ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 296, No. 2, August 1, pp. 497-504, 1992
Additional Binding Sites for the Pyruvate Dehydrogenase Kinase but Not for Protein X in the Assembled Core of the Mammalian Pyruvate Dehydrogenase Complex: Binding Region for the Kinase Lin Li, Gary A. Radke, Kazuo Ono, and Thomas Department
of Biochemistry,
Kansas State University,
E. Roche’
Manhattan.
Kansas 66502
Received December 13, 1991, and in revised form April 3, 1992
A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of -60 dihydrolipoyl transacetylase (EZ) subunits, -6 protein X subunits, and -2 pyruvate dehydrogenase kinase heterodimers (KKb). Using a preparation of resolved kinase in which K, 4 Kb, EP-X-KCKt, subcomplex additionally bound at least 15 catalytic subunits of the kinase (K,) and a much lower level of Kb. The binding of K, to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-K,Kt, subcomplex. This pattern of binding K, but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the K, subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the
1 To whom correspondence should be addressed. * Abbreviations used: PDC, pyruvate dehydrogenase complex; El, pyruvate dehydrogenase component; E2, dihydrolipoyl acetylatransferase component; E21, COOH-terminal, transacetylase-catalyzing, oligomerforming inner domain of E2; E2s, El-binding domain of E2; E2L,, NHterminal lipoyl domain; E2r,, , inner lipoyl domain of E2; E2L, fragment containing E2~r, E2Lz. and COOH-linked hinge regions of these domains; E3, dihydrolipoyl dehydrogenase component; K,, catalytic subunit of Ela kinase; Kb, basic subunit of the kinase; X, protein X or component X, XI, inner domain of protein X; McAb, monoclonal antibody; H, McAb heavy chain; L, McAb light chain; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid. 0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
kinase (M. Rahmatullah et aE., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugationyielded an EaL-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2. 0 1992 Academic PWS, 1~.
The mammahan pyruvate dehydrogenase compiex (PDC)’ has a structural core composed of two lipoylbearing components-the dihydrolipoyl transacetylase (E2) and protein X. These components bind the other cardinal components (components required for the overall reaction catalyzed by the complex) as well as the regulatory enzymes (l-8). The E2 component contains a COOH-terminal inner domain (Ear) and an extended NHa-terminal structure (9) composed of three domains with connecting hinge regions between each of the four domains (10, 11). Connected to E2r is a domain (designated E2s) that binds the pyruvate dehydrogenase (El) component (4, B), then two lipoyl domains with the outermost (NH*-terminal) domain being designated E2L1 and the lipoyl domain between E2L1 and E2s domains being designated E2Lz. The protein X component also contains a lipoyl domain and an inner domain structure (XI) (4, 5,12, 13). Mammalian protein X binds the dihydrolipoyl dehydrogenase (E3) component (4, 6, 7). The X component of yeast PDC contains a linker region-connected three-domain structure, with a domain between the lipoyl domain and the inner domain that binds the E3 component of yeast PDC (13, 14). The pattern of proteolytic cleavage of the mammalian protein X (3) is consistent with such a three-domain structure. The inner domain of 491
Inc. reserved.
498
LI ET AL.
