Proc. Natl. Acad. Sci. USA Vol. 76, No. 4, pp. 1731-1735, April 1979
Biochemistry
Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis* [carbamoyl-phosphate synthase (glutamine)/aspartate carbamoyltransferase/dihydro-orotase/intracellular proteolysis] JEFFREY N. DAVIDSON AND DAVID PATTERSON Eleanor Roosevelt Institute for Cancer Research, Department of Biochemistry, Biophysics, and Genetics, University of Colorado Medical Center, Denver, Colorado 80262
Communicated by Theodore T. Puck, February 5,1979
A combined genetic, biochemical, and immuABSTRACT nological approach has clarified structural relationships involving the first three enzymes of de novo pyrimidine biosynthesis. A procedure involving antibody and protein A-Sepharose was used to isolate the enzymes carbamoyl-phosphate synthase [ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring), EC 2.7.2.91, aspartate transcarbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) from Chinese hamster ovary cell CHO-KI, the uridine-requiring auxotroph Urd-A, and selected UrdcA revertants. The enzymes of Urd-A and the Urd-A revertants were significantly altered in activity, native structure, and molecular weight from those of CHO-Ki. The results presented permit the conclusions that (i) these three enzymes reside in a single multifunctional 220,000-dalton polypeptide; (il) the aspartate transcarbamoyltransferase activity is located on a portion (;20,000 daltons) at one end of the polypeptide; (iiM) this portion may also be required for monomers to aggregate into the multimeric form present in mammalian cells; (iv) the mutations in Urd-A and the Urd-A revertants lie in the structural gene for this multifunctional protein; and (v) increased sensitivity to proteases could account for the alterations in the structure of these enzymes in the mutants.
Pyrimidine biosynthesis has been studied extensively in eukaryotes. In several organisms, the first three enzymes in the pathway-carbamoyl-phosphate synthase [CPSase; ATP:carbamate phosphotransferase (diphosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (ATCase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (DHOase; L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3)-copurify as a single multienzyme complex (1-5). In Syrian hamster cells the native enzyme is a trimer (4). When the enzyme complex purified from Syrian hamster cells or rat hepatoma cells is subjected to sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis, a single protein band is observed at 200,000-210,000 daltons (4, 6). The homogeneity of this protein has not been established. Because the three enzyme activities from rat hepatoma cells could be partially separated by sucrose gradient centrifugation in high-salt medium, Mori and Tatibana (5) concluded that the native enzyme must be composed of separate, nonidentical subunits of similar molecular weight, whereas Coleman et al. (4) concluded from binding studies with phosphonoacetyl-laspartate, an inhibitor of ATCase, and from experiments with cells that coordinately overproduce CPSase, ATCase, and DHOase that the native enzyme is composed of identical
multifunctional subunits. Thus, biochemical procedures alone have not yet resolved the question about the molecular integrity or separateness of the individual enzyme activities. Such resolution is a prerequisite to formulation of appropriate testable hypotheses regarding genetic and biochemical regulation of pyrimidine biosynthesis in mammalian cells. Recently, the isolation, from the Chinese hamster ovary (CHO) cell K1, of a mutant, Urd-A, that has decreased levels of the first three enzymes of pyrimidine biosynthesis was reported (7). Certain revertants of Urd-A (B48 and D20) have levels of CPSase and DHOase activities closer to those of CHO-K1 but still have a very low level of ATCase activity (7, 8). Although CPSase, ATCase, and DHOase from CHO-K1 cosediment through a glycerol gradient, the sedimentation pattern for these enzymes from Urd-A and the revertants B48 and D20 is altered considerably. The CPSase and DHOase activities still cosediment but at a much slower rate than the same enzymes from CHO-K1 (8). In addition, the ATCase activity from the mutants is clearly separated into two peaks, one lower and the other higher in the gradient than the CPSase/DHOase peak, but both sedimenting slower than the CPSase/ATCase/ DHOase complex of CHO-Ki (8). A combined biochemical and immunological approach to study the enzymes from CHO-Ki, Urd-A, and the revertants B48 and D20 is described here. By isolating the enzymes CPSase, ATCase, and DHOase from these