Purification and properties of branched-chain alpha-keto acid dehydrogenase kinase from bovine kidney

Branched-chain alpha-keto acid dehydrogenase (BCKDH) kinase was purified 5000-fold to apparent homogeneity from extracts of bovine kidney mitochondria. The kinase co-purified with the BCKDH complex. About 70% of the kinase was released by treatment of the complex with 1.5 M NaCl and 0.1% 2-mercaptoethanol at pH 7.4, followed by chromatography on Sephacryl S-400. The uncomplexed kinase was purified further by chromatography on Q Sepharose and Superose 12. The purified kinase is a monomer of apparent Mr approximately 43,000. BCKDH kinase exhibited little activity, if any, toward pyruvate dehydrogenase.     

Purification and properties of the catalytic subunit of the branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney mitochondria

The catalytic subunit of the branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase (Damuni, Z., Merryfield, M.L., Humphreys, J.S., and Reed, L.J., (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4335-4338) has been purified over 50,000-fold from extracts of bovine kidney mitochondria. The apparently homogeneous protein consists of a single polypeptide chain with an apparent Mr = approximately 33,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BCKDH phosphatase, with an apparent Mr = 460,000, was dissociated to its catalytic subunit with no apparent change in activity, at an early stage in the purification procedure by treatment with 6 M urea. The specific activity of the catalytic subunit was 1,500-2,500 units/mg. The catalytic subunit exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex but was inactive with phosphorylase a and with p-nitrophenyl phosphate. The catalytic subunit, like the Mr = 460,000 species, was inhibited by nanomolar concentrations of BCKDH phosphatase inhibitor protein, was unaffected by protein phosphatase inhibitor 1 and inhibitor 2, and was inhibited by nucleoside tri- and diphosphates but not by nucleoside monophosphates.     

Mechanism of activation of branched-chain alpha-keto acid dehydrogenase complex by exercise

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complex catalyzes the committed step of branched-chain amino acid catabolism, and its activity is regulated by the phosphorylation-dephosphorylation cycle. BCKDH kinase is responsible for inactivation of the complex by phosphorylation. In the present study, we examined acute exercise on the activity state of the complex as well as the amounts of bound and free forms of the kinase in rat liver and skeletal muscle. Acute exercise activated the complex in association with a decrease in the bound form of kinase in both liver and muscle. The free form of kinase in both tissues was slightly increased but the total amount of the kinase was not affected by acute exercise. The protein amount ratio of bound kinase to E1beta component of the complex was much higher in muscle than in the liver of rats, reflecting the low activity state of the complex in muscle. These results suggest that the amount of the bound kinase plays an important role in regulation of the activity state of the complex. We propose that the alteration in the amount of bound BCKDH kinase is a short-term regulatory mechanism for determining the activity of BCKDH complex.     

Purification and characterization of human liver branched-chain alpha-keto acid dehydrogenase complex

Human liver BCKADH complex was purified. On SDS-polyacrylamide gel electrophoresis, the purified enzyme complex gave three major bands having molecular weights of 51,000, 46,000, and 36,000, and one minor band with a molecular weight of 55,000. The minor band corresponded in molecular weight to lipoamide oxidoreductase which was purified separately. The purified BCKADH represented only approximately 20% of the maximum activity when assayed without addition of exogenous lipoamide oxidoreductase, indicating that lipoamide oxidoreductase component was readily dissociable from the complex. The BCKADH effectively oxidized all of KIV, KIC, and KMV, yielding apparent Km values in the range of 14-17 microM for those alpha-keto acids. Vmax values obtained were 0.86, 0.61, and 0.51 mumole NADH produced/min/mg of protein for KIV, KIC, and KMV, respectively, in the presence of excess amount of lipoamide oxidoreductase. This ratio of Vmax values was practically identical to those of specific activities obtained with respective branched-chain alpha-keto acids at each purification step. The enzyme complex also oxidized pyruvate and alpha-ketoglutarate to a lesser extent. Kinetic experiments gave Km values of 0.98 and 2.9 mM for pyruvate and alpha-ketoglutarate, respectively, with Vmax of 0.43 and 0.08 mumole NADH produced/min/mg of protein. NAD and CoASH were absolutely required for the reaction. Km values for NAD and CoASH were estimated to be 47 and 25 microM, respectively.     

Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex.

