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• W1975171610 abstract "Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 μm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k catalong with about a 2-fold decrease in the K m of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g.using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2–60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated ≥3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors. Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 μm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k catalong with about a 2-fold decrease in the K m of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g.using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2–60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated ≥3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors. pyruvate dehydrogenase complex pyruvate dehydrogenase kinase pyruvate dehydrogenase dihydrolipoyl acetyltransferase N-terminal lipoyl domain of E2 inner lipoyl domain of E2 dihydrolipoyl dehydrogenase E3-binding protein (formerly protein X) lipoyl domain of E3BP glutathione S-transferase polyacrylamide gel electrophoresis pyruvate dehydrogenase phosphatase 3-(N-morpholino)propanesulfonic acid dichloroacetate dithiothreitol thiamine pyrophosphate The pyruvate dehydrogenase complex (PDC)1 catalyzes the irreversible conversion of pyruvate to acetyl-CoA and NADH with the departure of CO2. The inactivation of PDC by phosphorylation (1.Linn T.C. Pettit F.H. Reed L.J. Proc. Natl. Acad. Sci. U. S. A. 1969; 62: 234-241Crossref PubMed Scopus (518) Google Scholar) limits the commitment of glucose-connected fuels to undergoing complete oxidation or to being transformed to fatty acids (2.Randel P.J. Biochem. Soc. Trans. 1986; 14: 799-806Crossref PubMed Scopus (240) Google Scholar). The fractional PDC activity is set by the competing steady state activities of two classes of dedicated enzymes, the pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). These highly regulated enzymes catalyze the phosphorylation and dephosphorylation, respectively, of the pyruvate dehydrogenase (E1) component of PDC (1.Linn T.C. Pettit F.H. Reed L.J. Proc. Natl. Acad. Sci. U. S. A. 1969; 62: 234-241Crossref PubMed Scopus (518) Google Scholar, 2.Randel P.J. Biochem. Soc. Trans. 1986; 14: 799-806Crossref PubMed Scopus (240) Google Scholar, 3.Patel M.S. Roche T.E. FASEB J. 1990; 4: 3224-3233Crossref PubMed Scopus (500) Google Scholar). PDC is regulated by at least four related PDK isoforms (4.Popov K.M. Kedishvili N.Y. Zhao Y. Shimomura Y. Crabb D.W. Harris R.A. J. Biol. Chem. 1993; 268: 26602-26606Abstract Full Text PDF PubMed Google Scholar, 5.Popov K.M. Kedishvili N.Y. Zhao Y. Gudi R. Harris R.A. J. Biol. Chem. 1994; 269: 29720-29724Abstract Full Text PDF PubMed Google Scholar, 6.Gudi R. Bowker-Kinley M.M. Kedishvili N.Y. Zhao Y. Popov K.M. J. Biol. Chem. 1995; 270: 28989-28994Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 7.Rowles J. Scherer S.W. Xi T. Majer M. Nickle D.C. Rommens J.M. Popov K.M. Harris R.A. Riebow N.L. Xia J. Tsui L.-C. Bogardus C. Prochazka M. J. Biol. Chem. 1996; 271: 22376-22382Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and two PDP isoforms, with related catalytic subunits (8.Huang B. Gudi R. Wu P. Harris R.A. Hamilton J. Popov K.M. J. Biol. Chem. 1998; 273: 17680-17688Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The PDK isozymes and the related branched chain dehydrogenase kinase (9.Popov K.M. Zhao Y. Shimomura Y. Kuntz M.J. Harris R.A. J. Biol. Chem. 1992; 267: 13127-13130Abstract Full Text PDF PubMed Google Scholar) form a unique family of serine kinases. They are distantly related to bacterial histidine