cell types with an antibody and protein A-Sepharose procedure and analyzing them on NaDodSO4/polyacrylamide gels, the subunit structure of the enzyme can be shown to be a multifunctional polypeptide with all three enzyme activities. EXPERIMENTAL Cell Extract. Cells to be radioactively labeled were grown on 150-mm petri dishes to midlogarithmic phase in F-12 (9) containing 10% (vol/vol) fetal calf serum and 30 AM uridine. Then the medium was changed to F-12 without methionine but containing 10% dialyzed fetal calf serum and 30,gM uridine to which 15-20,uM [35S]methionine [278-473 mCi/mmol (1 Ci = 3.7 X 101" becquerels); Amersham] was added. Cells were labeled for 16 hr at 370C. For unlabeled preparations, cells were grown to middle or late logarithmic phase. Cells were removed from dishes by trypsinization (10) and broken as described (8). The supernatant after centrifugation at 30,000 X g was used in all experiments. Glycerol Gradient Centrifugation. The cell-free extract Abbreviations: CPSase, carbamoyl-phosphate synthase, EC 2.7.2.9; ATCase, aspartate transcarbamoyltransferase, EC 2.1.3.2; DHOase, dihydro-orotase, EC 3.5.2.3; CHO, Chinese hamster ovary; NaDodSO4, sodium dodecyl sulfate. * This is paper no. 4 in a series. Paper no. 3 is ref. 8.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertsement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1731
1732
Biochemistry: Davidson and PattersonPProc. Natl. Acad. Sci. USA 76 (1979)
from 1-3 X 107 cells was centrifuged on 8-30% (vol/vol) glycerol gradients in an SW 50.1 rotor as described (8). CPSase, ATCase, and DHOase activities were assayed as described (7, 8). Enzyme Isolation. A rapid procedure was developed for isolating enzyme from cell extracts or gradient fractions by using rabbit antiserum (kindly provided by G. R. Stark) raised against CPSase/ATCase/DHOase enzyme from Syrian hamster. To cell-free extract from 107 cells, 10,1l of antiserum (5 ,gl precipitates 1 ,tg of enzyme) was added and the mixture was incubated for 12-16 hr at 4VC. Then, 10 pl of a protein ASepharose (Pharmacia) slurry in phosphate-buffered saline was added. Samples were incubated at 40C for 30 min with occasional shaking. The enzyme-antibody-protein A-Sepharose complex was washed six times with phosphate-buffered saline. Then, the pellet was extracted three times with 0.5 ml of 1 M acetic acid which dissociates the enzyme-antibody complex from the protein A-Sepharose. The pooled volumes were lyophilized to dryness and the powder was resuspended in NaDodSO4 sample buffer (11). NaDodSO4/Polyacrylamide Slab Gel Electrophoresis. Electrophoresis was performed according to Laemmli (11) with a linear acrylamide gradient of 5-20% (wt/vol). Gels were stained for protein by the procedure of Fairbanks et al. (12). The amounts of stained proteins were determined by scanning gel strips in an Acta spectrophotometer (Beckman) at 565 nm. When samples contained [a5S]methionine, gels were impregnated with 2,5-diphenyloxazole (13), dried, and exposed to Cronex 4 film (Du Pont) at -70'C. The standards used to estimate molecular weights were spectrin (I = 240,000; II = 220,000), ferritin (dodecamer = 220,000; monomer = 18,500), Escherichia coli RNA polymerase (f3 = 165,000; (3' = 155,000; a7 = 39,000), albumin (67,000), and gamma globulin (heavy chain = 50,000; light chain = 22,500). RESULTS Enzyme Isolation. The antibody-protein A-Sepharose procedure successfully removed most of the CPSase, ATCase, and DHOase activities from a CHO-K1 cell-free extract and could remove these activities from the mutants (Table 1). Thus, this simple procedure can be used to isolate the CPSase, ATCase, and DHOase enzymes directly from total soluble protein. Analysis of Enzyme in Cell-Free Extracts. When the antigen-antibody complex made with CHO-K1 cell extract was denatured and subjected to NaDodSO4/polyacrylamide gel electrophoresis, several proteins appeared (Fig. 1A). The two most prominent proteins were rabbit immunoglobulin (heavy and light chains). One protein band consistently isolated from CHO-K1 cell extract migrated electrophoretically the same distance as half ferritin and spectrin II. Urd-A revertants B48 Table 1. Enzyme activities removed by binding to antibody-protein A-Sepharose % activity removed* Strain CPSase ATCase DHOase CHO-K1 61t 98 98 t t 82 Urd-A D20 93 63 36
* Cell extract was assayed for enzyme activities before and after treatment with antiserum and protein A-Sepharose. t The percentage of CPSase activity removed is probably closer to 98%; the discrepancy is due to an artifact in the assay procedure
(8).