mammalian protein X associates with the inner domain of the transacetylase (3) and together these form an oligomer apparently composed of about 60 E2r and about 6 Xr domains. The location for binding of the XI domain to the dodecahedron structure formed by association of E2r domain has not been demonstrated. The initial process of resolution of the bovine kidney complex yields a subcomplex (M, N 4 X 106)that contains about two pyruvate dehydrogenase kinase dimers (K,K,) in addition to the E2 and X subunits. A further step can remove the kinase and some protein X (15, 16). Reassociation of the kinase with the E2-X subcomplex causes a large increase in kinase activity (15-18). Here we evaluate whether resolved kinase and resolved protein X (functional in the binding of the E3 component) can bind to the assembled EB-X-K,Kb subcomplex, the assembled E2 oligomer greatly reduced in the content of protein X (6), or the EB-Xr-K,(KJ subcomplex in which the lipoyl domain of protein X was selectively removed (5). The addition of resolved X to the latter XI-containing subcomplex not only allowed the possibility of binding additional protein X but the possible exchange of XI domains and protein X to be evaluated. The particular fraction of resolved kinase employed was one with a high K:Kb ratio, allowing the dependence of binding and activation of the catalytic subunits (K,) on the presence of the basic subunit (Kb) to be evaluated. Rahmatullah et al. (8) have shown that selective and complete release of an E2L fragment (composed of E2L1 and E2L2 plus the first and second hinge regions) also released the kinase subunits (K,K,). That suggests the binding site for the kinase resides in the E2L fragment and probably involves an association with one of the two lipoyl domains. Furthermore, assuming all E2L regions of E2 subunits are equivalent, this predicts that there should be a capacity for binding more than the two to three K,K,, dimers associated with the purified complex. Here we evaluate whether there is a higher binding capacity that explains the capacity of the subcomplex to greatly enhance the activity of a large number of kinase molecules (18). We eliminate the possibility (18) that activation and binding may have depended on the presence of protein X in the kinase fraction. Finally, we determine whether the kinase remains tightly associated with the E2L fragment. EXPERIMENTAL
PROCEDURES
Materials. Minor modifications of standard procedures were used to prepare the bovine kidney pyruvate dehydrogenase complex (19), the EZ-X-KKb subcomplex and El component (l), and the X, KK, fraction along with the E2-X subcomplex (15,16). The E2 oligomer with essentially all the protein X and kinase subunits removed was prepared by
3 After the minimum period required for cleavage of protein X, a portion (~40%) of the Kb subunit as well as a small portion of the E2 subunits ( to interact with intact E2 seems to be required. Since protein X was first characterized as a new component of the mammalian pyruvate dehydrogenase complex (12, 31), the nature of its tight association with the E2 component has not been understood. The lack of binding of protein X to the assembled E2 oligomer either results because protein X must bind during assembly or protein X was damaged during the reaction of E2-X-kinase subcomplex with the mercurial agent used in its release. That preparation procedure involves reacting reduced lipoyl moieties with p-hydroxymercuriphenyl sulfonate and, after isolation of the X, K,K,, fraction, exhaustive dialysis with dithiothreitol and EDTA to remove the mercurial agent (16). The protein X, which constituted less than 30% of the protein of this fraction, bound E3 efficiently (cf. Results). Thus, protein X was native in the region of its structure that binds E3. Its lipoyl domain was also in a native state since it served as an efficient substrate for El-catalyzed reductive acetylation reaction (16). That reaction requires structural integrity of the lipoyl domain and does not occur with noncognate lipoyl domains (e.g., 32). We could not establish whether the inner domain of protein X, which binds to E2’s inner domain (3), was in a native state since there is no other ‘T. E. Roche, S. L. Powers-Greenwood, W. F. Shi, W. B. Zhang, S. Z. Ren, E. D. Roche, D. J. Cox, and C. M. Sorensen, submitted for publication. ’ K. Ono, G. A. Radke, and T. E. Roche, unpublished observation.
504
LI ET AL.
known function to be evaluated. It is very interesting that coexpression of yeast E2 and yeast protein X under conditions that led to excess production of protein X did not result in an increased level of incorporation of X into yeast complex (14). Lawson et al. (14) did report that mixing of dilute E2 with crude fractions containing protein X led to reconstitution of some PDC activity. That clearly supports binding of a few protein X molecules, but the possibility of some partial assembly or disassembly of dilute E2 under these conditions was not eliminated. The basis for the binding of only about 6 protein X to 60 subunits of E2 remains an interesting problem since the lowest symmetry element in the dodecahedron structure formed by assembled E2 is 12 faces. We have found that the catalytic subunit of the kinase is specifically and tightly bound to the lipoyl domain region of E2 subunits. Since the activity of only a couple molecules of kinase can rapidly inactivate the bovine kidney complex, it is not surprising that about this level of kinase is found in the isolated complex. The finding of an expanded capacity to bind the kinase suggests that low number results from control in the production of kinase rather than an inherent limitation in the number of binding sites. The structural feature that limits the assembled E2 oligomer to binding only one kinase per three subunits needs to be determined and may give new insights into the nature of kinase binding and function. As in the case of protein X binding which appears to be limited to five to six per core, these results suggest some form of existing or induced asymmetry in the assembled core. ACKNOWLEDGMENTS We thank Connie Schmidt for help in preparation of the manuscript. This work was supported by National Institutes of Health Grant DK8320 and by Kansas State Agriculture Experiment Station Contribution 92298J.
