibitory effects on the enzyme. The polyanion dextran sulfate (Fig. lD), however, was a more potent enzyme inhibitor than heparin, since greater than 75% inhibition of enzyme activity resulted with concentrations as low as 2 pg/ml. In experiments designed to explore the mechanism of heparin inhibition, it was observed that increasing concentrations of Mn2+ had no effect on inhibition, indicating that heparin was not acting simply as a chelator of divalent cations. Kinetic analyses utilizing Lineweaver-Burk plots revealed that the inhibitory effect of heparin on R-l polymerase was noncompetitive with regard to the binding of poly rA.(dT),,-,, (our
414
BRENNESSEL,
BUHRER, AND GOTTLIEB
100
100
0’ 75
75
E 50 z o 25
50
Y L 5
25
75
75
$2 50
53
25
25 I
2
3456
GLYCOSAMINOGLYCAN
(-log
I
2
3456
CONCENTRATION
,uq/ml
I
FIG, 1. The effect of glycosaminoglycans on the myeloma ribopolymer-transcribing DNA Polymerase: (A) hyaluronic acid; (B) heparin; (C) chondroitin sulfate: (D) dextran sulfate. Samples of glycosaminoglycans were preincubated with 1 unit of enzyme in a total volume of 20 ~1 for 30 min at room temperature and, 30 ~1 of the remaining assay mixture was added. After a 60-min incubation at 37”C, radioactivity incorporated into trichloroacetic acid precipitates was determined as described in the Materials and Methods section.
unpublished observations). Moreover, the inhibitory effects of heparin on the R-l polymerase could be completely reversed by increasing the KC1 concentration of the enzyme assay reaction mixture from 50 mM to 150 mM (Table 1). Fully active enzyme generated by this procedure could once again be inhibited by heparin if the KC1 concentration was reduced to 50 mM. The above observations suggested that the interaction of heparin with the R-l DNA polymerase was ionic in nature. Furthermore, the fact that fully active enzyme could be recovered by increasing the KC1 concentration of the reaction mixture indicated that heparin could, in principle, be useful in the isolation of the R-l polymerase, and perhaps other DNA polymerases as well. Isolation of Polymerases by Use of Heparin depharose
Columns of heparin-Sepharose were utilized for the isolation of DNA polymerase activities from MOPC-21 tumor cells. As shown in Fig. 2, the ribopolymer-transcribing activity of the MOPC-21 tumor eluted at 0.4 to 0.45 M KU. MOPC-21 DNA polymerase activity, which utilized activated DNA as template-primer, eluted from heparin-Sepharose at 0.28 to 0.32 M KCl. This activity most likely represents the DNA-directed polymerase activities which can be further separated on DEAE-cellulose (1,2). A summary of the results of the heparin-Sepharose isolation procedure for each of the MOPC-21 tumor enzymes studied appears in Table 2.
ISOLATION
OF MYELOMA TABLE
REVERSIBILITY
OF HEPARIN
DNA POLYMERASE
415
I INHIBITION
BY
KCI”
Ratio of counts per minute for heparin-treated and control samples
KC1 concentration (I-m) 50” 150’ 50”
82.4 104.0 60.2
(1Assays for the R-l polymerase were performed in duplicate as described in Materials and Methods. After 20 min of incubation with 50 mM KCI, aliquots were removed from the reaction mixture, and KC1 concentration was increased to 150 mM. After an additional 20 min of incubation. aliquots were again removed, and KCI concentration was reduced to 50 mM by dilution. Aliquots were removed after an additional 20 min of incubation. Concentrations of all other reagents in the enzyme assay were kept constant. Net incorporation of [“H]TTP was calculated for each 20 min of incubation, and results were compared to a parallel incubation which did not contain heparin. The final concentration of heparin in the assay was 200 pgiml. b First 20-min incubation period. (’ Second 20-min incubation period. ‘r Third 20-min incubation period.