kinases, sharing five conserved motifs (4.Popov K.M. Kedishvili N.Y. Zhao Y. Shimomura Y. Crabb D.W. Harris R.A. J. Biol. Chem. 1993; 268: 26602-26606Abstract Full Text PDF PubMed Google Scholar, 5.Popov K.M. Kedishvili N.Y. Zhao Y. Gudi R. Harris R.A. J. Biol. Chem. 1994; 269: 29720-29724Abstract Full Text PDF PubMed Google Scholar, 6.Gudi R. Bowker-Kinley M.M. Kedishvili N.Y. Zhao Y. Popov K.M. J. Biol. Chem. 1995; 270: 28989-28994Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The bacterial kinases form a stable histidine-phosphate intermediate and generally transfer this phosphate to an aspartic acid side chain (10.Stock J.B. Ninfa A.J. Stock A.M. Microbiol. Rev. 1989; 53: 450-490Crossref PubMed Google Scholar, 11.Parkinson J.S. Kafoid E.C. Annu. Rev. Genet. 1992; 26: 71-112Crossref PubMed Scopus (1244) Google Scholar). The mitochondrial kinases phosphorylate serine substrates without forming a stable histidine phosphate; indeed, slow (relative to kinase turnover) autophosphorylation of a serine and no autophosphorylation of the conserved histidine was found with the branched chain kinase (12.Davie J.R. Wynn R.M. Meng M. Huang Y. Aalund G. Chuang D.T. Lau K.S. J. Biol. Chem. 1995; 270: 19861-19867Abstract Full Text PDF PubMed Scopus (51) Google Scholar). The availability of the individual PDK isoforms allows determination of their unique functional and regulatory properties, a step toward understanding how the required tissue-specific regulation of PDC activity is achieved. Kinase-catalyzed inactivation of PDC plays a key role in limiting glucose oxidation when more abundant fatty acids are used to provide oxidative energy (2.Randel P.J. Biochem. Soc. Trans. 1986; 14: 799-806Crossref PubMed Scopus (240) Google Scholar). This routinely occurs in many tissues but is particularly important during starvation (2.Randel P.J. Biochem. Soc. Trans. 1986; 14: 799-806Crossref PubMed Scopus (240) Google Scholar, 13.Kerbey A.L. Randle P.J. Cooper R.H. Whitehouse S. Pask H.T. Denton R.M. Biochem. J. 1976; 154: 327-348Crossref PubMed Scopus (271) Google Scholar, 14.Denyer G.S. Kerbey A.L. Randle P.J. Biochem. J. 1986; 239: 347-354Crossref PubMed Scopus (56) Google Scholar, 15.Hutson N.J. Randle P.J. FEBS Lett. 1978; 92: 73-76Crossref PubMed Scopus (52) Google Scholar, 16.Sugden M.C. Orfali K.A. Fryer L.G.D. Holness M.J. Priestman D.A. J. Mol. Cell. Cardiol. 1997; 29: 1867-1875Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 17.Randle P.J. Priestman D.A. Patel M.S. Roche T.E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 151-161Crossref Google Scholar, 18.Kerbey A.L. Richardson L.J. Randle P.J. FEBS Lett. 1984; 176: 115-119Crossref PubMed Scopus (27) Google Scholar), when limited glucose must be conserved for glucose-utilizing tissues such as brain. PDC is similarly down-regulated due to high PDK activity in the diabetic state (2.Randel P.J. Biochem. Soc. Trans. 1986; 14: 799-806Crossref PubMed Scopus (240) Google Scholar, 15.Hutson N.J. Randle P.J. FEBS Lett. 1978; 92: 73-76Crossref PubMed Scopus (52) Google Scholar, 16.Sugden M.C. Orfali K.A. Fryer L.G.D. Holness M.J. Priestman D.A. J. Mol. Cell. Cardiol. 1997; 29: 1867-1875Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 17.Randle P.J. Priestman D.A. Patel M.S. Roche T.E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 151-161Crossref Google Scholar, 18.Kerbey A.L. Richardson L.J. Randle P.J. FEBS Lett. 1984; 176: 115-119Crossref PubMed Scopus (27) Google Scholar, 19.Stanley W.C. Lopaschuk G.D. McCormack J.G. Cardiovas. Res. 1997; 34: 25-33Crossref PubMed Scopus (406) Google Scholar, 20.Sugden M.C. Holness M.J. Biochem. Soc. Trans. 