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

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.

[1]  L. Reed,et al.  Regulation of pyruvate dehydrogenase kinase activity by protein thiol-disulfide exchange. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[2]  E. Fischer,et al.  Activation of skeletal muscle phosphorylase phosphatase. Effects of proteolysis and divalent cations. , 1982, Biochemistry.

[3]  P. Cuatrecasas,et al.  Insulin stimulates the release from liver plasma membranes of a chemical modulator of pyruvate dehydrogenase. , 1981, Biochemical and biophysical research communications.

[4]  L. Reed,et al.  Use of trypsin and lipoamidase to study the role of lipoic acid moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes of Escherichia coli. , 1981, Biochemistry.

[5]  F. Kiechle,et al.  Partial purification from rat adipocyte plasma membranes of a chemical mediator which simulates the action of insulin on pyruvate dehydrogenase. , 1981, The Journal of biological chemistry.

[6]  M. Czech,et al.  Characterization of a pyruvate dehydrogenase activator released by adipocyte plasma membranes in response to insulin. , 1981, The Journal of biological chemistry.

[7]  R. Hansford.  Effect of micromolar concentrations of free Ca2+ ions on pyruvate dehydrogenase interconversion in intact rat heart mitochondria. , 1981, The Biochemical journal.

[8]  W. Merlevede,et al.  ATP x Mg-dependent protein phosphatase from rabbit skeletal muscle. I. Purification of the enzyme and its regulation by the interaction with an activating protein factor. , 1980, The Journal of biological chemistry.

[9]  P. Cohen,et al.  The broad specificity protein phosphatase from mammalian liver , 1980, FEBS letters.

[10]  F. Kiechle,et al.  Insulin stimulation of pyruvate dehydrogenase in an isolated plasma membrane-mitochondrial mixture occurs by activation of pyruvate dehydrogenase phosphatase. , 1980, The Journal of biological chemistry.

[11]  J. Mccormack,et al.  Role of calcium ions in the regulation of intramitochondrial metabolism. Properties of the Ca2+-sensitive dehydrogenases within intact uncoupled mitochondria from the white and brown adipose tissue of the rat. , 1980, The Biochemical journal.

[12]  N. J. Edgell,et al.  Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2+-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria. , 1980, The Biochemical journal.

[13]  J. Haiech,et al.  Ligand binding to macromolecules: determination of binding parameters by combined use of ligand buffers and flow dialysis; application to calcium-binding proteins. , 1980, Analytical biochemistry.

[14]  L. Jarett,et al.  Pyruvate dehydrogenase activation in adipocyte mitochondria by an insulin-generated mediator from muscle. , 1979, Science.

[15]  K. Cheng,et al.  Generation by insulin of a chemical mediator that controls protein phosphorylation and dephosphorylation. , 1979, Science.

[16]  A. L. Kerbey,et al.  Role of multi‐site phosphorylation in regulation of pig heart pyruvate dehydrogenase phosphatase , 1979, FEBS letters.

[17]  K Feldmann,et al.  New devices for flow dialysis and ultrafiltration for the study of protein--ligand interactions. , 1978, Analytical biochemistry.

[18]  L. Reed,et al.  Peptides derived from pyruvate dehydrogenase as substrates for pyruvate dehydrogenase kinase and phosphatase. , 1977, Biochemical and biophysical research communications.

[19]  P. Munk,et al.  Sedimentation Equilibrium of Polymers in Good Solvents , 1976 .

[20]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[21]  C. Mukherjee,et al.  Activation of pyruvate dehydrogenase in adipose tissue by insulin. Evidence for an effect of insulin on pyruvate dehydrogenase phosphate phosphatase. , 1975, The Biochemical journal.

[22]  T. Linn.  Studies on the apparent instability of bovine kidney α-ketoglutarate dehydrogenase complex , 1974 .

[23]  D. DeRosier,et al.  Automatic measurement of intereference photographs from the ultracentrifuge , 1972 .

[24]  T. Roche,et al.  Function of calcium ions in pyruvate dehydrogenase phosphatase activity. , 1972, Biochemical and biophysical research communications.

[25]  F. Hucho,et al.  α-Keto acid dehydrogenase complexes: XVII. Kinetic and regulatory properties of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase from bovine kidney and heart , 1972 .

[26]  R. Denton,et al.  Stimulation by calcium ions of pyruvate dehydrogenase phosphate phosphatase. , 1972, The Biochemical journal.

[27]  O. Wieland,et al.  Purification and characterization of pyruvate-dehydrogenase phosphatase from pig-heart muscle. , 1972, European journal of biochemistry.

[28]  F. Hucho,et al.  α-Keto acid dehydrogenase complexes: XV. Purification and properties of the component enzymes of the pyruvate dehydrogenase complexes from bovine kidney and heart , 1972 .

[29]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[30]  S. Colowick,et al.  Binding of diffusible molecules by macromolecules: rapid measurement by rate of dialysis. , 1969, The Journal of biological chemistry.

[31]  B. Davis.  DISC ELECTROPHORESIS – II METHOD AND APPLICATION TO HUMAN SERUM PROTEINS * , 1964, Annals of the New York Academy of Sciences.

[32]  L. Ornstein,et al.  DISC ELECTROPHORESIS. I. BACKGROUND AND THEORY. , 1964, Annals of the New York Academy of Sciences.

[33]  D. A. Yphantis.  EQUILIBRIUM ULTRACENTRIFUGATION OF DILUTE SOLUTIONS. , 1964, Biochemistry.

[34]  Oliver H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[35]  G. Scatchard,et al.  THE ATTRACTIONS OF PROTEINS FOR SMALL MOLECULES AND IONS , 1949 .

[36]  A. Gornall,et al.  Determination of serum proteins by means of the biuret reaction. , 1949, The Journal of biological chemistry.

[37]  L. Reed,et al.  Pyruvate dehydrogenase complex from bovine kidney and heart. , 1982, Methods in enzymology.

[38]  L. Reed,et al.  Regulation of mammalian pyruvate dehydrogenase complex by a phosphorylation-dephosphorylation cycle. , 1981, Current topics in cellular regulation.

[39]  J. Williamson,et al.  Calcium homeostasis and compartmentation in liver. , 1980, Advances in experimental medicine and biology.

[40]  P. Cohen.  The role of cyclic-AMP-dependent protein kinase in the regulation of glycogen metabolism in mammalian skeletal muscle. , 1978, Current topics in cellular regulation.

[41]  E. Stellwagen,et al.  Measurement of protein concentration with interferences optics. , 1969, Analytical biochemistry.

[42]  K. Burton.  [23] d-Amino acid oxidase from kidney: R·CHNH2COOH+O2+H2O →R·CO·COOH+NH3+H2O2 , 1955 .