Affinity chromatography of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 196, No. 1, August, pp. 134-137, 1979

Affinity Chromatography of Bacillus subtilis Glutamine Phosphoribosylpyrophosphate Amidotransferasel JOSEPH Y. WONG AND ROBERT L. SWITZER Department

of Biochemistry,

University

Urbana, Illinois

of Illinois,

61801

Received February 28, 1979 Purified glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis bound to affinity adsorbents containing immobilized adenine nucleotides. Although the enzyme probably bound via an allosteric site at which AMP acts most effectively, 50 times more enzyme was bound by NG-(aminohexyl)-ATP-agarose than by NYaminohexyl)-AMP-agarose. The enzyme could be efficiently and specifically eluted from NB-(aminohexyl)-ATPagarose with the substrate phosphoribosylpyrophosphate, which antagonizes AMP inhibition in kinetic experiments. Elution could also be effected by 0.5 M KC1 or by chelation of Mg*+ ions. The usefulness of these techniques in purification of partially purified amidotransferase was demonstrated.

Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) has received considerable attention as a probable focal point for the regulation of purine nucleotide biosynthesis (l-3). Studies of the interaction of amidotransferase with substrates and al!osteric inhibitors have been hampered by the difficulty of purifying this enzyme from many sources and the tendency of the enzyme to lose its sensitivity to allosteric inhibition during purification (4-6). We have recently developed procedures for purifying amidotransferase from Bacillus subtilis cells (‘7, S), and we have conducted kinetic studies of the interaction of this enzyme with allosteric inhibitors (9). In the course of these studies it was observed that partially purified amidotransferase could be further purified by affinity chromatography on N6-(aminohexyl)-AMP-agarose and N6(aminohexyl)-ATP-agarose. A more complete study of the specificity of adsorption and elution of amidotransferase, presented in this paper, provides insight into ligand binding by the enzyme. Furthermore, methods are provided for purifying amidotransferase that may be of use to investigators studying this enzyme from other sources. 1 This work was supported by Grant AI 11121from the U. S. Public Health Service. 0003-9861/79/090134-04$02.00/O

Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

MATERIALS

AND METHODS

Materials. All nucleotides and P-Rib-PP were obtained from Sigma Chemical Company. The purity of the P-Rib-PP was determined to be 74% by enzymatic assay (10); concentrations given are based on this value. NG-(Aminohexyl)-AMP-agarose (AGAMP, type II), NG-(aminohexyl)-ATP-agarose (AGATP, type II), ATP-adipic acid hydrazide-agarose (AGATP, type IV), and GTP-adipic acid hydrazide-agarose (AGGTP, type IV) were purchased from P-L Biochemicals. N6-[(6Aminohexyl)-carbamoylmethyl]-ATP-Sepharose was prepared by Simon Rosenzweig of this laboratory according to the procedure of Lindberg and Mosbach (11); this material was shown to contain 1 pmol of ATP/ml Sepharose by phosphate analysis (12). Blue dextranSepharose was prepared by Katharine Gibson using the procedure of Ryan and Vestling (13); this adsorbent was shown to bind P-Rib-PP synthetase from Salmo(14). Glutamine P-Rib-PP amidonella typhimwium transferase was purified from B. subtilis as described elsewhere (7, 8; Wong, Meyer, Turnbough, and Switzer, in preparation). The partially purified amidotransferase used to evaluate purification was taken from the DEAE-cellulose step of the purification procedure. Methods. A 0.39 x 5-cm column was packed with the indicated absorbent and equilibrated at 4°C with 20 mM Tris/HCl buffer, pH 7.9, containing 5 mM MgCl, and 5 mM dithiothreitol. Purified amidotransferase (2 to 9 units) was applied to the column and allowed to equilibrate with the adsorbent for 5 to 10 min at 4°C. The column was then washed with 5 to 10 column vol of the equilibrating buffer. Elution was attempted with 7

134

AFFINITY

CHROMATOGRAPHY

135

OF AMIDOTRANSFERASE

TABLE I BINDING OF AMIDOTRANSFERASETO AFFINITY ADSORBENTS” Amidotransferase

Adsorbent N*-(Aminohexyl)-ATP-agarose Ns-(Aminohexyl)-AMP-agarose

pm01 AMP or ATPI ml agarose

Bed volume (ml)

Adsorbed (units)

Recovered by elution 6)

4.9 5.2

0.6 0.6

112 2.1

96 66

(2Purified amidotransferase was applied to a column containing the indicated adsorbent until enzyme activity was detected in the pass-through fluid. The column was then washed with 10 column vol of equilibrating buffer, and the amount of amidotransferase bound was determined by elution with 1 M KC1 in equilibrating buffer. to 10 column vol of the indicated ligand or eluant dissolved in the equilibrating buffer. When P-Rib-PP was used as the eluant, 30 column vol of eluting buffer was used. The flow rate was about 8 to 12 ml/h. At the end of each trial, the column was washed with 0.5 or 1.0 M KC1 in the equilibrating buffer to remove any amidotransferase not eluted by prior treatment. This wash was followed by 10 ml of 50 mM Tris/HCl, pH 7.9, containing 0.1 mM EDTA, 10 mM MgCl,, and 0.02% sodium aside. Amidotransferase was assayed by the iixed time spectrophotometric procedure previously described (7), except that the buffer was 25 mM potassium phosphate, pH 7.5, containing 5 mM MgCl, and 3 mM P-Rib-PP. RESULTS