1995; 23: 314-320Crossref PubMed Scopus (6) Google Scholar, 21.Sugden M.C. Orfali K.A. Holness M.J. J. Nutr. 1995; 125: 1746-1752PubMed Google Scholar, 22.Wu P. Sato J. Zhao Y. Jaskiewicz J. Popov K.M. Harris R.A. Biochem. J. 1998; 329: 197-201Crossref PubMed Scopus (263) Google Scholar). In both cases, this occurs with PDK overexpression (1.Linn T.C. Pettit F.H. Reed L.J. Proc. Natl. Acad. Sci. U. S. A. 1969; 62: 234-241Crossref PubMed Scopus (518) Google Scholar, 14.Denyer G.S. Kerbey A.L. Randle P.J. Biochem. J. 1986; 239: 347-354Crossref PubMed Scopus (56) Google Scholar, 15.Hutson N.J. Randle P.J. FEBS Lett. 1978; 92: 73-76Crossref PubMed Scopus (52) Google Scholar, 16.Sugden M.C. Orfali K.A. Fryer L.G.D. Holness M.J. Priestman D.A. J. Mol. Cell. Cardiol. 1997; 29: 1867-1875Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 17.Randle P.J. Priestman D.A. Patel M.S. Roche T.E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 151-161Crossref Google Scholar, 18.Kerbey A.L. Richardson L.J. Randle P.J. FEBS Lett. 1984; 176: 115-119Crossref PubMed Scopus (27) Google Scholar, 19.Stanley W.C. Lopaschuk G.D. McCormack J.G. Cardiovas. Res. 1997; 34: 25-33Crossref PubMed Scopus (406) Google Scholar, 20.Sugden M.C. Holness M.J. Biochem. Soc. Trans. 1995; 23: 314-320Crossref PubMed Scopus (6) Google Scholar, 21.Sugden M.C. Orfali K.A. Holness M.J. J. Nutr. 1995; 125: 1746-1752PubMed Google Scholar, 22.Wu P. Sato J. Zhao Y. Jaskiewicz J. Popov K.M. Harris R.A. Biochem. J. 1998; 329: 197-201Crossref PubMed Scopus (263) Google Scholar). When fatty acid oxidation is not used by a tissue or when glucose is being converted to fatty acids, the activity of PDC must be regulated very differently. Consequently, PDC limits the nearly exclusive use of glucose as an oxidative energy source in neural tissues and facilitates the conversion of glucose to fatty acids in adipose tissue when there is surplus glucose. Thus, it seems likely that different PDK (and PDP) isoforms have developed to meet the distinct tissue-specific and metabolic state-specific requirements for proper tuning of PDC activity. Variation in the distribution of specific mRNAs for the PDK isoforms supports this conclusion (22.Wu P. Sato J. Zhao Y. Jaskiewicz J. Popov K.M. Harris R.A. Biochem. J. 1998; 329: 197-201Crossref PubMed Scopus (263) Google Scholar, 23.Bowker-Kinley M.M. Davis W.I. Wu P. Harris R.A. Popov K.M. Biochem. J. 1998; 329: 191-196Crossref PubMed Scopus (447) Google Scholar, 24.Wu P. InsKeep K. Jaskiewicz J. Popov K.M. Harris R.A. Diabetes. 1998; 47 (abstr.): 425Google Scholar). The organization of PDC plays an important role in supporting PDC activity and in the regulation of PDC by PDKs and PDPs. The core structure of PDC is formed by association of 60 dihydrolipoyl acetyltransferase (E2) subunits. E2 is a segmented protein with four domains connected by mobile linker regions (25.Thekkumkara T.J. Ho L. Wexler I.D. Pons G. Lui T.-C. Patel M.S. FEBS Lett. 1988; 240: 45-48Crossref PubMed Scopus (74) Google Scholar, 26.Perham R.N. Biochemistry. 1991; 30: 8501-8512Crossref PubMed Scopus (365) Google Scholar). Twenty trimers of the C-terminal inner domain of E2 assemble at the vertices of dodecahedron; these trimers catalyze the transacetylation reaction (27.Reed L.J. Hackert M.L. J. Biol. Chem. 1990; 265: 8971-8974Abstract Full Text PDF PubMed Google Scholar,28.Wagenknecht T. Grassucci R. Radke G.A. Roche T.E. J. Biol. Chem. 1991; 266: 24650-24656Abstract Full Text PDF PubMed Google Scholar). Via linker regions, this inner core domain is first connected to an E1-binding domain and then two lipoate-bearing domains, an inner domain (L2) and an N-terminal domain (L1). The dihydrolipoyl dehydrogenase-binding proteins (E3BP) has a similar segmented structure (29.Harris R.A. Bowker-Kinley M.M. Wu P. Jeng J. Popov K.M. J. Biol. Chem. 1997; 272: 19746-19751Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30.Rahmatullah M. Gopalakrishnan S. