Atorvastatin accelerates extracellular nucleotide degradation in human endothelial cells

HMG-CoA reductase inhibitors (statins) exert pleiotropic effects in the cardiovascular system beyond its cholesterol-lowering action. We aimed to investigate how atorvastatin affects extracellular nucleotide degradation in human endothelial cells, as increased activity of this pathway would facilitate conversion of pro-inflammatory nucleotides into anti-inflammatory adenosine. Primary cultures of human endothelial cells were treated with 1 μM, 10 μM and 100 μM atorvastatin for 24 h. Enzyme assays were performed as well as intact cell studies, to evaluate capacity of cells to degrade ATP to adenosine. Atorvastatin significantly increased ATP breakdown and adenosine formation in the medium of intact cells in a dose-dependent manner. The activities of ATPase, ADPase and ecto-5′-nucleotidase (eN) in cell homogenates following Atorvastatin treatment were also increased while no change was observed in the lactate dehydrogenase activity. We suggest a new mechanism of protective effect of atorvastatin by activation of endothelial enzymes involved in extracellular nucleotide degradation in human endothelial cells.     

Metal binding to pyridoxal derivatives. An NMR study of the interaction of Eu(III) with pyridoxal phosphate and pyridoxamine phosphate

The solution conformations of pyridoxal-5′ -phosphate and pyridoxamine-5′-phosphate have been investigated using Eu(III) as a nuclear magnetic resonance shift probe. Binding of Eu(III) to pyridoxal phosphate results in the formation of two complexes, at the phosphate group and theo-hydroxy-aldehyde moiety, which are in slow exchange on the nuclear magnetic resonance time-scale. The lanthanide-induced pseudo contact shifts calculated using the McConnell-Robertson equation (J. Chem. Soc. (1950), 22, 1561) are in good agreement with the experimentally observed values for both pyridoxal phosphate and pyridoxamine phosphate and lead to a family of closely related conformations. Contribution No. 130 from the Molecular Biophysics Unit.     

Sequence analysis of the gene encoding a novel l-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii: similarity to the group II amino acid decarboxylases

The gene (ddc) encoding a novel enzyme, l-2,4-diaminobutyrate decarboxylase (DABA-DC; EC 4.1.1.-) in Acinetobacter baumannii was sequenced, and an open reading frame of 1,530 nucleotides was detected. The sequence of 20 N-terminal amino acids of purified DABA-DC and of its proteolytic peptide fragments coincided with those deduced from the nucleotide sequence determined. Comparison of the predicted amino acid sequence of the A. baumannii enzyme with those of other pyridoxal 5′-phosphate-dependent decarboxylases revealed significant similarity to the group II amino acid decarboxylases and conservation of the putative pyridoxal 5′-phosphate-binding domain. Received:20 February 1996 / Accepted 15 April 1996     

Effects of haloperidol, clotrimazole, and pyridoxal-5′-phosphate on synaptic transmission in smooth muscles of the human colon

In strips of smooth muscles of the human colon, haloperidol (Hal) and clotrimazole (Clo), in contrast to pyridoxal-5′-phosphate (PP), suppressed spontaneous electrical and contractile activities of these strips and also post-inhibitory excitation developing after inhibitory synaptic potentials (ISPs). Haloperidol, Clo, PP, and PP applied against the background of the action of Nω-nitro-L-arginine noticeably changed the parameters of ISPs. The pattern of effect of Hal on synaptic inhibition in smooth muscles was preserved against the background of the action of PP, and that of PP was preserved against the background of the action of Hal. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 408–411, July–October, 2007.     

Enzyme-catalyzed side reactions with molecular oxygen may contribute to cell signaling and neurodegenerative diseases

A link between neurodegeneration and well-characterized enzymatic and non-enzymatic reactions that produce reactive oxygen species (ROS) from O₂ is well established. Several enzymes that contain pyridoxal 5′-phosphate (PLP) or thiamine diphosphate (ThDP) catalyze side reactions (paracatalytic reactions) in the presence of ambient O₂. These side reactions produce oxidants such as hydrogen peroxide [H₂O₂] or extremely reactive peracids [RC(O)OOH]. We hypothesize that although these enzymes normally produce oxidants at low or undetectable levels, changes in substrate levels or disease-induced structural alterations may enhance interactions with O₂, thereby generating higher levels of reactive oxidants. These oxidants may damage the enzymes producing them, alter nearby macromolecules and/or destroy important metabolites/coenzymes. We propose that paracatalytic reactions with O₂ catalyzed by PLP-dependent decarboxylases and by ThDP-dependent enzymes within the α-keto acid dehydrogenase complexes may contribute to normal cellular signaling and to cellular damage in neurodegenerative diseases. Special issue dedicated to John P. Blass.     

The role of nickel in acetyl-CoA synthesis by the bifunctional enzyme CO dehydrogenase/acetyl-CoA synthase: enzymology and model chemistry

 CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) is one of the four known nickel enzymes. It is a bifunctional protein that catalyzes the oxidation of CO to CO₂ at a nickel iron-sulfur cluster (Cluster C) and a remarkable condensation reaction between a methyl group (donated from a methylated corrinoid iron-sulfur protein), carbon monoxide, and coenzyme A to form acetyl-CoA at a separate nickel iron-sulfur cluster (Cluster A). This review focuses on the current understanding of the structure and function of Cluster A and on related model chemistry. It describes studies that uncovered the first example of a biological organometallic reaction sequence. The mechanism of acetyl-CoA synthesis includes enzymebound methylnickel, iron-carbonyl, and acylmetal intermediates. Discovery of the methylnickel species constituted the first example of an alkylnickel species in biology and unveiled a new biological role for nickel. Received: 10 April 1996 / Accepted: 4 July 1996     