Since AMP is the most effective inhibitor of B. subtilis amidotransferase of a wide variety of nucleotides tested (91, we reasoned that the enzyme would bind to an affinity column containing AMP attached to the resin by a flexible arm. This prediction was fulfilled; the enzyme bound to N6(aminohexyl)-AMP-agarose (Table I). Surprisingly, however, the enzyme bound much more efficiently to Ns-(aminohexyl)-ATPagarose. Binding was also very dependent on the mode of attachment of the ligand to the agarose resin, because amidotransferase did not bind at all to Ng-[(6-aminohexyl)carbamoylmethyl]ATP-Sepharose or to ATP-adipic acid hydrazide-agarose. GTPadipic acid hydrazide-agarose and blue dextran-sepharose also did not bind the enzyme. The nature of the association between amidotransferase and NQminohexyl)-ATP-

agarose was further examined by determining the ability of ligands of the enzyme to elute the enzyme from the affinity adsorbent. Table II shows that concentrations of AMP or ATP up to 10 mM failed to elute the enzyme efficiently, as did 3 mM ADP, GMP, GDP, or GTP. Complete conversion of amidotransferase into a conformation that is fully inhibited by allosteric ligands is not TABLE II ELUTION OF AMID~TRANSFERASEFROM NVAMINOHEXYL)-ATP-AGAROSE Eluant m-f)

Amidotransferase eluted (96)

AMP (3) AMP (10) ADP (3) ATP (3, lO=) GMP (3) GDP (3) GTP (3) ADP (1.5) + GMP (1.5) P-Rib-PP (0.45) P-Rib-PP (1.8) Glutamine (15) Glutamine (15) + P-Rib-PP (0.45) KP, (10) =‘I (40) KC1 (40) KC1 (500, 1000) EDTA (6)b

1 8 0 0 0 8 0 0 17 87 0 12 54 100 1 99 89

a MgCI, content of the eluting buffer was increased to 10 mrd. b MgCl, was omitted from the eluting buffer.

136

WONG AND SWITZER

sufficient to accomplish elution, because the enzyme was not eluted by excess AMP or by completely inhibitory levels of two strongly synergistic inhibitors, ADP plus GMP (9). The substrate glutamine did not elute the amidotransferase, but modest concentrations of the other substrate, P-Rib-PP, brought the enzyme off the column in good yield. Ionic character to the association was indicated by elution of the enzyme by 0.5 or 1.0 M KCl. Elution of the enzyme by 6 mM EDTA indicated a requirement for Mg2+ for binding. This was confirmed in a separate experiment in which the capacity of N6(aminohexyl)-ATP-agarose for amidotransferase was examined using 20 lllM Tris/HCl buffer without MgC12;only 6% as much enzyme was bound as in the presence of 5 mM MgCl,. The enzyme was eluted by 10 to 40 mM Pi, but this may have been due to chelation of Mg2+. The potential utility of affinity chromatography for purification of amidotransferase was demonstrated by adsorption of a sample of partially purified enzyme (2.3 mg, 7.8 units/mg, shown to be free of ATP or AMP hydrolyzing activity) to a 2.0 ml column of N6-(aminohexyl)-ATP-agarose, washing with buffer and 40 mM KC1in buffer, and eluting with 2.5 mM P-Rib-PP as described under Materials and Methods. The fractions eluted with P-Rib-PP had an average specific activity of 18.7 units. The results of electrophoretic analysis (Fig. 1) showed that many, but not all, contaminating proteins were removed or partially removed by the procedure. Recovery of activity was about 50%. DISCUSSION

The specificity of affinity chromatography of amidotransferase displayed a number of properties that could not have been predicted from kinetic studies (9). We believe that the results can be best accounted for by suggesting that the enzyme binds to the adsorbents at an allosteric, nucleotide-binding site, rather than at the active site. AMP is a much more effective allosteric inhibitor than any other nucleotide. The ability of amidotransferase to bind to N6-(aminohexyl)-AMP-agarose and to be eluted from

FIG. 1. Gel electrophoretic analysis of partially purified amidotransferase before and after affinity chromatography on NG-(6-aminohexyl)-ATP-agarose. Electrophoresis was carried out in the presence of 0.1% sodium dodecyl sulfate as described by Laemmli (16), except that l-mm slab gels containing a gradient from 15 to 7.5% acrylamide were used. (a) Partially purified amidotransferase, 5.7 pg. (b), (c), and (d) Fractions eluted from the affinity column with P-Rib-PP were concentrated in dialysis bags with Aquacide, protein determined by the method of Lowry (li’), and samples analyzed by gel electrophoresis. Protein analyzed was 3.5, 3.0, and 3.5 pg, respectively. The arrow marks the position of migration of pure amidotransferase.