Branched-chain 2-oxo acid dehydrogenase complex was resolved into component E1 and E2-kinase subcomplex by gel filtration in the presence of 1 M-NaC1. Essentially all the original activity of the complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process. The specific activities of E1 and E2 were 25.1 and 19.0 units/mg respectively. Non-phosphorylated active E1 has an approx. 6-fold higher affinity for E2 than does phosphorylated E1. The components of the branched-chain 2-oxo acid dehydrogenase complex do not crossreact with the respective components from the pyruvate dehydrogenase complex. The significance of these results and of the tight association of the kinase with E2 are discussed.     

Amino acid sequence surrounding the lipoic acid cofactor of bovine kidney 2-oxoglutarate dehydrogenase complex

The 2-oxoglutarate dehydrogenase complex was succinylated using 2-oxo[5-14C]glutarate in the presence of N-ethylmaleimide to label the lipoic acid cofactor of the transuccinylase (E2) component. Following peptic digestion, 14C-lipoate-containing peptides were purified and subjected to automated Edman degradation and amino acid analysis. The amino acid sequence surrounding the lipoyllysine residue is reported.     

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Characterization of the inner transacylase core

Limited proteolysis has been used to probe the subunit structure (Mr = 52,000) of the dihydrolipoyl transacylase (E2) component of the branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Digestion of the complex at 0 degrees C with a low concentration of trypsin produces an inner E2 core that retains the activity for the transacylation reaction and is completely dissociated from the decarboxylase (E1) component. The trypsinized E2 maintains the highly assembled structure and migrates faster than the native E2 in the Sepharose 4B column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the inner E2 core consists of two lipoate-free tryptic fragments, i.e. fragment A and fragment B with Mr = 26,000 and 22,000, respectively. Both fragments apparently fail to bind the E1 component. Fragment A is converted into fragment B by increasing trypsin concentrations. Fragment B is a stable limit polypeptide containing the intersubunit-binding sites for E2. The assemblage of fragment B confers the cubelike appearance of the inner E2 core in electron micrographs. Activity measurements indicate that the larger fragment A, but not fragment B, possesses transacylation activity. It is likely that a critical portion of the active site is present in the 4,000-dalton fragment that is lost during the conversion of fragment A to B.     

Subunit structure of dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex from bovine kidney

The pyruvate dehydrogenase complex has been isolated from bovine kidney mitochondria under special anti-proteolytic conditions yielding preparations with a specific activity of up to 20 U/mg protein. Dihydrolipoamide acetyltransferase resolved from the complex was subjected to limited proteolysis resulting in the formation of two major fragments with apparent molecular weights of 36000 and 28000. The fragments were isolated by extraction from dodecyl sulfate polyacrylamide gels and were both shown to possess enzymatic activity for acetyl transfer. Acetylation studies indicated that each fragment contains one protein-bound lipoyl group. It is concluded that the kidney dihydrolipoamide acetyltransferase subunit consists of two homologous if not identical domains. A model is suggested where the acetyltransferase core of the mammalian pyruvate dehydrogenase complex is made up of 30 polypeptide chains whose 60 domains could be arranged in pentagonal dodecahedron symmetry quite similar as proposed for the 60 subunit structure of the acetyltransferase core.     

Multi-site phosphorylation of bovine kidney branched-chain 2-oxoacid dehydrogenase complex

The phosphorylation of NADP-specific isocitrate dehydrogenase in a wild-type and in an adenylate cyclase deletion mutant of Escherichia coli has been investigated. The results obtained clearly indicate that cyclic AMP is not required for the phosphorylation reaction per se, not is it for the synthesis or possible activation of the phosphoprotein kinase in this organism. This data are in contrast to results observed in Salmonella typhimurium, and indicate that important differences exist in the phosphorylation of the isocitrate dehydrogenase in these two organisms.     

Isolation of a branched chain alpha-keto acid dehydrogenase from rabbit liver

An enzyme catalysing a series of reactions resulting in the oxidative decarboxylation of branched chain alpha-keto acids and production of NADH, was extracted from rabbit liver mitochondria with the aid of NaClO4. Purification yielded a product which appeared homogeneous on electrophoresis. The enzyme is active on three substrates alpha-ketoisocaproate, alpha-keto-beta-methyl valerate, and alpha-ketoisovalerate.     