t Activities before and after precipitation were very low.
A1
2
3
1
B
3
2
240,000 220.000
_- _ _
165.000 155.000
- 220,000 165.000 - 155.000 -
67.00050.000-
* 39.000
- 67.000
-
U
22.500- t
_
Il
-
50.000
- 39.000
_- 22.500
FIG. 1. NaDodSO/polyacrylamide gel electrophoresis of denatured enzyme-antibody complex. (A) CHO-K1 (lane 1), mixture of CHO-K1 and B48 (lane 2), and B48 (lane 3). (B) D20 (lane 1), Ade-C (lane 2), and hybrid Ade-C/D20 (lane 3). Arrows point at 220,000and 200,000-dalton forms.
and D20 did not have this 220,000-dalton protein but did have a new band, not present in CHO-K1, at 200,000 daltons. When a mixture of denatured antigen-antibody complex from CHO-KI and B48 was analyzed by electrophoresis, the two forms clearly separated. Neither protein form was observed in Urd-A unless protein from 6 times more cells was applied to the gel (see Fig. 2B); then, only the 200,000-dalton protein appeared. In order to determine dominance, hybrids were constructed as described (8) between Ade-C, a purine-requiring auxotroph, and a Urd-A revertant (D20). The Ade-C/D20 hybrid showed codominance (both forms were present) (Fig. 1B). The antibody-protein A-Sepharose procedure, therefore, removes predominantly one 220,000-dalton protein from CHO-K1 cell-free extracts and one 200,000-dalton protein from Urd-A and the revertants B48 and D20. Ade-C/D20 hybrids express both proteins. Analysis of Enzyme in Glycerol Gradient Fractions. In order to correlate the protein bands on gels with the CPSase, ATCase, and DHOase activities, cell-free extracts were centrifuged on glycerol gradients and enzyme was assayed and isolated from the fractions collected. On the basis of previous results with glycerol gradients (8), DHOase activity can be used to monitor the sedimentation of the CPSase/ATCase/DHOase complex from CHO-K1 and of the CPSase/DHOase complex from Urd-A and D20 (the ATCase activity sedimenting elsewhere in the gradient). Fig. 2 shows a comparison between the presence of DHOase activity and the presence of immunologically purified protein in glycerol gradients of CHO-K1, Urd-A, D20, and the Ade-C/D20 hybrid. For CHO-K1 the amount and position of 220,000-dalton protein in the gradient corresponded exactly to the sedimentation of DHOase activity. There was no 200,000-dalton protein. When an equivalent amount of Urd-A cell extract was used, little DHOase activity was observed and no 220,000- or 200,000-dalton protein was
Biochemistry: Davidson and Patterson 4901
Proc. Natl. Acad. Sci. USA 76 (1979)
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FIG. 2. Comparison of DHOase activity of gradient fractions to amounts of 220,000- and 200,000-dalton polypeptides. Extract from 3 X 107 cells of CHO-K1 (A), UrdcA (B), D20 (C), or hybrid Ade-C/D20 (D) was applied to glycerol gradients. Gradient fractions were assayed for DHOase activity (x-X). Enzyme isolated from fractions was subjected to NaDodSO4/polyacrylamide gel electrophoresis. Coomassie blue absorbance (A ) of gel sections corresponding to 220,000 (O- - -0) and 200,000 daltons (o.....o) was determined (arrows point to these bands). As a reference, enzyme from 1 X 107 cells of CHO-Ki is shown in lane a of A, B, and C, of B48 in lane b of A and C, and of hybrid Ade-C/D20 in lane b of D and from 6 X 107 cells of Urd-A in lane b of B. The DHOase scale for Urd-A (B) is expanded 5-fold.
