PROTEIN
A.
DISULFIDE
ISOMERASE
B. 1234
12
56789
3
97.4 -
66.2 42.7 42.7 31.0 31.0 21.5
FIG. 2. Purification of recombinant PDI. SDS-PAGE on 10% acrylamide gels of samples of the PDI preparation at various stages of purification is shown. (A) Lane 1, molecular weight markers; lane 2, SDS-solubilized proteins from cells lacking the plasmid pETPDI.2; lane 3, SDS-solubilized proteins from pETPDI.2/BL21(DE3) induced with IPTG; lane 4, pooled fractions after Triton X-100 solubilization; lane 5, pooled fractions after carboxymethyl-Sephadex chromatography showing little purification from this step; lane 6, pooled fractions after chromatography on DEAE-Sephacel;
EXPRESSION
197
PROTEIN
A.
DISULFIDE
ISOMERASE
B. 1234
12
56789
3
97.4 -
66.2 42.7 42.7 31.0 31.0 21.5
FIG. 2. Purification of recombinant PDI. SDS-PAGE on 10% acrylamide gels of samples of the PDI preparation at various stages of purification is shown. (A) Lane 1, molecular weight markers; lane 2, SDS-solubilized proteins from cells lacking the plasmid pETPDI.2; lane 3, SDS-solubilized proteins from pETPDI.2/BL21(DE3) induced with IPTG; lane 4, pooled fractions after Triton X-100 solubilization; lane 5, pooled fractions after carboxymethyl-Sephadex chromatography showing little purification from this step; lane 6, pooled fractions after chromatography on DEAE-Sephacel;
EXPRESSION
197
198
GILBERT
Thiolldisulfide redox behavior. PDI isolated from bovine liver contains free cysteine residues that are inaccessible to modification by iodoacetamide (9,21) and which react very slowly with DTNB (data not shown) in the native enzyme. However, reaction of the native enzyme (both recombinant and bovine) for 3 hr with [3H] NEM (8.0 mM, 1.8 X lo6 cpm/pmol) at pH 8 resulted in the incorporation of 0.61 * 0.07 eq of NEM into the protein with no significant (~10%) effect on the activity of the enzyme in catalysis of the oxidative renaturation of RNase. Carmichael et al. (21) made similar observations with the bovine liver enzyme. Under denaturing conditions (4-6 M guanidinium hydrochloride, pH 7.5), titration of the free cysteines with DTNB showed 1.4 + 0.3 (n = 7) free cysteine residues for the liver enzyme and 1.8 * 0.2 (n = 6) free cysteine residues for the recombinant protein. The number of free cysteines in the two proteins was somewhat variable from preparation to preparation, ranging between values of 1.1 and 2.2. While little is known about the redox environment that exists in the lumen of the endoplasmic reticulum, the cytoplasmic environment of E. coli is highly reducing. In vitro, this would be sufficient to fully reduce the disulfides of PDI, suggesting that the protein is synthesized and folded in the reduced form in E. coli and that the disulfide bonds arise from autoxidation during purification. A similar fate is likely to have befallen the enzyme isolated from bovine liver since the isolated form of PDI is not an effective catalyst of the oxidative folding of RNase; a partially or fully reduced redox isomer of PDI appears to be the more effective catalyst (20). For optimum catalytic efficiency in oxidative protein folding, it would appear that a partially reduced form of PDI would be essential; however, this form of the enzyme does not survive isolation. At least two and possibly all of the disulfide bonds present in PDI can be used to oxidize fully reduced RNase to the native enzyme (20). Either PDI has multiple active sites or one active site is capable of transferring oxidizing equivalents from multiple disulfide bonds. Future experiments will delineate the relationships between the multiple disulfide bonds of PDI. ACKNOWLEDGMENT We thank Dr. pPDI-100 cDNA
Jeffery Edman, clone of the rat
UCSF, PDI.
for
his generous
gift
of the
ET
AL.
REFERENCES 1. Bergman, L. W., and Kuehl, W. M. (1979) J. Biol. Chem. 254, 5690-5694. 2. Peters, T., and Davidson, L. K. (1982) J. Biol. Chem. 257,88478853. 3. Freedman, R. B. (1989) Cell 67, 1069-1072. 4. Schmid, F. X. (1986) C. H. W., and Timasheff, demic Press, San Diego. 5. Ellis, R. J. (1987) Nature 6. Pelham, H. R. B. (1986) 7. Rothman, 8. Gething, 939-950.
in
“Methods S. N., Eds.),
in Enzymology” (Him., Vol. 131, pp. 70-82, Aca-
328,378-379. Cell 46.959-961.
