Flora H. Pettit

α-Keto acid dehydrogenase complexes: XV. Purification and properties of the component enzymes of the pyruvate dehydrogenase complexes from bovine kidney and heart☆

Abstract Procedures are described for isolation of the pyruvate and α-ketoglutarate dehydrogenase complexes in good yield and in a highly purified state from bovine kidney and heart mitochondria. Procedures are presented for separation of the kidney and heart pyruvate dehydrogenase complexes into their component enzymes and for purification of the individual enzymes. The nonphosphorylated and phosphorylated forms of the pyruvate dehydrogenase component of both the kidney and heart complexes were crystallized. Pyruvate dehydrogenase kinase, a regulatory enzyme, is tightly bound to the dihydrolipoyl transacetylase component of the kidney and heart complexes. The kinase was separated and purified from the kidney transacetylase. A second regulatory enzyme, pyruvate dehydrogenase phosphatase, appears to be loosely associated with the kidney and heart pyruvate dehydrogenase complexes. The kidney and heart pyruvate dehydrogenase phosphatases were purified 400- to 1000-fold from mitochondrial extracts. The heart phosphatase is close to being homogeneous. The molecular weight of the two phosphatases was estimated by gel filtration to be about 100,000. Preliminary data indicate that the molar concentration of the kinase and the phosphatase is about an order of magnitude less than that of their protein substrate, pyruvate dehydrogenase.

Regulation of pyruvate dehydrogenase kinase and phosphatase by acetyl-CoA/CoA and NADH/NAD ratios

Summary The interconversion of the active, nonphosphorylated form of pyruvate dehydrogenase and its inactive, phosphorylated form is modulated by acetyl-CoA/CoA and NADH/NAD molar ratios. An increase in either ratio increases the proportion of the phosphorylated form of pyruvate dehydrogenase. The activity of pyruvate dehydrogenase kinase is stimulated by acetyl-CoA and by NADH and is inhibited by CoA and by NAD. NADH inhibits pyruvate dehydrogenase phosphatase, and this inhibition is reversed by NAD.

Function of phosphorylation sites on pyruvate dehydrogenase

Abstract Evidence is presented that dephosphorylation of the three phosphorylation sites on bovine kidney pyruvate dehydrogenase by pyruvate dehydrogenase phosphatase is random. The relative rates of dephosphorylation were in the order site 2 > site 3 > site 1. Phosphorylation site 2, and possibly site 3, function, in addition to site 1, as inactivating sites. However, the presence of phosphoryl groups at sites 2 and 3 did not significantly affect the rate of dephosphorylation at site 1 or the rate of reactivation of the enzyme by the phosphatase. The rate-limiting step in the reactivation of phosphorylated pyruvate dehydrogenase is apparently the dephosphorylation at site 1.

[65] Pyruvate dehydrogenase complex from bovine kidney and heart

Publisher Summary In eukaryotic cells, the pyruvate dehydrogenase complex is located in mitochondria, within the inner membrane-matrix compartment. The complex consists of three catalytic components: pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. These three enzymes, acting in sequence, catalyze the overall reaction. The Activity of this multienzyme complex is assayed spectrophotometrically by the measurement of nicotinamide adenine dinucleotide dehydrogenase (NADH) production. The assay is used routinely at all the levels of purity of the enzyme complex, beginning with mitochondrial extracts prepared by the freeze-thaw procedure . However, the assay is less satisfactory with crude extracts that contain lactate dehydrogenase or NADH oxidase. With these preparations, 14 CO 2 production from pyruvate is measured, or the overall reaction is coupled with the arylamine acetyltransferase-catalyzed reaction. The acetylation of an appropriate arylamine is measured spectrophotometrically. The α subunit of pyruvate dehydrogenase and the subunit of dihydrolipoyl transacetylase are very sensitive to proteolysis. The steps involved in the purification of pyruvate dehydrogenase are also discussed in the chapter.

Peptides derived from pyruvate dehydrogenase as substrates for pyruvate dehydrogenase kinase and phosphatase

Abstract Evidence is presented that phosphopeptides produced by tryptic digestion of phosphorylated pyruvate dehydrogenase are effective substrates for pyruvate dehydrogenase phosphatase and that the dephosphopeptides can serve as substrates for pyruvate dehydrogenase kinase. These findings indicate that the phosphatase and the kinase do not require an intact tertiary structure in pyruvate dehydrogenase, but apparently recognize components of the local primary sequence around the phosphorylation sites.