REFERENCES 1. Linn, T. C., Pelley, J. W., Pettit, F. H., Hucho, F., Randall, D. D., and Reed, L. S. (1972) Arch. Biochem. Biophys. 148,327-342. 2. Pettit, F. H., Rocbe, T. E., and Reed, L. J. (1972) Bioc&rrz. Biophys. Res. Commun. 49, 563-571. 3. Rahmatullah, M., Gopalakrishnan, S., Radke, G. A., and Roche, T. E. (1989) J. Biol. Chem. 264, 12451251. 4. Rahmatullah, M., Gopalakrishnan, S., Andrews, P. C., Chang, C. L., Radke, G. A., and Roche, T. E. (1989) J. Biol. Chem. 264, 2221-2227. 5. Rahmatullah, 8110.
M., and Roche, T. E. (1988) J. Bial. &em. 263,8106-
6. Powers-Greenwood, S. L., Rahmatullah, M., Radke, G. A., and Roche, T. E. (1989) J. Biol. Chem. 264, 3655-3657. 7. Gopalakrishnan, S., Rahmatullah, M., Radke, G. A., PowersGreenwood, S. L., and Roche, T. E. (1989) B&hem. Biophys. Res. Commun. 160,715-721. 8. Rahmatullah, M., Radke, G. A., Powers-Greenwood, S. L., Andrews, P. C., and Roche, T. E. (1990) J. Biol. Chcm. 265, 14,512-14,517. 9. Bleile, D. M., Hackert, M. L., Pettit, F. H., and Reed, L. J. (1981) J. Biol. Chem. 256, 514-519. 10. Coppel, R. L., McNeilage, L. J., Surh, C. D., Van de Water, J., Spithill, T. W., Whittingham, S., and Gershwin, M. E. (1988) Z+OC. Natl. Acad. Sci. USA 85, 7317-7321. T. J., Ho, L., Wexler, I. D., Pons, G., Liu, T. C., and 11. Thekkumkara, Patel, M. S. (1988) FEBS Lett. 240, 45-58. 12. Jilka, J. M., Rahmatullah, M., Kazemi, M., and Roche, T. E. (1986) J. Biol. Chem. 261,1858-1867. 13. Nu, Y-D., Stoops, J. K., and Reed, L. J. (1990) Biochemistry 29, 8614-8619. 14. Lawson, J. E., Behal, R. H., and Reed, L. J. (1991) Biochemistry 30,2834-2839. 15. Stepp, L. R., Pettit, F. H., Yeaman, S. J., and Reed, L. J. (1983) J. Biol. Chem. 258,9454-9458. M., and Roche, T. E. (1987) J. Biol. Chem. 262, 16. Rahmatullah, 10,265-10,271. 17. Hucho, F., Randall, D. D., Roche, T. E., Burgett, M. W., Pelley, J. W., and Reed, L. J. (1972) Arch. Biochem. Biophys. 157, 328340. 18. Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261,6515-6523. 19. Laemmli, U. K. (1970) Nature 227,680-685. 20. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Biochem.
105,361-353. M., and Roche, T. E. (1985) J. Biol. Chem. 260, 21. Rahmatullah, 10,146-10,152. 22. Pratt, M. L., and Roche, T. E. (1979) J. Biol. Chem. 254, 71917196. P. J., Tsai, C. S., Eley, M. H., Roche, T. E., and Reed, 23. Butterworth, L. J. (1975) J. Biol. Chem. 250, 1921-1925. A., and Gershwin, M. E. (1990) J. Zm24. Surh, C. D., Ahmed-Ansari, munol. 144,2647-2652. 25. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sidman, J. G., Smith, J. A., and Struhl, K. (1988) Current Protocols in Immunology, Suppl. 1, Chap. II, Breene/Wiley-Interscience, New York. 26. Nau, D. R. (1989) Biochromatography 4,4-18. 27. Cress, M. C., and Ngo, T. T. (1989) Am. Biotechnol. Lab. 7, 16-19. 28. Patek M., and Roche, T. E. (1990) FASEB J. 4, 3224-3233. 29. Li, L. (1988) M. S. thesis, Kansas State University, Manhattan, KS. 30. Liu, S., and Roche, T. E. (1992) FASEB J. 6, A460. 31. DeMarcucci, D. L., and Lindsay, J. G. (1985) Eur. J. Biochem. 149, 641-648. 32. Perham, R. N., and Packman, L. C. (1989) Ann. Reu. N. Y. Acad. Sci. 573, l-20. 33. Fried, V. A., Ando, M. E., and Bell, A. J. (1985) Anal. Biochem.