N6-(aminohexyl)-ATP-agarose by AMP-although inefficiently in both cases-indicates probable binding at the AMP site. The complete discrimination of amidotransferase between N6-(aminohexyl)- and N6-[(fkminohexyl)- carbamoylmethyl] - ATP - Sepharose would not be expected if binding were due to

AFFINITY

CHROMATOGRAPHY

OF AMIDOTRANSFERASE

137

attachment of the ribose-phosphate portion readily lost by the enzyme from some of ATP at the P-Rib-PP binding region of sources (4, 5). the active site. The dramatic effect of modification at the N6 position of adenine ACKNOWLEDGMENTS indicates involvement of adenine in binding. We are grateful to Simon Rosenzweig and Katharine (A similar, but opposite, discrimination between these two adsorbents by phospho- Gibson for providing samples of affinity adsorbents fructokinase has been reported by Ramadoss which they had prepared by chemical synthesis. et al. (15)) Finally, the efficient elution of amidotransferase from N6-(aminohexyl)REFERENCES ATP-agarose by P-Rib-PP, presumably by 1. WYNGAARDEN, J. B. (1972) Curr. Top. Cell. an allosteric transition, is consistent with Regul. 5, 135-1’76. the antagonism of AMP inhibition by P-Rib2. WYNGAARDEN, J. B. (19’72) in The Enzymes of PP that has been observed kinetically (9). Glutamine Metabolism (Prusiner, S., and StadtThe 50-fold greater capacity for amidoman, E. R., eds.), pp. 365-386, Academic transferase of N6-(6-aminohexyl)-ATP-agaPress, New York. rose, as compared to N6-(6-aminohexyl)3. HENDERSON, J. F. (19’72) Regulation of Purine Biosynthesis, pp. 39-195, Amer. Chemical Sot., AMP-agarose, and the poor ability of alloWashington, D. C. steric inhibitors to elute the enzyme suggest that a site adjacent to the allosteric site may 4. CASKEY, C. T., ASHTON, D. M., AND WYNGAARDEN, J. B. (1964) J. Viol. Chem. 239, be involved in binding to the adsorbent. The 2570-2579. elution of the enzyme by P-Rib-PP could 5. HARTMAN, S. C. (1963) J. Biol. Chem. 238,3024reflect allosteric alterations at both the AMP 3035. site and adjacent areas. The presence of 6. HILL, D. L., AND BENNETT, L. L., JR. (1969) Mg2+ is required for AMP to inhibit amidoBiochemistry 8, 122-130. transferase (9), and the present studies 7. WONG, J. Y., MEYER, E., AND SWITZER, R. L. show that Mg2+ is required for binding at (1977) J. Biol. Chem. 252, 7424-7426. the AMP site. Concentrations of allosteric 8. WONG, J. Y. (1978) Ph.D. thesis, Univeristy of Illinois, Urbana. ligands that are sufficient to inhibit amidotransferase fully do not suffice to elute the 9. MEYER, E., AND SWITZER, R. L. (1979) J. Biol. Chem., in press. enzyme. Furthermore, 10 mM AMP, which completely converts amidotransferase from 10. SADLER, W. C., AND SWITZER, R. L. (1977) J. Biol. Chem. 252, 8504-8511. a tetramer to a dimer in solution (7), does 11. LINDBERG, M., AND MOSBACH, K. (1975) Eur. J. not result in elution of the enzyme from the B&hem. 53, 481-486. affinity adsorbent. 12. AMES, B. N. (1966) in Methods in Enzymology Finally, we wish to point out that affinity (Neufeld, E. F., and Ginsburg, V., eds.), Vol. 8, chromatography of amidotransferase propp. 115-118, Academic Press, New York. vides a gentle, rapid, and specific means of 13. RYAN, L. D., AND VESTLING, C. S. (1974) Arch. Biochem. Biophys. 160, 279-284. purifying this enzyme, which has proven SWITZER, R. L., AND GIBSON, K. J. (1978) in Meth14. very difficult to purify from mammalian ods in Enzymology (Hoffee, P. A., and Jones, sources. The results of our experiments M. E., eds.), Vol. 51, pp. 3-11, AcademicPress, show that some flexibility in adapting our New York. methods to purification of the enzyme from 15. RAMADOSS, G. S., LUBY, L. J., AND UYEDA, K. other sources will be required, however. It (1976) Arch. Biochem. Biophys. 175, 487-494. seems likely that use of an affinity adsorbent 16. LAEMMLI, U. K. (1970) Nature (London) 227, that binds amidotransferase at an allosteric 680-685. site offers means of purifying the enzyme in 17. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, a form that retains its sensitivity to allo265-275. steric inhibition, a characteristic that is