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 1245-1251Abstract Full Text PDF PubMed Google Scholar); about 12 E3BP associates via its C-terminal domain with the inner core of E2 (29.Harris R.A. Bowker-Kinley M.M. Wu P. Jeng J. Popov K.M. J. Biol. Chem. 1997; 272: 19746-19751Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30.Rahmatullah M. Gopalakrishnan S. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 1245-1251Abstract Full Text PDF PubMed Google Scholar, 31.Lawson J.E. Behal R.H. Reed L.J. Biochemistry. 1991; 30: 2834-2839Crossref PubMed Scopus (64) Google Scholar, 32.Maeng C.-Y. Yazdi M.A. Niu X.-D. Lee H.Y. Reed L.J. Biochemistry. 1994; 33: 13801-13807Crossref PubMed Scopus (42) Google Scholar). E3BP then contains an E3-binding domain and a lipoyl domain (designated L3) set off by linker regions. The assembled E2-E3BP are estimated to bind 6–12 E3 and 20–30 E1 α2β2 tetramers (3.Patel M.S. Roche T.E. FASEB J. 1990; 4: 3224-3233Crossref PubMed Scopus (500) Google Scholar). Bovine PDK and PDP activities have also been shown to be markedly enhanced via binding to this central E2-E3BP core structure via the lipoyl domain region of E2 (30.Rahmatullah M. Gopalakrishnan S. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 1245-1251Abstract Full Text PDF PubMed Google Scholar, 34.Rahmatullah M. Roche T.E. J. Biol. Chem. 1988; 263: 8106-8110Abstract Full Text PDF PubMed Google Scholar, 35.Rahmatullah M. Radke G.A. Powers-Greenwood S.L. Andrews P.C. Roche T.E. J. Biol. Chem. 1990; 265: 14512-14517Abstract Full Text PDF PubMed Google Scholar). By using recombinant constructs of the L1 and L2 domains of E2, unspecified isoforms of bovine PDK were shown to bind preferentially to the L2 domain via an interaction that requires the lipoyl prosthetic group (36.Liu S. Baker J.C. Roche T.E. J. Biol. Chem. 1995; 270: 793-800Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 37.Yang D. Gong X. Yakhnin A. Roche T.E. J. Biol. Chem. 1998; 273: 14130-14137Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Here we begin the characterization of purified human PDKs and evaluate the functional activation and the changes in regulatory properties of two kinase isoforms, PDK2 and PDK3, as a result of their specific interactions with the lipoyl domains of human E2 and human E3BP. Our ability to produce individual lipoyl domains and E2–60-mer structures with or without E3BP allow us to sort the direct effects of individual lipoyl domains and unique contributions of the assembled complexes to activated PDK function and regulation. Acetyl-CoA and NADH, common products of the PDC reaction and the catabolism of fatty acids, stimulate bovine PDK activity resulting in the feed-back throttling down of PDC activity (2.Randel P.J. Biochem. Soc. Trans. 1986; 14: 799-806Crossref PubMed Scopus (240) Google Scholar, 38.Pettit F.H. Pelley J.W. Reed L.J. Biochem. Biophys. Res. Commun. 1975; 65: 575-582Crossref PubMed Scopus (194) Google Scholar, 39.Roche T.E. Liu S. Ravinddran S. Baker J.C. Wang L. Patel M.S. Roche T, E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 33-52Crossref Google Scholar). Increases in the NADH/NAD+ and acetyl-CoA/CoA ratio stimulate PDK activities by increasing the proportion of reduced and acetylated lipoyl groups on the lipoyl domain of E2 (39.Roche T.E. Liu S. Ravinddran S. Baker J.C. Wang L. Patel M.S. Roche T, E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 33-52Crossref Google Scholar, 40.Cate R.L. Roche T.E. J. Biol. Chem. 1978; 253: 496-503Abstract Full Text PDF PubMed Google Scholar, 41.Rahmatullah M. Roche T.E. J. Biol. Chem. 1985; 260: 10146-10152Abstract Full Text PDF PubMed Google Scholar, 42.Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 43.Popov K.M. FEBS Lett. 1997; 419: 197-200Crossref PubMed Scopus (23) Google Scholar). Pyruvate and ADP, serving as signals indicating abundant substrate and low energy, act synergistically to inhibit PDK activity by direct binding to PDKs (44.Hucho F. Randall D.D. Roche T.E. Burgett M.W. Pelley J.W. Reed L.J. Arch. Biochem. Biophys. 1972; 151: 328-340Crossref PubMed Scopus (157) Google Scholar,45.Pratt M.L. Roche T.E. J. Biol. Chem. 1979; 254: 7191-7196Abstract Full Text PDF PubMed Google Scholar). Evidence has been presented that sensitivity to these effectors varies with the particular PDK isoform (23.Bowker-Kinley M.M. Davis W.I. Wu P. Harris R.A. Popov K.M. Biochem. J. 1998; 329: 191-196Crossref PubMed Scopus (447) Google Scholar). By using an all human system, we establish that marked changes in catalytic efficiency and effector responsiveness of human PDK2 and PDK3 occur as a consequence of their association with E2. Recombinant human E2, K46AE2, and K173AE2 were prepared as described previously (37.Yang D. Gong X. Yakhnin A. Roche T.E. J. Biol. Chem. 1998; 273: 14130-14137Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 46.Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar). Recombinant human E2-E3BP was prepared as described elsewhere. 2X. Gong, A. Yakhnin, J. C. Baker, X. Yan, and T. E. Roche, manuscript in preparation. Recombinant human E1 (47.Korotchkina L.G. Tucker M.M. Thekkumkara T.J. Madhusudhan K.T. Pons G. Kim H. Patel M.S. Protein Expression Purif. 1995; 6: 79-90Crossref PubMed Scopus (37) Google Scholar) was engineered with the polyhistidine purification tag easily removed by PreScission protease treatment; this E1 was prepared by modification of the method by Korotchkina et al.(47.Korotchkina L.G. Tucker M.M. Thekkumkara T.J. Madhusudhan K.T. Pons G. Kim H. Patel M.S. Protein Expression Purif. 1995; 6: 79-90Crossref PubMed Scopus (37) Google Scholar).2 The acetyltransferase-catalyzing inner dodecahedron core of E2, E2I, was prepared by tryptic removal of the exterior E1 binding and lipoyl domain region from bovine kidney E2, followed by pelleting the E2I through sucrose layers (42.Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Homogeneous lipoyl domain constructs were prepared in a fully lipoylated state either as free domains or fused to glutathioneS-transferase (GST) as described elsewhere (49.Liu S. Baker J.C. Andrews P.C. Roche T.E. Arch. Biochem. Biophys. 1995; 316: 926-940Crossref PubMed Scopus (29) Google Scholar) by log phase expression in Escherichia coli BL21 that prevented modifications of the lipoyl domains introduced by E. coliJM109 (33.Gong X. Peng T. Yakhnin A. Zolkiewski M. Quinn J. Yeaman S.J. Roche T.E. J. Biol. Chem. 1999; 275: 13645-13653Abstract Full Text Full Text PDF Scopus (17) Google Scholar). The lipoyl domain constructs of E2 used include the following: outer lipoyl domain, L1; inner lipoyl domain, L2; bilipoyl structure, L1-L2; and the lipoyl domain of E3BP, designated L3. The specific design for constructing, expressing, and preparing purified GST-L3 and, from this, preparing free L3 (residues 1–98) will be described elsewhere.2 Porcine heart E3 and thrombin were from Sigma, [γ-32P]ATP from NEN Life Science Products, and TALON affinity purification resin fromCLONTECH. Human E3 expression system was provided by M. Patel, and human E3 was prepared as described previously (50.Liu T.