For the ribopolymer-transcribing (R-l) activity of MOPC-21 tumors, this procedure represents a considerable improvement over previous methods used to prepare the R-l enzyme, with respect to yield, extent of purification, and ease of isolation.
I 0
00 20 40 60 80 100 120 140 160 180 200 220 240 ELUTION
VOLUME
FIG. 2. Elution of DNA polymerase enzymes from heparin-Sepharose. DNA polymerase enzymes were applied to and eluted from columns of heparin-Sepharose as described in the Materials and Methods section. Ten-microliter aliquots of the fractions were assayed for enzyme activity with (0) “nicked” DNA and with (0) poly rA.oligo dT as template-primers, as described in the Materials and Methods section. The KCI gradient is indicated.
416
BRENNESSEL,
BUHRER, TABLE
PURIFICATION
OF POLYMERASE
AND GOTTLIEB 2
ENZYMES
WITH HEPARIN-SEPHAROSE
Stage of purification
Total units of enzyme activity (X 10-S)
Specific activity
Purification (-fold)
Recovery (%I
Ribopolymer-directed polymerase Crude homogenate Soluble fraction of homogenate 0.4 M (NH&SO, eluate from DEAE Dialyzed 0.4 M (NH&SO4 eluate Heparin-Sepharose peak
114 74 48 8.1 8.2
8.7 21 59 25 2,680
-
100 65 43 7.1 7.2
“Nicked” DNA-directed polymerase Crude homogenate Soluble fraction of homogenate 0.4 M (NH&SO, eluate from DEAE Dialyzed 0.4 M (NH&SO, eluate Heparin-Sepharose peak
396 I8 30 75 59
31 5.1 36 237 29,800
2.4 6.7 2.9 307
-
100 4.5 7.5 19 14
1.2 7.8 980
To effect separation of DNA polymerases which were highly active on “nicked” DNA and the ribopolymer-directed enzyme, it was also possible to add heparin-Sepharose directly to the soluble fraction of the tumor homogenate (Table 3). By this procedure, it was found that 85% of the protein in the homogenate did not bind to the heparin-Sepharose. Although 80% of the polymerase activity on “nicked” DNA was present in the nonbinding fraction, virtually all of the ribopolymer-transscribing activity bound to the heparin-Sepharose and could be subsequently eluted with increasing concentrations of KCl. Over 50% of the activity of the ribopolymer-directed enzyme was thus recovered in the combined 0.5 M and 1.O M KC1 eluates, and a partial purification of this enzyme was achieved at a very early stage of the isolation procedure. TABLE BATCHWISE
SEPARATION
OF
3
MOPC-21 DNA POLYMERASES
WITH
HEPARIN-SEPHAROSE
Enzyme activity (% recovery) Fraction Nonbinding Wash 0.3 M KCI eluate 0.5 M KCI eluate 1.O M KCI eluate
“Nicked” 80.6 1.7 16.0 12.2 5.1
DNA
Poly rA.(dT),,-,,
Protein” (% recovery)
2.7 0.0 1.5 27.3 26.7
4 Protein was determined by use of fluorescamine (Fluram, Roche Diagnostics).
84.6 23.3 16.4 9.5 4.7
ISOLATION
OF MYELOMA
DNA POLYMERASE
417
The rapidity of isolation and separation of these DNA polymerase enzymes on heparin-Sepharose should simplify the study of other eukaryotic polymerases. Moreover, heparin-Sepharose may be useful in determining the subunit structure and mechanism of action of eukaryotic DNA polymerases, particularly since the inhibition of at least one cellular DNA polymerase enzyme by heparin can be correlated with a dissociation of the polymeric form of the enzyme into a smaller active species (5). The comparison of the size of the heparin-Sepharose-isolated enzymes to their size when isolated by conventional methods may help to determine the structure and function of these important cellular enzymes. ACKNOWLEDGMENT Supported in part by a grant from the National Cancer Institute (CA-19164) and grants from the Edward G. Schleider Foundation and Metzger-Price Fund. We are greatly indebted to Ms. J. Chang for expert technical assistance in the execution of these studies.
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