visible. With D20, the amount and position of 200,000-dalton protein correlated exactly with the sedimentation of DHOase activity. In the case of the hybrid Ade-C/D20, the amount and position of 220,000-dalton protein corresponded to the deeper sedimenting DHOase activity (i.e., CPSase/ATCase/DHOase peak) whereas the amount and position of 200,000-dalton protein corresponded to the shallower sedimenting DHOase activity (i.e., CPSase/DHOase peak). Thus, the 220,000-dalton protein observed on gels corresponds to an enzyme complex having CPSase, ATCase, and DHOase activities, whereas the 200,000-dalton protein corresponds to an enzyme complex having only CPSase and DHOase activities. Analysis of Enzyme in Labeled Cells. To increase the sensitivity of analyzing these enzymes in CHO-Ki and the mutants, enzyme was isolated from cells grown in the presence of
[s5S]methionine, subjected to NaDodSO4/polyacrylamide gel electrophoresis, and identified by autoradiography. For CHO-Ki, radioactive protein appeared as a major band at 220,000 daltons and a minor band at 200,000 daltons (Fig. 3). The opposite result was observed for D20: a major band at 200,000 daltons and a minor band at 220,000 daltons. The D20 cell extract also contained an immunologically crossreacting protein band not present in CHO-Ki cell extract at 19,00021,000 daltons (heavy arrow). When radioactive CHO-K1 cell extract was treated with trypsin prior to enzyme isolation, the resulting gel pattern was similar to that of D20 (Fig. 3) in that a major protein band appeared at about 200,000 daltons. Therefore, CHO-KI cells have a small amount of protein similar in molecular weight to the CPSase/DHOase enzyme of D20, and D20 cells have a small amount of protein similar in
1734
Biochemistry: Davidson and Patterson dv.:",%
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Biochemistry
Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis* [carbamoyl-phosphate synthase (glutamine)/aspartate carbamoyltransferase/dihydro-orotase/intracellular proteolysis] JEFFREY N. DAVIDSON AND DAVID PATTERSON Eleanor Roosevelt Institute for Cancer Research, Department of Biochemistry, Biophysics, and Genetics, University of Colorado Medical Center, Denver, Colorado 80262
Communicated by Theodore T. Puck, February 5,1979
A combined genetic, biochemical, and immuABSTRACT nological approach has clarified structural relationships involving the first three enzymes of de novo pyrimidine biosynthesis. A procedure involving antibody and protein A-Sepharose was used to isolate the enzymes carbamoyl-phosphate synthase [ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring), EC 2.7.2.91, aspartate transcarbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) from Chinese hamster ovary cell CHO-KI, the uridine-requiring auxotroph Urd-A, and selected UrdcA revertants. The enzymes of Urd-A and the Urd-A revertants were significantly altered in activity, native structure, and molecular weight from those of CHO-Ki. The results presented permit the conclusions that (i) these three enzymes reside in a single multifunctional 220,000-dalton polypeptide; (il) the aspartate transcarbamoyltransferase activity is located on a portion (;20,000 daltons) at one end of the polypeptide; (iiM) this portion may also be required for monomers to aggregate into the multimeric form present in mammalian cells; (iv) the mutations in Urd-A and the Urd-A revertants lie in the structural gene for this multifunctional protein; and (v) increased sensitivity to proteases could account for the alterations in the structure of these enzymes in the mutants.