146,271-276.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 296, No. 2, August 1, pp. 497-504, 1992
Additional Binding Sites for the Pyruvate Dehydrogenase Kinase but Not for Protein X in the Assembled Core of the Mammalian Pyruvate Dehydrogenase Complex: Binding Region for the Kinase Lin Li, Gary A. Radke, Kazuo Ono, and Thomas Department
of Biochemistry,
Kansas State University,
E. Roche’
Manhattan.
Kansas 66502
Received December 13, 1991, and in revised form April 3, 1992
A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of -60 dihydrolipoyl transacetylase (EZ) subunits, -6 protein X subunits, and -2 pyruvate dehydrogenase kinase heterodimers (KKb). Using a preparation of resolved kinase in which K, 4 Kb, EP-X-KCKt, subcomplex additionally bound at least 15 catalytic subunits of the kinase (K,) and a much lower level of Kb. The binding of K, to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-K,Kt, subcomplex. This pattern of binding K, but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the K, subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the
1 To whom correspondence should be addressed. * Abbreviations used: PDC, pyruvate dehydrogenase complex; El, pyruvate dehydrogenase component; E2, dihydrolipoyl acetylatransferase component; E21, COOH-terminal, transacetylase-catalyzing, oligomerforming inner domain of E2; E2s, El-binding domain of E2; E2L,, NHterminal lipoyl domain; E2r,, , inner lipoyl domain of E2; E2L, fragment containing E2~r, E2Lz. and COOH-linked hinge regions of these domains; E3, dihydrolipoyl dehydrogenase component; K,, catalytic subunit of Ela kinase; Kb, basic subunit of the kinase; X, protein X or component X, XI, inner domain of protein X; McAb, monoclonal antibody; H, McAb heavy chain; L, McAb light chain; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid. 0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
kinase (M. Rahmatullah et aE., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugationyielded an EaL-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2. 0 1992 Academic PWS, 1~.
The mammahan pyruvate dehydrogenase compiex (PDC)’ has a structural core composed of two lipoylbearing components-the dihydrolipoyl transacetylase (E2) and protein X. These components bind the other cardinal components (components required for the overall reaction catalyzed by the complex) as well as the regulatory enzymes (l-8). The E2 component contains a COOH-terminal inner domain (Ear) and an extended NHa-terminal structure (9) composed of three domains with connecting hinge regions between each of the four domains (10, 11). Connected to E2r is a domain (designated E2s) that binds the pyruvate dehydrogenase (El) component (4, B), then two lipoyl domains with the outermost (NH*-terminal) domain being designated E2L1 and the lipoyl domain between E2L1 and E2s domains being designated E2Lz. The protein X component also contains a lipoyl domain and an inner domain structure (XI) (4, 5,12, 13). Mammalian protein X binds the dihydrolipoyl dehydrogenase (E3) component (4, 6, 7). The X component of yeast PDC contains a linker region-connected three-domain structure, with a domain between the lipoyl domain and the inner domain that binds the E3 component of yeast PDC (13, 14). The pattern of proteolytic cleavage of the mammalian protein X (3) is consistent with such a three-domain structure. The inner domain of 491
Inc. reserved.
498
LI ET AL.
