Affinity chromatography of glutathione reductase: Bound by immobilized GSSG, eluted by NADPH

ANALYTICAL

BIOCHEMISTRY

82, 586-590 (1977)

Affinity Chromatography Bound by Immobilized

of Glutathione Reductase: GSSG, Eluted by NADPH1,2

Glutathione reductase bound to an affinity matrix of immobilized GSSG was eluted by its coenzyme NADPH rather than by its substrate GSSG or by NADH. NADP+ could also elute the enzyme, but a high concentration was needed to release enzyme activity in a sharp peak. This chromatographic system exhibits an unusual form of biospecificity in which the enzyme is bound to an immobilized substrate but released by its soluble cofactor.

A number of affinity chromatographic systems have been utilized for binding yeast GSSG reductase (EC 1.6.4.2). In the two in which the enzyme was bound by immobilized GSSG, the enzyme was not eluted with soluble GSSG, but rather with NaCl(l,2). Salt has also been used by Lowe et al. (3) to recover the enzyme bound to an immobilized analog of NADP+. In an attempt to demonstrate biospecific chromatography of GSSG reductase, we decided to study the effect of the cofactor NADPH as a potential eluting agent. This cofactor has been used successfully to elute GSSG reductase from a column of an immobilized adenine nucleotide derivative (4,5), but it has not been used with immobilized GSSG derivatives. There have been previous reports of coenzymes affecting affinity chromatographic systems. For example, O’Carra and Barry achieved successful adsorption of LDH (EC 1.1.1.27) to an immobilized pyruvate analog only when free NADH was present (6). Successful elution of a number of lactate dehydrogenases from 8-(6-aminohexyl)-amino-adenine nucleotide-Sepharoses has also been demonstrated by use of a reduced NADpyruvate adduct (7). Similarly, citrate synthase (EC 4.1.3.7) adsorbed to ATP- Sepharose was eluted by CoA and oxaloacetate in combination but not alone (8)) and phosphofructokinase (EC 2.7.1.11) bound to an immobilized adenine nucleotide derivative was eluted only when fructose-6-phos1 This paper is dedicated to the late William Farnsworth Loomis. Dr. Loomis, known primarily to readers of this journal for his classical work on the uncoupling of phosphorylation from oxidation and on glutathione as a feeding activator in hydra, in 1953 was the first recognized biochemist to enter full-time into research on simple aquatic invertebrates. He selected the freshwater hydraas his experimental organism. His novel and quantitative researches stimulated biologists to use this animal for research in receptor activation, developmental biology, behavior, and symbiosis. As biochemists now turn to new research organisms for their studies, they will learn much from Loomis’ pioneering studies about how to develop a new organism for laboratory investigations. Original thinkers are rare upon the biological scene. W. F. Loomis was one. *Supported by NIH Grant No. NS11171. 586 Copyright All rghts

0 1977 by Academic Press. Inc. of reproduction in any form reserved

ISSN 0003.2697

SHORT

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COMMUNICATIONS

phate and ADP were present together (9). In this paper we describe the retention of GSSG reductase by immobilized GSSG and elution of the enzyme by the free coenzyme. Yeast GSSG reductase was obtained from Boehringer-Mannheim Corp. (New York, N.Y.) and was assayed by the method of Racker (10) in the presence of 0.05 M phosphate, pH 7.4. GSSG, NADH, NAD+, NADPH, and NADP+ were obtained from Sigma (St. Louis, MO.). Polyethylene columns, 0.9 cm in diameter and 0.6-1.0 cm high, were used for chromatography. All operations were carried out at room temperature (21-25°C) in the presence of 0.05 M phosphate (Na+), pH 7.4. Each fraction was 1.1 ml and was collected at a flow rate of 1 drop/lo- 15 sec. As soon as the fractions were collected they were stored at 4°C. Prior to chromatography the reductase was dialyzed at 4°C against the buffer for 18-24 hr; 2-4 units of enzyme activity were used for each experiment. GSSG was covalently linked through its amino group(s) (2) to cellulose activated with the linking reagent s-triazine trichloride (sTT) (11). The amount of matrix-bound GSSG, referred to as GSSG(N)-sTT-cellulose, was determined as described elsewhere (Danner er al., 1977). The columns described in the paper contained a total of 0.3-0.6 pmole of GSSG. GSSG reductase applied to a column of GSSG(N)-sTT-cellulose was retained by that column. That the enzyme bound to the immobilized GSSG derivative and was not nonspecifically absorbed to the triazine nucleus was shown by the inability of a control column, consisting of sTT-cellulose without GSSG, to retain the reductase (2). Elution of the enzyme from GSSG(N)-sTT-cellulose was carried out with 3.3 mM GSSG followed by 0.1 mM NADPH. Figure 1 shows that, although 6% of the activity was recovered in a diffuse band after applying GSSG, 7% of reductase activity was eluted in a sharp peak using soluble

60

Buffer

GSSG + Buffer

NADPH+ Buffer

I

I

I

Fraction FIG. 1. Chromatography GSSG and NADPH.