Pyrimidine biosynthesis has been studied extensively in eukaryotes. In several organisms, the first three enzymes in the pathway-carbamoyl-phosphate synthase [CPSase; ATP:carbamate phosphotransferase (diphosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (ATCase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (DHOase; L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3)-copurify as a single multienzyme complex (1-5). In Syrian hamster cells the native enzyme is a trimer (4). When the enzyme complex purified from Syrian hamster cells or rat hepatoma cells is subjected to sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis, a single protein band is observed at 200,000-210,000 daltons (4, 6). The homogeneity of this protein has not been established. Because the three enzyme activities from rat hepatoma cells could be partially separated by sucrose gradient centrifugation in high-salt medium, Mori and Tatibana (5) concluded that the native enzyme must be composed of separate, nonidentical subunits of similar molecular weight, whereas Coleman et al. (4) concluded from binding studies with phosphonoacetyl-laspartate, an inhibitor of ATCase, and from experiments with cells that coordinately overproduce CPSase, ATCase, and DHOase that the native enzyme is composed of identical
multifunctional subunits. Thus, biochemical procedures alone have not yet resolved the question about the molecular integrity or separateness of the individual enzyme activities. Such resolution is a prerequisite to formulation of appropriate testable hypotheses regarding genetic and biochemical regulation of pyrimidine biosynthesis in mammalian cells. Recently, the isolation, from the Chinese hamster ovary (CHO) cell K1, of a mutant, Urd-A, that has decreased levels of the first three enzymes of pyrimidine biosynthesis was reported (7). Certain revertants of Urd-A (B48 and D20) have levels of CPSase and DHOase activities closer to those of CHO-K1 but still have a very low level of ATCase activity (7, 8). Although CPSase, ATCase, and DHOase from CHO-K1 cosediment through a glycerol gradient, the sedimentation pattern for these enzymes from Urd-A and the revertants B48 and D20 is altered considerably. The CPSase and DHOase activities still cosediment but at a much slower rate than the same enzymes from CHO-K1 (8). In addition, the ATCase activity from the mutants is clearly separated into two peaks, one lower and the other higher in the gradient than the CPSase/DHOase peak, but both sedimenting slower than the CPSase/ATCase/ DHOase complex of CHO-Ki (8). A combined biochemical and immunological approach to study the enzymes from CHO-Ki, Urd-A, and the revertants B48 and D20 is described here. By isolating the enzymes CPSase, ATCase, and DHOase from these cell types with an antibody and protein A-Sepharose procedure and analyzing them on NaDodSO4/polyacrylamide gels, the subunit structure of the enzyme can be shown to be a multifunctional polypeptide with all three enzyme activities. EXPERIMENTAL Cell Extract. Cells to be radioactively labeled were grown on 150-mm petri dishes to midlogarithmic phase in F-12 (9) containing 10% (vol/vol) fetal calf serum and 30 AM uridine. Then the medium was changed to F-12 without methionine but containing 10% dialyzed fetal calf serum and 30,gM uridine to which 15-20,uM [35S]methionine [278-473 mCi/mmol (1 Ci = 3.7 X 101" becquerels); Amersham] was added. Cells were labeled for 16 hr at 370C. For unlabeled preparations, cells were grown to middle or late logarithmic phase. Cells were removed from dishes by trypsinization (10) and broken as described (8). The supernatant after centrifugation at 30,000 X g was used in all experiments. Glycerol Gradient Centrifugation. The cell-free extract Abbreviations: CPSase, carbamoyl-phosphate synthase, EC 2.7.2.9; ATCase, aspartate transcarbamoyltransferase, EC 2.1.3.2; DHOase, dihydro-orotase, EC 3.5.2.3; CHO, Chinese hamster ovary; NaDodSO4, sodium dodecyl sulfate. * This is paper no. 4 in a series. Paper no. 3 is ref. 8.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertsement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1731