mammalian protein X associates with the inner domain of the transacetylase (3) and together these form an oligomer apparently composed of about 60 E2r and about 6 Xr domains. The location for binding of the XI domain to the dodecahedron structure formed by association of E2r domain has not been demonstrated. The initial process of resolution of the bovine kidney complex yields a subcomplex (M, N 4 X 106)that contains about two pyruvate dehydrogenase kinase dimers (K,K,) in addition to the E2 and X subunits. A further step can remove the kinase and some protein X (15, 16). Reassociation of the kinase with the E2-X subcomplex causes a large increase in kinase activity (15-18). Here we evaluate whether resolved kinase and resolved protein X (functional in the binding of the E3 component) can bind to the assembled EB-X-K,Kb subcomplex, the assembled E2 oligomer greatly reduced in the content of protein X (6), or the EB-Xr-K,(KJ subcomplex in which the lipoyl domain of protein X was selectively removed (5). The addition of resolved X to the latter XI-containing subcomplex not only allowed the possibility of binding additional protein X but the possible exchange of XI domains and protein X to be evaluated. The particular fraction of resolved kinase employed was one with a high K:Kb ratio, allowing the dependence of binding and activation of the catalytic subunits (K,) on the presence of the basic subunit (Kb) to be evaluated. Rahmatullah et al. (8) have shown that selective and complete release of an E2L fragment (composed of E2L1 and E2L2 plus the first and second hinge regions) also released the kinase subunits (K,K,). That suggests the binding site for the kinase resides in the E2L fragment and probably involves an association with one of the two lipoyl domains. Furthermore, assuming all E2L regions of E2 subunits are equivalent, this predicts that there should be a capacity for binding more than the two to three K,K,, dimers associated with the purified complex. Here we evaluate whether there is a higher binding capacity that explains the capacity of the subcomplex to greatly enhance the activity of a large number of kinase molecules (18). We eliminate the possibility (18) that activation and binding may have depended on the presence of protein X in the kinase fraction. Finally, we determine whether the kinase remains tightly associated with the E2L fragment. EXPERIMENTAL
PROCEDURES
Materials. Minor modifications of standard procedures were used to prepare the bovine kidney pyruvate dehydrogenase complex (19), the EZ-X-KKb subcomplex and El component (l), and the X, KK, fraction along with the E2-X subcomplex (15,16). The E2 oligomer with essentially all the protein X and kinase subunits removed was prepared by
3 After the minimum period required for cleavage of protein X, a portion (~40%) of the Kb subunit as well as a small portion of the E2 subunits ( to interact with intact E2 seems to be required. Since protein X was first characterized as a new component of the mammalian pyruvate dehydrogenase complex (12, 31), the nature of its tight association with the E2 component has not been understood. The lack of binding of protein X to the assembled E2 oligomer either results because protein X must bind during assembly or protein X was damaged during the reaction of E2-X-kinase subcomplex with the mercurial agent used in its release. That preparation procedure involves reacting reduced lipoyl moieties with p-hydroxymercuriphenyl sulfonate and, after isolation of the X, K,K,, fraction, exhaustive dialysis with dithiothreitol and EDTA to remove the mercurial agent (16). The protein X, which constituted less than 30% of the protein of this fraction, bound E3 efficiently (cf. Results). Thus, protein X was native in the region of its structure that binds E3. Its lipoyl domain was also in a native state since it served as an efficient substrate for El-catalyzed reductive acetylation reaction (16). That reaction requires structural integrity of the lipoyl domain and does not occur with noncognate lipoyl domains (e.g., 32). We could not establish whether the inner domain of protein X, which binds to E2’s inner domain (3), was in a native state since there is no other ‘T. E. Roche, S. L. Powers-Greenwood, W. F. Shi, W. B. Zhang, S. Z. Ren, E. D. Roche, D. J. Cox, and C. M. Sorensen, submitted for publication. ’ K. Ono, G. A. Radke, and T. E. Roche, unpublished observation.
504
LI ET AL.
known function to be evaluated. It is very interesting that coexpression of yeast E2 and yeast protein X under conditions that led to excess production of protein X did not result in an increased level of incorporation of X into yeast complex (14). Lawson et al. (14) did report that mixing of dilute E2 with crude fractions containing protein X led to reconstitution of some PDC activity. That clearly supports binding of a few protein X molecules, but the possibility of some partial assembly or disassembly of dilute E2 under these conditions was not eliminated. The basis for the binding of only about 6 protein X to 60 subunits of E2 remains an interesting problem since the lowest symmetry element in the dodecahedron structure formed by assembled E2 is 12 faces. We have found that the catalytic subunit of the kinase is specifically and tightly bound to the lipoyl domain region of E2 subunits. Since the activity of only a couple molecules of kinase can rapidly inactivate the bovine kidney complex, it is not surprising that about this level of kinase is found in the isolated complex. The finding of an expanded capacity to bind the kinase suggests that low number results from control in the production of kinase rather than an inherent limitation in the number of binding sites. The structural feature that limits the assembled E2 oligomer to binding only one kinase per three subunits needs to be determined and may give new insights into the nature of kinase binding and function. As in the case of protein X binding which appears to be limited to five to six per core, these results suggest some form of existing or induced asymmetry in the assembled core. ACKNOWLEDGMENTS We thank Connie Schmidt for help in preparation of the manuscript. This work was supported by National Institutes of Health Grant DK8320 and by Kansas State Agriculture Experiment Station Contribution 92298J.