J. E. (1989) Cell 59,591-601. M. J., McCammon, K., and Sambrook,
9. Freedman, them. Sot.
R. B., Brockway, Trans. 12,929-932.
B. E., and
J. (1986)
Lambert,
Cell 46,
N. (1984)
Bio-
10. Morin, J. E., and Dixon, J. E. (1985) in “Methods in Enzymology” (Meister, A., Ed.), Vol. 113, pp. 541-547, Academic Press, San Diego. 11. Kivirikko, K. I., Myllyla, J. 3, 1609-1617. 12. Geetha Habib, M., Noiva, (1988) Cell 54, 1053-1060. 13. Obata, (1988)
T., Kitagawa, J. Biol. Chem.
R., and Pihlajaniemi, R., Kaplan,
T. (1989)
FASEB
H. A., and Lennarz,
S., Gong, Q. H., Pastan, 263,782-785.
14. Wetterau, J. R., Combs, K. A., Spinner, (1990) J. Biol. Chem. 265,9801-9807.
W. J.
I., and Cheng,
S. Y.
S. N., and Joiner,
B. J.
15.
Edman, J. C., Ellis, L., Blather, R. W., Roth, R. A., and Rutter, W. J. (1985) Nature 317, 267-270. 16. Yamauchi, K., Yamamoto, T., Hayashi, H., Koya, S., Takikawa, H., Toyoshima, K., and Horiuchi, R. (1987) Biochem. Biophys. Res. Commun. 146,1485-1492.
17.
Morris, J. I., and Varandani, 949, 169-180.
18.
Freedman, R. B., Hawkins, (1988) Biochem. Sot. Trans.
P. T. (1988) H.
C., Murant,
B&him.
S. J., and
Acta
Reid,
L.
16, 96-99.
19. 20.
Gilbert, H. F. (1989) Biochemistry 28,7298-7305. Lyles, M. L., and Gilbert, H. F. (1991) Biochemistry
21.
Carmichael, Biol. Chem.
22.
Studier, F. W., Rosenberg, A. H., Dunn, J. J., and J. W. (1990) in “Methods in Enzymology” (Goeddel, Vol. 185, pp. 60-89, Academic Press, San Diego.
D. F., Keefe, 254,8386-8390.
Biophys.
M.,
Pace,
M., and Dixon,
30,613-619. J. E. (1979)
J.
Dubendorff, D. V., Ed.),
23. Hillson, D. A., Lambert, N., and Freedman, R. B. (1984) in “Methods in Enzymology” (Wold, F., and Moldave, K., Eds.), Vol. 107, pp. 281-294, Academic Press, San Diego. 24. Lyles, M. L., and Gilbert, H. F. (1991) Biochemistry 30,619-625. 25. Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975) Nature 258,598-599. 26. Bradford, M. M. (1976) Anal. Bzbchem. 72, 248-254.
A.
DISULFIDE
ISOMERASE
B. 1234
12
56789
3
97.4 -
66.2 42.7 42.7 31.0 31.0 21.5
FIG. 2. Purification of recombinant PDI. SDS-PAGE on 10% acrylamide gels of samples of the PDI preparation at various stages of purification is shown. (A) Lane 1, molecular weight markers; lane 2, SDS-solubilized proteins from cells lacking the plasmid pETPDI.2; lane 3, SDS-solubilized proteins from pETPDI.2/BL21(DE3) induced with IPTG; lane 4, pooled fractions after Triton X-100 solubilization; lane 5, pooled fractions after carboxymethyl-Sephadex chromatography showing little purification from this step; lane 6, pooled fractions after chromatography on DEAE-Sephacel;
EXPRESSION
197
PROTEIN
A.
DISULFIDE
ISOMERASE
B. 1234
12
56789
3
97.4 -
66.2 42.7 42.7 31.0 31.0 21.5
FIG. 2. Purification of recombinant PDI. SDS-PAGE on 10% acrylamide gels of samples of the PDI preparation at various stages of purification is shown. (A) Lane 1, molecular weight markers; lane 2, SDS-solubilized proteins from cells lacking the plasmid pETPDI.2; lane 3, SDS-solubilized proteins from pETPDI.2/BL21(DE3) induced with IPTG; lane 4, pooled fractions after Triton X-100 solubilization; lane 5, pooled fractions after carboxymethyl-Sephadex chromatography showing little purification from this step; lane 6, pooled fractions after chromatography on DEAE-Sephacel;
EXPRESSION
197
198
GILBERT
Thiolldisulfide redox behavior. PDI isolated from bovine liver contains free cysteine residues that are inaccessible to modification by iodoacetamide (9,21) and which react very slowly with DTNB (data not shown) in the native enzyme. However, reaction of the native enzyme (both recombinant and bovine) for 3 hr with [3H] NEM (8.0 mM, 1.8 X lo6 cpm/pmol) at pH 8 resulted in the incorporation of 0.61 * 0.07 eq of NEM into the protein with no significant (~10%) effect on the activity of the enzyme in catalysis of the oxidative renaturation of RNase. Carmichael et al. (21) made similar observations with the bovine liver enzyme. Under denaturing conditions (4-6 M guanidinium hydrochloride, pH 7.5), titration of the free cysteines with DTNB showed 1.4 + 0.3 (n = 7) free cysteine residues for the liver enzyme and 1.8 * 0.2 (n = 6) free cysteine residues for the recombinant protein. The number of free cysteines in the two proteins was somewhat variable from preparation to preparation, ranging between values of 1.1 and 2.2. While little is known about the redox environment that exists in the lumen of the endoplasmic reticulum, the cytoplasmic environment of E. coli is highly reducing. In vitro, this would be sufficient to fully reduce the disulfides of PDI, suggesting that the protein is synthesized and folded in the reduced form in E. coli and that the disulfide bonds arise from autoxidation during purification. A similar fate is likely to have befallen the enzyme isolated from bovine liver since the isolated form of PDI is not an effective catalyst of the oxidative folding of RNase; a partially or fully reduced redox isomer of PDI appears to be the more effective catalyst (20). For optimum catalytic efficiency in oxidative protein folding, it would appear that a partially reduced form of PDI would be essential; however, this form of the enzyme does not survive isolation. At least two and possibly all of the disulfide bonds present in PDI can be used to oxidize fully reduced RNase to the native enzyme (20). Either PDI has multiple active sites or one active site is capable of transferring oxidizing equivalents from multiple disulfide bonds. Future experiments will delineate the relationships between the multiple disulfide bonds of PDI. ACKNOWLEDGMENT We thank Dr. pPDI-100 cDNA
Jeffery Edman, clone of the rat
UCSF, PDI.