1732
Biochemistry: Davidson and PattersonPProc. Natl. Acad. Sci. USA 76 (1979)
from 1-3 X 107 cells was centrifuged on 8-30% (vol/vol) glycerol gradients in an SW 50.1 rotor as described (8). CPSase, ATCase, and DHOase activities were assayed as described (7, 8). Enzyme Isolation. A rapid procedure was developed for isolating enzyme from cell extracts or gradient fractions by using rabbit antiserum (kindly provided by G. R. Stark) raised against CPSase/ATCase/DHOase enzyme from Syrian hamster. To cell-free extract from 107 cells, 10,1l of antiserum (5 ,gl precipitates 1 ,tg of enzyme) was added and the mixture was incubated for 12-16 hr at 4VC. Then, 10 pl of a protein ASepharose (Pharmacia) slurry in phosphate-buffered saline was added. Samples were incubated at 40C for 30 min with occasional shaking. The enzyme-antibody-protein A-Sepharose complex was washed six times with phosphate-buffered saline. Then, the pellet was extracted three times with 0.5 ml of 1 M acetic acid which dissociates the enzyme-antibody complex from the protein A-Sepharose. The pooled volumes were lyophilized to dryness and the powder was resuspended in NaDodSO4 sample buffer (11). NaDodSO4/Polyacrylamide Slab Gel Electrophoresis. Electrophoresis was performed according to Laemmli (11) with a linear acrylamide gradient of 5-20% (wt/vol). Gels were stained for protein by the procedure of Fairbanks et al. (12). The amounts of stained proteins were determined by scanning gel strips in an Acta spectrophotometer (Beckman) at 565 nm. When samples contained [a5S]methionine, gels were impregnated with 2,5-diphenyloxazole (13), dried, and exposed to Cronex 4 film (Du Pont) at -70'C. The standards used to estimate molecular weights were spectrin (I = 240,000; II = 220,000), ferritin (dodecamer = 220,000; monomer = 18,500), Escherichia coli RNA polymerase (f3 = 165,000; (3' = 155,000; a7 = 39,000), albumin (67,000), and gamma globulin (heavy chain = 50,000; light chain = 22,500). RESULTS Enzyme Isolation. The antibody-protein A-Sepharose procedure successfully removed most of the CPSase, ATCase, and DHOase activities from a CHO-K1 cell-free extract and could remove these activities from the mutants (Table 1). Thus, this simple procedure can be used to isolate the CPSase, ATCase, and DHOase enzymes directly from total soluble protein. Analysis of Enzyme in Cell-Free Extracts. When the antigen-antibody complex made with CHO-K1 cell extract was denatured and subjected to NaDodSO4/polyacrylamide gel electrophoresis, several proteins appeared (Fig. 1A). The two most prominent proteins were rabbit immunoglobulin (heavy and light chains). One protein band consistently isolated from CHO-K1 cell extract migrated electrophoretically the same distance as half ferritin and spectrin II. Urd-A revertants B48 Table 1. Enzyme activities removed by binding to antibody-protein A-Sepharose % activity removed* Strain CPSase ATCase DHOase CHO-K1 61t 98 98 t t 82 Urd-A D20 93 63 36
* Cell extract was assayed for enzyme activities before and after treatment with antiserum and protein A-Sepharose. t The percentage of CPSase activity removed is probably closer to 98%; the discrepancy is due to an artifact in the assay procedure
(8).
t Activities before and after precipitation were very low.
A1
2
3
1
B
3
2
240,000 220.000
_- _ _
165.000 155.000
- 220,000 165.000 - 155.000 -
67.00050.000-
* 39.000
- 67.000
-
U
22.500- t
_
Il
-
50.000
- 39.000
_- 22.500
FIG. 1. NaDodSO/polyacrylamide gel electrophoresis of denatured enzyme-antibody complex. (A) CHO-K1 (lane 1), mixture of CHO-K1 and B48 (lane 2), and B48 (lane 3). (B) D20 (lane 1), Ade-C (lane 2), and hybrid Ade-C/D20 (lane 3). Arrows point at 220,000and 200,000-dalton forms.
