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

Abstract

Branched-chain a-keto acid dehydrogenase (BCKDH) phosphatase was purified about 8000-fold from extracts of bovine kidney mitochondria. The highly purified phosphatase exhibited a molecular weight of -460,000, as estimated by gel-permeation chromatography. Another form of the phosphatase, with an apparent molecular weight of =230,000, was also detected under conditions of high dilution. In contrast to pyruvate dehydrogenase phosphatase, BCKDH phosphatase was active in the absence of divalent cations.BCKDH phosphatase was inactive toward 32P-labeled phosphorylase a, but exhibited -10% maximal activity with 32p-labeled pyruvate dehydrogenase complex. BCKDH phosphatase activity was inhibited by GTP, GDP, ATP, ADP, UTP, UDP, CTP, and CDP. Half-maximal inhibition occurred at about 60, 200, 200, 400, 100, 250, 250, and 400 jIM, respectively. These inhibitions were reversed completely by 2 mM Mg2e. GTP was replaceable by guanosine 5'-(i,y-imido)triphosphate. GMP, AMP, UMP, CMP, NAD, and NADH showed little effect, if any, on BCKDH phosphatase activity at concentrations up to 1 mM. Heparin showed half-maximal inhibition at 2 g/mIl. This inhibition was only partially (30%) reversed by 2 mM Mg2+. CoA and various acyl-CoA compounds exhibited half-maximal inhibition at 150-300 ,uM. These inhibitions were not reversed by 2 mM M&+. BCKDH phosphatase activity was stimulated 1.5-to 3-fold by protamine, poly(L-lysine), and poly(L-arginine) at 3.6 pg/mI.The mammalian branched-chain a-keto acid dehydrogenase (BCKDH) complex, like the analogous pyruvate dehydrogenase complex, is located within the mitochondrial inner membrane-matrix compartment and is subject to regulation by phosphorylation and dephosphorylation (1)(2)(3)(4)(5)(6). BCKDH is inactivated by phosphorylation of its a subunit (Mr, 46,000), catalyzed by a protein kinase that copurifies with the BCKDH complex (7-10). Although this protein kinase has not been isolated in a homogeneous state, its properties (9,11,12) indicate that it is different from pyruvate dehydrogenase kinase (13). The existence of a specific BCKDH phosphatase has been uncertain. Previous attempts to detect and purify a BCKDH phosphatase have been unsuccessful (8,14). Also contributing to this uncertainty are conflicting reports (2,4,8,14) concerning the activity of pyruvate dehydrogenase phosphatase toward the phosphorylated BCKDH complex. Dephosphorylation and reactivation of phosphorylated BCKDH complex by a broad-specificity cytosolic phosphatase have been reported (14). Although this mechanism is not considered to be physiologically relevant because of the mitochondrial localization of the BCKDH complex, cytosolic phosphatases could interfere in the assay for BCKDH phosphatase unless well-washed mitochondria are used. Activation of phosphorylated BCKDH complex by a protein factor without accompanying dephosphorylation has been observed (15). The molecular basis of this activation is not known. Low levels of a Mg2+-dependent phosphatase activity were detected in purified preparations of BCKD...

Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids

“…The BCOD complex comprises multiple copies of three catalytic components : a thiamine pyrophosphate-dependent branched-chain 2-oxoacid decarboxylase (E1) that contains two E1α and two E1β subunits ; a dihydrolipoyl transacylase (E2) ; and a dihydrolipoyl dehydrogenase (E3) [1,2]. In addition, the complex has a set of regulatory enzymes : a specific kinase [3] and a specific phosphatase [4], which control enzyme activity through reversible phosphorylation (inactivation)\dephosphorylation (activation) of E1 [5].…”

Premature termination of transcription and alternative splicing in the human transacylase (E2) gene of the branched‐chain α‐ketoacid dehydrogenase complex

“…The branched-chain corn plex consists of three catalytic components: a branched-chain c~-keto acid decarboxylase or El, a dihydrolipoyl transacylase or E2 and a dihydrolipoyl dehydrogenase or E3 [2,3]. The E3 component is common to all three a-kate acid dehydrogenase complexes., The branched-chain complex also has two regulatory enzymes, a specific kinase and a specific phosphatase [4][5][6][7][8]. The E2 subunit forms a 24-mar cubic core to which El, E3, the kinase and the phosphatase are attached through non-covalent interactions.…”

Maple syrup urine disease. Complete primary structure of the E1 beta subunit of human branched chain alpha-ketoacid dehydrogenase complex deduced from the nucleotide sequence and a gene analysis of patients with this disease.

“…On the other hand, the E3 component is common among the three ketoacid dehydrogenase complexes, BCKDH, pyruvate dehydrogenase (PDH), and a-ketoglutarate dehydrogenase (4,5). The BCKDH complex also contains two specific regulatory enzymes, a kinase (6)(7)(8) and a phosphatase (9,10), responsible for regulation ofthe catalytic activity through phosphorylation and dephosphorylation.…”