The reaction of diethyl pyrocarbonate with pyruvate kinase (Short Communication)

Diethyl pyrocarbonate inactivates muscle pyruvate kinase with the substitution of 3-4 histidine residues per subunit. Phosphoenolpyruvate, ATP and ADP to a lesser extent, and Mg(2+) and pyruvate to a small extent, protect against inactivation.     

Lack of temperature-sensitivity of rat liver pyruvate kinase (Short Communication)

Rat liver pyruvate kinase, prepared by Sephadex treatment of a 10(5)g supernatant in phosphate buffer, is quite stable and gives reproducible results when a variety of parameters are altered in the enzyme assay. Incubation of this preparation at 25 degrees C or 0-2 degrees C has no effect on the activation by fructose 1,6-diphosphate or inhibition by ATP or alanine.     

Specificity of glycerol kinase (Short Communication)

The activity of a number of alcohols was examined as substrates or inhibitors of glycerol kinase (ATP-glycerol phosphotransferase; EC 2.7.1.30) from Candida mycoderma. On the basis of these and other results, a modified model is proposed to account for the substrate specificity of the enzyme.     

Activation of pyruvate dehydrogenase in perfused rat heart by dichloroacetate (Short Communication)

The activity of pyruvate dehydrogenase was assayed in extracts of rat hearts perfused in vitro with media containing glucose and insulin±acetate±dichloroacetate. Dichloroacetate (100μm, 1mm or 10mm) increased the activity of pyruvate dehydrogenase in perfusions with glucose or glucose+acetate. Evidence is given that dichloroacetate may facilitate the conversion of pyruvate dehydrogenase from an inactive (phosphorylated) form into an active (dephosphorylated) form.     

Dexamethasone effects on liver pyruvate kinase

Dexamethasone in the medium perfusin isolated rabbit livers caused a fast-acting and reversible effect on liver pyruvate kinase. The effect was to lower th assayable V activity (units/g tissue) without changing the concentration (nmol/g enzyme protein). In effect, glucocorticoid lowered the specific activity (units/nmol of enzyme) by direct action on liver. The effect on liver pyruvate kinase is mediated by a relatively stable alteration; 30 min after perfusate (with steroid) was replaced by perfusate (without steroid), the effect remained strongly evident.     

Aging effects on the liver aldolase of rabbits (Short Communication)

A chemical method is used to determine the amount of aldolase sequence in rabbit livers. It is demonstrated that the aldolase sequences of the livers of young rabbits are more efficient catalytically than are those from the livers of old rabbits. The presence of altered residues in the aldolase sequences of old animals may account for these observations.     

Properties and mechanism of action of creatine kinase from ox smooth muscle. Anion effects compared with pyruvate kinase

1. An improved purification procedure for the brain-type creatine kinase from ox smooth muscle is described. 2. Michaelis constants show the characteristic dependence on the concentration of the second substrate: the derived constants are compared with those for the enzyme from ox brain. 3. Inhibition by iodoacetamide gives a biphasic curve and the total extent of the reaction depends on the enzyme concentration. The rate of inhibition at pH8.6 is not affected by creatine plus MgADP or by a range of simple anions. Addition of creatine plus MgADP plus either NO(3) (-) or Cl(-) ions affords 71.5 and 44% protection respectively. ADP could be replaced by 2-deoxy-ADP but not by alphabeta-methylene ADP, XDP, IDP, GDP or CDP. Nucleotides that did not protect would not act as substrates. 4. Difference-spectra measurements support the interpretation that addition of NO(3) (-) ions to the enzyme-creatine-MgADP complex causes further conformational changes in the enzyme accompanying the formation of a stable quaternary enzyme-creatine-NO(3) (-)-MgADP complex that simulates an intermediate stage in the transphosphorylation reaction. However, the enzyme structure is partially destabilized by quaternary-complex formation. IDP apparently fails to act as a substrate because it cannot induce the necessary conformational change. This behaviour is compared with that of rabbit skeletal muscle creatine kinase. 5. With pyruvate kinase from rabbit muscle, anions activate in the absence of an activating cation and either inhibit or have no effect in its presence. 6. Both activation and inhibition were competitive with respect to the substrate, phosphoenolpyruvate, and curved double-reciprocal plots were obtained. The results may be interpreted in terms of co-operatively induced conformational changes, and this is supported by difference-spectra measurements. However, the Hill coefficient of 1 was not significantly altered. 7. Inhibition by lactate plus pyruvate is less than additive, indicating that both bind to the same site on the enzyme, whereas that by lactate plus NO(3) (-) is additive, indicating binding at separate sites. It is inferred that a quaternary enzyme-pyruvate-NO(3) (-)-MgADP complex could form, but no evidence was obtained to suggest that it possessed special properties comparable with those found with creatine kinase. The implications of these findings for the unidirectional nature of the mechanism of pyruvate kinase is discussed. 8. Lactate or alpha-hydroxybutyrate could not act instead of pyruvate to form a stable quaternary complex, although both activate the K(+)-free enzyme. Only the former inhibits the K(+)-activated enzyme. The activating cation both lowers the Michaelis constant for phosphoenolpyruvate and tightens up the specificity of its binding site.     

Nuclear protein kinase activity in glucagon-stimulated perfused rat livers (Short Communication)

A simple method of purification of alpha-mannosidase from jack-bean meal is described which yields a product free of beta-N-acetylglucosaminidase activity.     