REFERENCES 1. Linn, T. C., Pelley, J. W., Pettit, F. H., Hucho, F., Randall, D. D., and Reed, L. S. (1972) Arch. Biochem. Biophys. 148,327-342. 2. Pettit, F. H., Rocbe, T. E., and Reed, L. J. (1972) Bioc&rrz. Biophys. Res. Commun. 49, 563-571. 3. Rahmatullah, M., Gopalakrishnan, S., Radke, G. A., and Roche, T. E. (1989) J. Biol. Chem. 264, 12451251. 4. Rahmatullah, M., Gopalakrishnan, S., Andrews, P. C., Chang, C. L., Radke, G. A., and Roche, T. E. (1989) J. Biol. Chem. 264, 2221-2227. 5. Rahmatullah, 8110.
M., and Roche, T. E. (1988) J. Bial. &em. 263,8106-
6. Powers-Greenwood, S. L., Rahmatullah, M., Radke, G. A., and Roche, T. E. (1989) J. Biol. Chem. 264, 3655-3657. 7. Gopalakrishnan, S., Rahmatullah, M., Radke, G. A., PowersGreenwood, S. L., and Roche, T. E. (1989) B&hem. Biophys. Res. Commun. 160,715-721. 8. Rahmatullah, M., Radke, G. A., Powers-Greenwood, S. L., Andrews, P. C., and Roche, T. E. (1990) J. Biol. Chcm. 265, 14,512-14,517. 9. Bleile, D. M., Hackert, M. L., Pettit, F. H., and Reed, L. J. (1981) J. Biol. Chem. 256, 514-519. 10. Coppel, R. L., McNeilage, L. J., Surh, C. D., Van de Water, J., Spithill, T. W., Whittingham, S., and Gershwin, M. E. (1988) Z+OC. Natl. Acad. Sci. USA 85, 7317-7321. T. J., Ho, L., Wexler, I. D., Pons, G., Liu, T. C., and 11. Thekkumkara, Patel, M. S. (1988) FEBS Lett. 240, 45-58. 12. Jilka, J. M., Rahmatullah, M., Kazemi, M., and Roche, T. E. (1986) J. Biol. Chem. 261,1858-1867. 13. Nu, Y-D., Stoops, J. K., and Reed, L. J. (1990) Biochemistry 29, 8614-8619. 14. Lawson, J. E., Behal, R. H., and Reed, L. J. (1991) Biochemistry 30,2834-2839. 15. Stepp, L. R., Pettit, F. H., Yeaman, S. J., and Reed, L. J. (1983) J. Biol. Chem. 258,9454-9458. M., and Roche, T. E. (1987) J. Biol. Chem. 262, 16. Rahmatullah, 10,265-10,271. 17. Hucho, F., Randall, D. D., Roche, T. E., Burgett, M. W., Pelley, J. W., and Reed, L. J. (1972) Arch. Biochem. Biophys. 157, 328340. 18. Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261,6515-6523. 19. Laemmli, U. K. (1970) Nature 227,680-685. 20. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Biochem.
105,361-353. M., and Roche, T. E. (1985) J. Biol. Chem. 260, 21. Rahmatullah, 10,146-10,152. 22. Pratt, M. L., and Roche, T. E. (1979) J. Biol. Chem. 254, 71917196. P. J., Tsai, C. S., Eley, M. H., Roche, T. E., and Reed, 23. Butterworth, L. J. (1975) J. Biol. Chem. 250, 1921-1925. A., and Gershwin, M. E. (1990) J. Zm24. Surh, C. D., Ahmed-Ansari, munol. 144,2647-2652. 25. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sidman, J. G., Smith, J. A., and Struhl, K. (1988) Current Protocols in Immunology, Suppl. 1, Chap. II, Breene/Wiley-Interscience, New York. 26. Nau, D. R. (1989) Biochromatography 4,4-18. 27. Cress, M. C., and Ngo, T. T. (1989) Am. Biotechnol. Lab. 7, 16-19. 28. Patek M., and Roche, T. E. (1990) FASEB J. 4, 3224-3233. 29. Li, L. (1988) M. S. thesis, Kansas State University, Manhattan, KS. 30. Liu, S., and Roche, T. E. (1992) FASEB J. 6, A460. 31. DeMarcucci, D. L., and Lindsay, J. G. (1985) Eur. J. Biochem. 149, 641-648. 32. Perham, R. N., and Packman, L. C. (1989) Ann. Reu. N. Y. Acad. Sci. 573, l-20. 33. Fried, V. A., Ando, M. E., and Bell, A. J. (1985) Anal. Biochem.
146,271-276.