Activation of hepatic branched-chain alpha-keto acid dehydrogenase complex by tumor necrosis factor-alpha in rats

Tumor necrosis factor-alpha (TNFalpha) promotes oxidation of branched-chain amino acids (BCAA). BCAA catabolism is regulated by branched-chain alpha-keto acid dehydrogenase (BCKDH) complex, which is regulated by phosphorylation-dephosphorylation of the E1alpha subunit at Ser293. BCKDH kinase is responsible for inactivation of the complex by phosphorylation. In the present study, we examined the effects of TNFalpha administration on hepatic BCKDH complex and kinase in rats. Rats were intravenously administered with 25 or 50 microg TNFalpha/kg body weight 4 h prior to sacrifice. The TNFalpha treatment at both doses elevated the activity state (percentage of the active form) of BCKDH complex from 22% to 69% and 86%, respectively, and the amount of phospho-Ser293 on the E1alpha subunit in each group of rats corresponded inversely to the activity state of BCKDH complex. The TNFalpha treatment of rats significantly decreased the activity as well as the bound form of BCKDH kinase. These results suggest that the decrease in the bound form of kinase is involved in the mechanism responsible for TNFalpha-induced activation of the BCKDH complex.     

Purification, resolution, and reconstitution of rat liver branched-chain alpha-keto acid dehydrogenase complex

Branched-chain alpha-keto acid dehydrogenase (BCKADH) was solubilized as an enzyme complex from rat liver mitochondria by sonic treatment. Dehydrogenase (E1) and dihydrolipoyltransacylase (E2) components of the complex were purified in an associated form and resolved into individual components in the presence of 1 M NaCl, while lipoamide dehydrogenase (E3) component was dissociated from the complex during purification. Analysis by gel electrophoresis in dodecyl sulfate revealed the E1 comprised two different subunits with apparent molecular weights of 36,000 and 45,500, presumably in an equal molar ratio, while E2 consisted of a single subunit with an apparent molecular weight of 51,000. The BCKADH complex was reconstituted by combining E1, E2, and E3, and the formation of the complex was confirmed by analysis by sucrose density gradient centrifugation. The reconstituted enzyme complex oxidized not only alpha-ketoisovalerate (KIV), alpha-ketoisocaproate (KIC), and alpha-keto-beta-methylvalerate (KMV), but also pyruvate and alpha-ketoglutarate. Apparent Km values were 10-12 microM for the branched-chain alpha-keto acids, 2.2 mM for pyruvate, and 2.5 mM for alpha-ketoglutarate.     

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Mapping of the lipoyl-bearing domain by limited proteolysis

To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain alpha-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C] alpha-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), L2 (Mr = 24,500), L3 (Mr = 21,000), L4 (Mr = 15,000) to L5 (Mr = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserum against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E2 core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L1 to L5), with the L1 site in the hinge region, and the L5 site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L1 and L4 tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E2 chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L5, the limit polypeptide carrying the single lipoyl residue.     

Homotropic regulation of dihydrolipoyl dehydrogenase from bovine kidney

Dihydrolipoyl dehydrogenase from bovine kidney catalyzes NAD-linked redox reaction of lipoamide. Hates of the catalyzed reaction were studied in both directions. Saturation curves for NAD and lipoamide are nonhyperbolic, suggesting homotropic cooperative interactions of these substrates with the enzyme. The cooperative effect was analyzed by Hill plots according to the diagnostic procedure of Levitzki and Koshland. Dihydrolipoyl dehydrogenase is subject to homotropic regulation in which NAD acts as a negative cooperative effector, whereas lipoamide acts as a positive cooperative effector. At high concentrations, dihydrolipoamide normalizes the saturation curve of NAD, while NADH tends to enhance the cooperative interaction of lipoamide with the enzyme.     

Amino acid sequence at the major phosphorylation site on bovine kidney branched-chain 2-oxoacid dehydrogenase complex

The N-terminal part of native one-chain tissue plasminogen activator from melanoma cells is not homogeneous. The protein chain starts at two different positions, in all probability representing a processing difference in the N-terminus. Both 'long' L-chains and 3-residue shorter S-chains are present in the preparations. In addition, results compatible with a positional Ser/Gly microheterogeneity were obtained at a single position (position L-4 which is equal to S-1). The N-terminal tripeptide difference seems to be coupled to the possible microheterogeneity: L-chains contain Ser in this position, while S-chains appear to contain predominantly Gly.