Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney

Abstract

Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2.

Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs

“…Ca 2ϩ stimulation of PDP1 arises in part because PDP1 binds to E2 via an interaction that requires micromolar concentrations of Ca 2ϩ (38) and in part because Ca 2ϩ decreases the K m of PDP1 for Mg 2ϩ (61). PDP1 comprises a catalytic and a regulatory subunit (42,56). The catalytic subunit (PDP1c) is in the phosphatase 2C class (28).…”

An Essential Role of Glu-243 and His-239 in the Phosphotransfer Reaction Catalyzed by Pyruvate Dehydrogenase Kinase

“…Phosphorylation of the dehydrogenase component of the complex (E1 component) by dedicated pyruvate dehydrogenase kinase (PDK) renders the entire complex inactive (3). Phospho-PDC can be re-activated through the action of another dedicated enzyme, pyruvate dehydrogenase phosphatase (4). In mammals, both kinase and phosphatase components exist in several isozymic forms (four for PDK (5) and two for pyruvate dehydrogenase phosphatase (6)), which are likely to contribute to the tissue-specific regulation of PDC.…”

Isoenzymes of Pyruvate Dehydrogenase Phosphatase

“…The phosphoenzyme can be reactivated only via dephosphorylation catalyzed by phosphatase (11). In contrast to PDK, PDP is loosely associated with PDC but becomes complex-bound upon stimulation (12). Both PDK and PDP can integrate a considerable number of different regulatory stimuli (reviewed in Ref.…”

“…The enzymatic activity of PDP depends on intramito-chondrial concentrations of Mg 2ϩ and Ca 2ϩ ions (14,15). The latter promotes the association of PDP with the complex (12) and, by this means, stimulates the rate of dephosphorylation. Another co-factor that may be important for the regulation of PDP is intramitochondrial NADH (16).…”