of GSSG

reductase

No. on GSSG(N)-sTT-cellulose:

elution

with

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SHORT COMMUNICATIONS

NADPH. Furthermore, if the NADPH (0.1 mM) was first mixed with the GSSG reductase, no binding of the enzyme to the column could be detected. The specificity of the adsorbed GSSG reductase toward the various forms of pyridine nucleotides at 0.1 mM was investigated (Fig. 2, lower graph) because of the known reactivity of the soluble enzyme toward these cofactors (12- 14). The data show that, whereas neither NAD+ nor NADH eluted significant enzyme activity, both NADP+ and NADPH did elute the enzyme. The NADP+ released 62% of the enzyme activity in a diffuse band; the remainder was eluted in a sharp peak by the further addition of NADPH. That this elution behavior was the result of interactions with the triphosphopyridine nucleotides and not due to a peculiarity of the column was shown by repeating the experiment with the same column, but eluting only with NADPH. The results (Fig. 2, upper graph) show that enzyme activity emerged as would be expected from examination of the results of Fig. 1. To further explore the elution of GSSG reductase with NADP+, the con-

a C ., 2

Buffer

NAD+

NADH

NADP+

NADPH

‘IL,d 2

4

6

6

IO

12

14

16

16 20

22

24

26 26

Fraction No. (IJmVfmction) FIG. 2. Chromatography of GSSG reductase on GSSG(N)-sTT-cellulose: various pyridine nucleotides.

elution with

SHORT COMMUNICATIONS

589

centration was increased to 1 mM. Rather than being released in a diffuse band as was effected by 0.1 mM NADP+, 67% of the reductase was recovered in a sharp peak (Fig. 3). In conclusion, we have shown that GSSG reductase bound to a derivative of the immobilized substrate GSSG was eluted by its coenzyme NADPH (Figs. 1 and 2). The elution patterns show that the bound enzyme, like the soluble enzyme (12), exhibits a specificity for NADPH relative to NADH (Fig. 2). A related finding by Lowe ef al. (3) demonstrated that the yeast reductase bound less well to an immobilized NAD+ derivative than to the corresponding NADP+ analog. NADPH is known to partially reduce soluble GSSG reductase (13,14). Such a reduction was apparently not necessary for the elutions described in this paper because NADP+ also eluted the enzyme (Figs. 2 and 3). Hence, it seems probable that interaction of the bound reductase with the triphosphopyridine nucleotides resulted in a conformational change in the enzyme leading to a decreased affinity for the GSSG(N)-sTT-cellulose. The recovery of enzyme activity by NADP+ in a sharp peak required a higher concentration than that of the reduced cofactor, NADPH; this result is in agreement with the observation by Bulger and Brandt (15) that NADP+ has less affinity for soluble GSSG reductase than does NADPH. The elution patterns described in this paper are consistent with data on the known reactivity of soluble GSSG reductase toward the pyridine nucleotides. It would, therefore, appear that the chromatographic system we describe exhibits a form of biochemical specificity in which the enzyme is bound to an immobilized substrate but released by its soluble cofactor.

2 FIG. 3. Chromatography NADP+ .

4

6 8 IO Fraction No.

of GSSG reductase on GSSG(N)-s’IT-cellulose:

elution with

590

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In summary, glutathione reductase bound to an affinity matrix of immobilized GSSG was eluted by its coenzyme NADPH rather than by its substrate GSSG or by NADH. NADP+ could also elute the enyzme, but a high concentration was needed to release enzyme activity in a sharp peak. This chromatographic system exhibits an unusual form of biospecificity in which the enzyme is bound to an immobilized substrate but released by its soluble cofactor. REFERENCES 1. Harding, J. J. (1973) J. Chromarogr. 77, 191-199. 2. Danner, J., Lenhoff, H. M., and Heagy, W. (1976)J. SolidPhase Biochem. 1, 177-188. 3: Lowe, C. R., Mosbach, K., and Dean, P. D. (1972) Biochem. Biophys. Res. Commun. 48, 1004- 1010. 4. Brodelius, P., Larsson, P.-O., and Mosbach, K. (1974) Eur. J. Biochem. 47, 81-89. 5. Mannervik, B., Jacobsson, K., and Boggaram, V. (1976) FEBS Left. 66,221-224. 6. O’Carra, P., and Barry, S. (1972) FEBS Let?. 21, 281-285. 7. Lee, C-Y, Lappi, D. A., Wiermuth, B., Everse, J., and Kaplan, N. 0. (1974)Arch. Biothem.

Biophys.

163,561-569.

8. Mukhejee, A., and Srere, P. A. (1976) J. Biol. Chem. 251, 1476-1480. 9. Ramadoss, C. S.,Luby, L. J., and Uyeda, K. (1976) Arch. Biochem. Biophys. 175,487494. 10. Racker, E. (1955)in Methods inEnzymology (Kaplan, N. O., andColowick, S. P., eds.), Vol. 2, pp. 722-725, Academic Press, New York. 11. Smith, N. L., and Lenhoff, H. M. (1974) Anal. Biochem. 61,392-415. 12. Racker, E. (1955) J. Biol. Chem. 217, 855-865. 13. Bulger, J. E., and Brandt, K. G. (1971) J. Biol. Chem. 246, 5570-5577. 14. Massey, V., and Williams, C. H. (1965) J. Biol. Chem. 240, 4470-4480. 15. Bulger, J. E., and Brandt, K. G. (1971) J. Biol. Chem. 246, 5578-5587. JEAN DANNER~ HOWARD M. LENHOFF~ WYRTA HEAGY~ Department of Developmental and Cell Biology and Molecular Biology and Biochemistry University of California Irvine, California 92717 Received March 7, 1977; accepted May 31, 1977

3 Present address: Department of Biochemistry and Molecular Biology, JHM Health Center, University of Florida, Gainesville, Florida 32610. 4 Send reprint requests to Dr. Howard M. Lenhoff, Faculty Research Facility, University of California, Irvine, California 92717. 5 Present address: National Institute of Mental Health, Betheseda, Maryland 20014.