-C. Korotchkina L.G. Hyatt S.L. Vettakkorumakankav N.N. Patel M.S. J. Biol. Chem. 1995; 270: 15545-15550Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). PreScission protease is a product of Amersham Pharmacia Biotech; BL21(DE3) E. coli and pET28a vector were obtained from Novagen. GroESL plasmid was kindly provided by Dr. Anthony Gatenby at DuPont (51.Goloubinoff P. Gatenby A.A. Lorimer G.H. Nature. 1989; 337: 44-47Crossref PubMed Scopus (527) Google Scholar). Synthetic DNA was obtained from Integrated DNA Technologies, Inc., Coralville, IA. The cloning vectors harboring the human PDK2 and PDK3 cDNA inserts were a kind gift from Kirill M. Popov and Robert A. Harris (6.Gudi R. Bowker-Kinley M.M. Kedishvili N.Y. Zhao Y. Popov K.M. J. Biol. Chem. 1995; 270: 28989-28994Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). For PDK2, PCR was performed with Pfu DNA polymerase (Stratagene) using primers that produced a 1.2-kilobase pair product encoding mature PDK2 with a 5′ NheI site and a 3′ HindIII site introduced. The product was ligated into pET28a vector at the NheI andHindIII sites using T4 DNA ligase (New England Biolabs) by standard procedures (52.$$$$$$ ref data missingGoogle Scholar). The resulting plasmid was co-transformed along with a plasmid encoding GroEL and GroES into E. coliBL21(DE3) made competent by the method of Inoue et al. (53.Inoue H. Nojima H. Okayama H. Gene (Amst.). 1990; 96: 23-28Crossref PubMed Scopus (1569) Google Scholar). Selection for the presence of both plasmids was done on LB plates containing 50 μg/ml kanamycin and 50 μg/ml chloramphenicol. The pET28a vector provides a start codon and encodes an N-terminal polyhistidine sequence followed by a sequence encoding a thrombin cleavage site. Along with this construct, which provided high quality PDK2, a second expression vector was constructed in which the thrombin cut site coding region was replaced by a region coding for the PreScission protease cleavage site. This was made by removing DNA from the plasmid between NcoI and NdeI sites and replacing it with a synthesized DNA segment to encoding a cut site specific for human rhinovirus 3C protease (54.Cordingley M.G. Callahan P.L. Sardana V.V. Garsky V.M. Colonno R.J. J. Biol. Chem. 1990; 265: 9062-9065Abstract Full Text PDF PubMed Google Scholar). For construction of PDK3 expression plasmid, a 270-base pair PCR fragment was generated using primers that matched the 5′SacI site at the beginning of the mature sequence and a 3′BamHI site matching the unique internal BamHI site in PDK3 cDNA. This PCR-amplified region of PDK3 was then recovered and used as a template for a second round of PCR that changed the 5′ restriction site to NheI and maintained the 3′BamHI site. After restriction treatment, this was ligated with the remaining PDK3 coding region produced by digestion withBamHI and XhoI and pET28a plasmid opened fromNheI to XhoI sites. This was followed by transformation and subsequent re-engineering, as described for PDK2, to produce two expression vectors, one encoding a thrombin cut site and the other a PreScission protease site, with each expressing N-terminal polyhistidine tags. All constructs were confirmed by DNA sequencing performed by the Automated Sequencing Facility at Kansas State University. PDK plasmid-containing bacteria were grown at 37 °C to mid-log phase (A 600 ≈0.6) in LB media containing 50 μg/ml kanamycin and 50 μg/ml chloramphenicol. Then expression was induced with 0.5 mmisopropyl-β-d-thiogalactopyranoside at 22–24 °C for 16 h. Bacteria were harvested by centrifugation at 4,000 ×g for 20 min at 4 °C and frozen at −80 °C. Following thawing, bacterial pellets were resuspended to 10% (w/v) in HN buffer (20 mm Hepes-Na, pH 8.0, 0.5 m NaCl, 1% (v/v) ethylene glycol). Ice water-cooled suspensions were sonicated by six repetitions of 50% pulsing at 250 watts for 30 s followed by at least 1 min of cooling. Supernatants were cleared by centrifugation at 10,000 × g for 20 min at 4 °C, and Pluronic-F68 was added to the supernatant to a level of 0.1% (w/v). 