Binding of thiamin thiazolone pyrophosphate to mammalian pyruvate dehydrogenase and its effects on kinase and phosphatase activities

Abstract The pyruvate dehydrogenase component of the bovine kidney pyruvate dehydrogenase complex has two thiamin-PP binding sites per α 2 β 2 tetramer. Titration of these binding sites with the transition state analog, thiamin thiazolone pyrophosphate, strongly inhibits phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase and ATP. The analog has little effect, if any, on dephosphorylation of phosphorylated pyruvate dehydrogenase by pyruvate dehydrogenase phosphatase. Phosphorylation of pyruvate dehydrogenase inactivates the enzyme, but does not significantly affect the thiamin-PP binding sites. It appears that phosphorylation produces a conformational change in pyruvate dehydrogenase that displaces a catalytic group (or groups) at the active center.

Structure, Function and Regulation of the Mammalian Pyruvate Dehydrogenase Complex

The mammalian pyruvate dehydrogenase complex is organized about a core of dihydrolipoyl transacetylase, to which are bound pyruvate dehydrogenase, dihydrolipoyl dehydrogenase, and two regulatory enzymes — a kinase and a phosphatase. Association of these enzymes into a complex alters the kinetic and regulatory properties of some, and possibly all, of the enzymes. The pyruvate dehydrogenase component has the subunit composition α2β2. The kinase, in a MgATP2−-dependent reaction, phosphorylates a seryl residue in one of the α-chains of pyruvate dehydrogenase, and thereby inactivates the tetramer. The phosphatase, which dephosphorylates and activates pyruvate dehydrogenase phosphate, requires Mg2+ and Ca2+ ions. Ca2+ ions increase binding of phosphatase to the transacetylase, thereby lowering the apparent Km of the phosphatase for pyruvate dehydrogenase phosphate. The kinase is inhibited by pyruvate and by ADP. It appears that its activity may be regulated by the intramitochondrial concentration of pyruvate and by the ATP/ADP ratio. The ATP/ADP ratio may also regulate the activity of the phosphatase by determiningg at least in part, the intramitochondrial concentrations of uncomplexed Mg2+ and Ca2+ ions. It appears that the kinase and phosphatase maintain steady state levels of activity of the complex and that these levels are modulated through the actions on the kinase and phosphatase of the factors mentioned and possibly by other factors yet to be determined.

Evidence for sulfite as an essential metabolite for human peripheral lymphocytes.

Sulfite has been identified as an essential metabolite by means of growth studies using a chemically-defined, protein-free medium for culture of human peripheral lymphocytes. Sulfite reduced the amount of cysteine required for optimum growth by at least four-fold. In some subjects, sulfite stimulated growth even in the presence of optimal amounts of cysteine indicating that lymphocytes of some individuals are unable to convert cysteine to sulfite in adequate amounts.

The effect of asparagine and adenine on the glutamine requirement for growth of human peripheral lymphocytes

Quantitative growth responses of lymphocytes directly isolated from individual subjects in a newly developed chemically-defined, protein-free medium are used to demonstrate that supplements of both L-asparagine and a purine source, but neither alone, significantly reduce the quantitative requirement for L-glutamine for growth. This system is useful for exploring individual differences in quantitative glutamine requirements and adequacy of asparagine and purine biosynthesis.

Chemically defined medium for the growth of lymphocytes.

Publisher Summary This chapter describes the composition and preparation of a chemically defined medium, CFBI 1000, that promotes growth of human lymphocytes in the absence of serum or added proteins. It also discusses other serum-free but not protein-free media used for serum-free experimentation. The CFBI 1000 medium is a buffered solution containing amino acids, vitamins, adenine, choline, glucose, inositol, pyruvate, EDTA, and the ions sodium, potassium, phosphate, chloride, sulfate, calcium, iron, and magnesium. This defined medium can be utilized to test effects of any metabolite not present in the medium for a deficit in biosynthesis or utilization. In addition, a defined medium also has broad applications in the study of the immune response mediated by the lymphocyte population (T and B cells). For media preparation, the composition of medium CFBI 1000 is presented in tabular form in the chapter. Medium CFBI 1000 is prepared from solid materials and a series of sterile stock solutions. The stock solutions are sterilized by filtration and stored in aliquots at -20° (except phenol red is stored at 5°, and MgSO 4 and CaCl 2 stock solutions are stored at 25°).