Ligand-induced effects on pyruvate dehydrogenase kinase isoform 2

Tryptophan fluorescence was used to analyze binding of ligands to human pyruvate dehydrogenase isoform 2 (PDHK2) and to demonstrate effects of ligand binding on distal structure of PDHK2 that is required for binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase. Ligand-altered binding of PDHK2 to L2 and effects of specific ligands on PDHK2 oligomeric state were characterized by analytical ultracentrifugation. ATP, ADP, and pyruvate markedly quenched the tryptophan fluorescence of PDHK2 and gave maximum quenching/L0.5 estimates: approximately 53%/3 microM for ATP; approximately 49%/15 microM for ADP; and approximately 71%/approximately 590 microM for pyruvate. The conversion of Trp-383 to phenylalanine completely removed ATP- and ADP-induced quenching and > or = 80% of the absolute decrease in fluorescence due to pyruvate. The W383F-PDHK2 mutant retained high catalytic activity. Pyruvate, added after ADP, quenched Trp fluorescence with an L0.5 of 3.4 microM pyruvate, > or = 150-fold lower concentration than needed with pyruvate alone. ADP-enhanced binding of pyruvate was maintained with W383F-PDHK2. Binding of PDHK2 dimer to L2 is enhanced when L2 are housed in oligomeric structures, including the glutathione S-transferase (GST)-L2 dimer, and further strengthened by reduction of the lipoyl groups (GST-L2(red)) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2(red) was modestly hindered by 200 microM level of ATP or ADP or 5.0 mM pyruvate; a marked change to nearly complete prevention of binding was observed with ATP or ADP plus pyruvate at only 100 microM levels, and these conditions caused PDHK2 dimer to associate to a tetramer. These changes should make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate. Ligand-induced changes that interfere with PDHK2 binding to GST-L2(red) may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.     

pH-controlled hydrogen-bonding (Short Communication)

The pH-dependence of the degree of hydrogen-bonding between a base and its conjugate acid is considered. When only a small proportion of the total base is complexed, the amount complexed is proportional to (1+coshp)(-1) where p=2.303 (pK(a)-pH), pK(a) being the dissociation constant of the conjugate acid. This represents sharp pH-dependence. As the proportion complexed increases, the curve broadens, eventually becoming flat-topped, with more than half the base complexed over the range of pH values pK(a)+/-logKC, approximately. (K is the complex association constant and C is the formal base concentration, including all forms.) There are similarities to the extent of mono-protonation of a dibasic acid.     

Inhibition by acetaldehyde of the pyruvate dehydrogenase complex from ox brain and ox kidney (Short Communication)

Low concentrations of acetaldehyde, similar to those that can occur in the brain of ethanol-treated animals, effectively inhibit the pyruvate dehydrogenase complex purified from ox brain or from ox kidney, although the precise mechanism of this inhibition remains to be defined.     

Virally mediated membrane changes: inverse effects on transport and diffusion (Short Communication)

SENDAI VIRUS MODIFIES THE CELL SURFACE IN TWO WAYS: (a) by inhibiting facilitated transport and (b) by enhancing passive diffusion.     

Specificity of anti-nucleoside antibodies (Short Communication)

The method of conjugation of a nucleoside or related compound to a carrier protein may have a significant effect on the specificity of the antibodies elicited. It is demonstrated, by means of the membrane-filtration assay, that anti-isopentenyladenosine antibodies produced by the ;periodate procedure' are much more reactive with the periodate-oxidized form of the nucleoside than with the parent compound. In addition, the simplicity and specificity of the assay used suggests its use as a sensitive radioimmunoassay for this multifunctional nucleoside.     

Porphyrins in egg shells (Short Communication)

T.l.c. of esterified egg-shell porphyrin shows a mixture containing protoporphyrin with admixture of significant amounts of coproporphyrin, pentacarboxylic porphyrin and uroporphyrin and other, unidentified, porphyrins. This points to porphyrin biosynthesis taking place in the oviduct epithelium.     

Polyphenol–protein interactions (Short Communication)

The association of some natural and synthetic polyphenols with beta-glucosidase was examined and some observations on the chemical nature of the complex were made.     

Crystallization of macromolecular heparin (Short Communication)

X-ray fibre-diffraction photographs were obtained from oriented films of the sodium salt of macromolecular heparin (molecular weight approx. 10⁶) prepared from rat skin. Two distinct molecular chain conformations corresponding to two different crystal lattices were observed as a function of relative humidity. The first conformation, obtained at 78% relative humidity, has a layer-line spacing of 1.73nm, which can be interpreted as an approximate twofold helix. On increasing the relative humidity to 84% a second phase with a layer-line repeat of 1.65nm is obtained with the reflexions indexing on a triclinic unit cell similar to that obtained previously (Nieduszynski & Atkins, 1973) for pig mucosal heparin.     

Tight linkage of pyruvate kinase (PKLR) and glucocerebrosidase (GBA) genes

Two polymorphisms, one in the liver-type pyruvate kinase gene (PKLR) and one in the glucocerebrosidase gene (GBA), both of which are on band q21 of chromosome 1, were found to be tightly linked. Each of three Gaucher disease mutations in 112 chromosomes studied was associated with a unique haplotype. With a conservative assumption about the length of time that the Gaucher disease mutation has been present in the Jewish population, we deduce that the genetic distance between these two loci is probably under 0.2 centimorgans. Four haplotypes are produced by these polymorphic loci, but two of these are relatively uncommon because the polymorphic sites are in linkage disequilibrium. Nonetheless these markers are potentially useful in the prenatal diagnosis of pyruvate kinase deficiency in families who have at least one affected child and may also be helpful in heterozygote detection in families with Gaucher disease where a specific mutation producing the disease in unknown.