1 ml of equilibrated TALON resin was added per 50 ml of supernatant, and this suspension was gently mixed for 60 min at 4 °C. The mixture was transferred to a column, and the gel resin was washed first with 4 column volumes of HN buffer + 0.1% Pluronic-F68 + 20 mmimidazole and then with 3 column volumes of HN buffer containing 0.1% Pluronic-F68 plus 25 mm imidazole. PDK was then eluted with buffer containing 100 mm imidazole. PDK containing fractions were pooled, and 1 mm dithiothreitol, 1 mm EDTA, and either 50 units of thrombin plus 2 mm Ca2+ or 100 units of PreScission protease were added for a 1- or a 3-h incubation on ice, respectively. Following thrombin digestion, EGTA was added to 2 mm and glycerol was then added to 20%. Following PreScission protease digestion, protease was removed by passing the mixture through a column with 0.25 ml of GSH-Sepharose equilibrated with HG buffer (16 mm Hepes-Na, pH 8.0, 0.5 mm EDTA, 0.1% Pluronic F-68, 1% ethylene glycol, 20% glycerol). PDK preparations were desalted on a Sephadex G-25 column equilibrated with HG buffer for PDK2 and HG buffer containing 0.15 m NaCl for PDK3. PDK preparations were stored unfrozen at −20 °C. Final PDK recovery was typically 5–10 mg per liter of bacterial growth media for PDK2 and 2–4 mg per liter of growth media with PDK3. Much greater purity was obtained using the Co2+-containing Talon system than with nickel-affinity columns. PDKs were stored for over 9 months in an unfrozen state at −20 °C with <30% loss of activity. However, aged preparations of PDK3 needed to be preincubated with E2 at 4 °C for 60 min to exhibit maximal activity. PDK2 could be concentrated to >10 mg/ml, whereas PDK3 had to be maintained at <0.3 mg/ml to avoid development of insoluble aggregates. PDK activity was measured in duplicate as the initial rate of incorporation of [32P]phosphate into E1 (42.Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 46.Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar) using 0.1 mm[γ-32P]ATP (150–500 cpm/pmol) at 30 °C, unless otherwise stated. For comparative purposes with assays of prior kinase preparations, kinase activities were evaluated in 60 mmTris-Hepes, pH 7.3, with no inorganic ions and in the three buffer formulations used with purified PDKs as follows: MOPS-K+buffer, 50 mm MOPS-K+, pH 7.3, 20 mm KxPO4, pH 7.3, 60 mmKCl, 0.4 mm DTT, 0.4 mm EDTA, 2 mmMgCl2 (42.Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 46.Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar); phosphate buffer, 20 mmKxPO4, pH 7.0, 2 mm MgCl2, 0.2 mm EDTA, 2 mm DTT, 0.1% Triton X-100, 0.1% Pluronic F-68 (55.Stepp L.R. Pettit F.H. Yeaman S.J. Reed L.J. J. Biol. Chem. 1983; 258: 9454-9458Abstract Full Text PDF PubMed Google Scholar); Tris buffer, 20 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 50 mm KCl, 1 mm β-mercaptoethanol (23.Bowker-Kinley M.M. Davis W.I. Wu P. Harris R.A. Popov K.M. Biochem. J. 1998; 329: 191-196Crossref PubMed Scopus (447) Google Scholar). Subsequently, the MOPS-K+ buffer system was used as described previously (42.Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar,46.Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar). With the standard levels of kinase and E1, elevated (E2-activated and stimulated) kinase initial velocities were highly linear for 2 min and lower kinase rates for over 3 min. Most dat" @default.
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• W1975171610 title "Marked Differences between Two Isoforms of Human Pyruvate Dehydrogenase Kinase" @default.
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