and D20 did not have this 220,000-dalton protein but did have a new band, not present in CHO-K1, at 200,000 daltons. When a mixture of denatured antigen-antibody complex from CHO-KI and B48 was analyzed by electrophoresis, the two forms clearly separated. Neither protein form was observed in Urd-A unless protein from 6 times more cells was applied to the gel (see Fig. 2B); then, only the 200,000-dalton protein appeared. In order to determine dominance, hybrids were constructed as described (8) between Ade-C, a purine-requiring auxotroph, and a Urd-A revertant (D20). The Ade-C/D20 hybrid showed codominance (both forms were present) (Fig. 1B). The antibody-protein A-Sepharose procedure, therefore, removes predominantly one 220,000-dalton protein from CHO-K1 cell-free extracts and one 200,000-dalton protein from Urd-A and the revertants B48 and D20. Ade-C/D20 hybrids express both proteins. Analysis of Enzyme in Glycerol Gradient Fractions. In order to correlate the protein bands on gels with the CPSase, ATCase, and DHOase activities, cell-free extracts were centrifuged on glycerol gradients and enzyme was assayed and isolated from the fractions collected. On the basis of previous results with glycerol gradients (8), DHOase activity can be used to monitor the sedimentation of the CPSase/ATCase/DHOase complex from CHO-K1 and of the CPSase/DHOase complex from Urd-A and D20 (the ATCase activity sedimenting elsewhere in the gradient). Fig. 2 shows a comparison between the presence of DHOase activity and the presence of immunologically purified protein in glycerol gradients of CHO-K1, Urd-A, D20, and the Ade-C/D20 hybrid. For CHO-K1 the amount and position of 220,000-dalton protein in the gradient corresponded exactly to the sedimentation of DHOase activity. There was no 200,000-dalton protein. When an equivalent amount of Urd-A cell extract was used, little DHOase activity was observed and no 220,000- or 200,000-dalton protein was
Biochemistry: Davidson and Patterson 4901
Proc. Natl. Acad. Sci. USA 76 (1979)
173
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FIG. 2. Comparison of DHOase activity of gradient fractions to amounts of 220,000- and 200,000-dalton polypeptides. Extract from 3 X 107 cells of CHO-K1 (A), UrdcA (B), D20 (C), or hybrid Ade-C/D20 (D) was applied to glycerol gradients. Gradient fractions were assayed for DHOase activity (x-X). Enzyme isolated from fractions was subjected to NaDodSO4/polyacrylamide gel electrophoresis. Coomassie blue absorbance (A ) of gel sections corresponding to 220,000 (O- - -0) and 200,000 daltons (o.....o) was determined (arrows point to these bands). As a reference, enzyme from 1 X 107 cells of CHO-Ki is shown in lane a of A, B, and C, of B48 in lane b of A and C, and of hybrid Ade-C/D20 in lane b of D and from 6 X 107 cells of Urd-A in lane b of B. The DHOase scale for Urd-A (B) is expanded 5-fold.
visible. With D20, the amount and position of 200,000-dalton protein correlated exactly with the sedimentation of DHOase activity. In the case of the hybrid Ade-C/D20, the amount and position of 220,000-dalton protein corresponded to the deeper sedimenting DHOase activity (i.e., CPSase/ATCase/DHOase peak) whereas the amount and position of 200,000-dalton protein corresponded to the shallower sedimenting DHOase activity (i.e., CPSase/DHOase peak). Thus, the 220,000-dalton protein observed on gels corresponds to an enzyme complex having CPSase, ATCase, and DHOase activities, whereas the 200,000-dalton protein corresponds to an enzyme complex having only CPSase and DHOase activities. Analysis of Enzyme in Labeled Cells. To increase the sensitivity of analyzing these enzymes in CHO-Ki and the mutants, enzyme was isolated from cells grown in the presence of
[s5S]methionine, subjected to NaDodSO4/polyacrylamide gel electrophoresis, and identified by autoradiography. For CHO-Ki, radioactive protein appeared as a major band at 220,000 daltons and a minor band at 200,000 daltons (Fig. 3). The opposite result was observed for D20: a major band at 200,000 daltons and a minor band at 220,000 daltons. The D20 cell extract also contained an immunologically crossreacting protein band not present in CHO-Ki cell extract at 19,00021,000 daltons (heavy arrow). When radioactive CHO-K1 cell extract was treated with trypsin prior to enzyme isolation, the resulting gel pattern was similar to that of D20 (Fig. 3) in that a major protein band appeared at about 200,000 daltons. Therefore, CHO-KI cells have a small amount of protein similar in molecular weight to the CPSase/DHOase enzyme of D20, and D20 cells have a small amount of protein similar in
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