for
his generous
gift
of the
ET
AL.
REFERENCES 1. Bergman, L. W., and Kuehl, W. M. (1979) J. Biol. Chem. 254, 5690-5694. 2. Peters, T., and Davidson, L. K. (1982) J. Biol. Chem. 257,88478853. 3. Freedman, R. B. (1989) Cell 67, 1069-1072. 4. Schmid, F. X. (1986) C. H. W., and Timasheff, demic Press, San Diego. 5. Ellis, R. J. (1987) Nature 6. Pelham, H. R. B. (1986) 7. Rothman, 8. Gething, 939-950.
in
“Methods S. N., Eds.),
in Enzymology” (Him., Vol. 131, pp. 70-82, Aca-
328,378-379. Cell 46.959-961.
J. E. (1989) Cell 59,591-601. M. J., McCammon, K., and Sambrook,
9. Freedman, them. Sot.
R. B., Brockway, Trans. 12,929-932.
B. E., and
J. (1986)
Lambert,
Cell 46,
N. (1984)
Bio-
10. Morin, J. E., and Dixon, J. E. (1985) in “Methods in Enzymology” (Meister, A., Ed.), Vol. 113, pp. 541-547, Academic Press, San Diego. 11. Kivirikko, K. I., Myllyla, J. 3, 1609-1617. 12. Geetha Habib, M., Noiva, (1988) Cell 54, 1053-1060. 13. Obata, (1988)
T., Kitagawa, J. Biol. Chem.
R., and Pihlajaniemi, R., Kaplan,
T. (1989)
FASEB
H. A., and Lennarz,
S., Gong, Q. H., Pastan, 263,782-785.
14. Wetterau, J. R., Combs, K. A., Spinner, (1990) J. Biol. Chem. 265,9801-9807.
W. J.
I., and Cheng,
S. Y.
S. N., and Joiner,
B. J.
15.
Edman, J. C., Ellis, L., Blather, R. W., Roth, R. A., and Rutter, W. J. (1985) Nature 317, 267-270. 16. Yamauchi, K., Yamamoto, T., Hayashi, H., Koya, S., Takikawa, H., Toyoshima, K., and Horiuchi, R. (1987) Biochem. Biophys. Res. Commun. 146,1485-1492.
17.
Morris, J. I., and Varandani, 949, 169-180.
18.
Freedman, R. B., Hawkins, (1988) Biochem. Sot. Trans.
P. T. (1988) H.
C., Murant,
B&him.
S. J., and
Acta
Reid,
L.
16, 96-99.
19. 20.
Gilbert, H. F. (1989) Biochemistry 28,7298-7305. Lyles, M. L., and Gilbert, H. F. (1991) Biochemistry
21.
Carmichael, Biol. Chem.
22.
Studier, F. W., Rosenberg, A. H., Dunn, J. J., and J. W. (1990) in “Methods in Enzymology” (Goeddel, Vol. 185, pp. 60-89, Academic Press, San Diego.
D. F., Keefe, 254,8386-8390.
Biophys.
M.,
Pace,
M., and Dixon,
30,613-619. J. E. (1979)
J.
Dubendorff, D. V., Ed.),
23. Hillson, D. A., Lambert, N., and Freedman, R. B. (1984) in “Methods in Enzymology” (Wold, F., and Moldave, K., Eds.), Vol. 107, pp. 281-294, Academic Press, San Diego. 24. Lyles, M. L., and Gilbert, H. F. (1991) Biochemistry 30,619-625. 25. Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975) Nature 258,598-599. 26. Bradford, M. M. (1976) Anal. Bzbchem. 72, 248-254.