The NAD-linked soluble hydrogenase from Alcaligenes eutrophus H16: detection and characterization of EPR signals deriving from nickel and flavin

 In this study we confirmed the previous observation that the cytoplasmic NAD-linked hydrogenase of Alcaligenes eutrophus H16 is EPR-silent in the oxidized state. We also demonstrated the presence of significant Ni-EPR signals when the enzyme was either reduced with the natural electron carrier NADH (5–10 mM) or carefully titrated with sodium dithionite to an intermediate, narrow redox potential range (–280 to –350 mV). Reduction with NADH under argon atmosphere led to a complex EPR spectrum at 80 K with g values at 2.28, 2.20, 2.14, 2.10, 2.05, 2.01 and 2.00. This spectrum could be differentiated by special light/dark treatments into three distinct signals: (1) the "classical" Ni-C signal with g values at 2.20, 2.14 and 2.01, observed with many hydrogenases in the reduced, active state; (2) the light-induced signal (Ni-L) with g values at 2.28, 2.10 and 2.05 and (3) a flavin radical (FMN semiquinone) signal at g = 2.00. The assignment of the Ni-EPR signal was clearly confirmed by EPR spectra of hydrogenase labeled with ⁶¹Ni (nuclear spin I = 3/2) yielding a broadening of the Ni spectra at all g values and a resolved ⁶¹Ni hyperfine splitting into four lines of the low field edge in the case of the light-induced Ni-EPR signal. The redox potentials determined at pH 7.0 for the described redox components were: for FMN –170 mV (midpoint potential, E_m, for appearance), –200 mV (EPR signal intensity maximum) and –230 mV (E_m for disappearance); for the Ni centre (Ni-C), –290 mV (E_m for appearance), –305 mV (signal intensity maximum) and –325 mV (E_m for disappearance). Exposure of the NADH-reduced hydrogenase to carbon monoxide led to an apparent Ni-CO species indicated by a novel rhombic EPR signal with g values at 2.35, 2.08 and 2.01. Received: 19 July 1995 / Accepted: 10 September 1995     

Overexpression and characterization of dimeric and tetrameric forms of recombinant serine hydroxymethyltransferase from Bacillus stearothermophilus

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5′-phosphate (PLP) dependent enzyme catalyzes the interconversion of L-Ser and Gly using tetrahydrofolate as a substrate. The gene encoding for SHMT was amplified by PCR from genomic DNA ofBacillus stearothermophilus and the PCR product was cloned and overexpressed inEscherichia coli. The purified recombinant enzyme was isolated as a mixture of dimer (90%) and tetramer (10%). This is the first report demonstrating the existence of SHMT as a dimer and tetramer in the same organism. The specific activities at 37°C of the dimeric and tetrameric forms were 6.7 U/mg and 4.1 U/mg, respectively. The purified dimer was extremely thermostable with aT _m of 85°C in the presence of PLP and L-Ser. The temperature optimum of the dimer was 80°C with a specific activity of 32.4 U/mg at this temperature. The enzyme catalyzed tetrahydrofolate-independent reactions at a slower rate compared to the tetrahydrofolate-dependent retro-aldol cleavage of L-Ser. The interaction with substrates and their analogues indicated that the orientation of PLP ring ofB. stearothermophilus SHMT was probably different from sheep liver cytosolic recombinant SHMT (scSHMT).     

The puzzle of the Krebs citric acid cycle: Assembling the pieces of chemically feasible reactions,and opportunism in the design of metabolic pathways during evolution

The evolutionary origin of the Krebs citric acid cycle has been for a long time a model case in the understanding of the origin and evolution of metabolic pathways: How can the emergence of such a complex pathway be explained? A number of speculative studies have been carried out that have reached the conclusion that the Krebs cycle evolved from pathways for amino acid biosynthesis, but many important questions remain open: Why and how did the full pathway emerge from there? Are other alternative routes for the same purpose possible? Are they better or worse? Have they had any opportunity to be developed in cellular metabolism evolution? We have analyzed the Krebs cycle as a problem of chemical design to oxidize acetate yielding reduction equivalents to the respiratory chain to make ATP. Our analysis demonstrates that although there are several different chemical solutions to this problem, the design of this metabolic pathway as it occurs in living cells is the best chemical solution: It has the least possible number of steps and it also has the greatest ATP yielding. Study of the evolutionary possibilities of each one-taking the available material to build new pathways-demonstrates that the emergence of the Krebs cycle has been a typical case of opportunism in molecular evolution. Our analysis proves, therefore, that the role of opportunism in evolution has converted a problem of several possible chemical solutions into asingle-solution problem, with the actual Krebs cycle demonstrated to be the best possible chemical design. Our results also allow us to derive the rules under which metabolic pathways emerged during the origin of life.     

Formation of aerial hyphae and conidia under orthophosphate-limited conditions inNeurospora crassa: Role of repressible cyclic phosphodiesterase

A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them. In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM.     

Lead inhibits type-I iodothyronine 5′-monodeiodinase in the Indian rock pigeonColumba livia: A possible involvement of essential thiol groups

A study has been made to reveal the mode of action of lead inhibiting type-I iodothyronine 5′-monodeiodinase (5′-D) activity in the Indian rock pigeon,Columba livia. Administration of lead acetate (6 mg/kg body weight/day) for 20 days decreased 5′-D activity and glutathione content in the liver and kidney homogenates. It also reduced the serum concentration of 3, 3′, 5-triiodothyronine (T₃) with a marginal increase in serum thyroxine (T4). Hepatic and renal lipid peroxidative process increased significantly following lead treatment, whereas the levels of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were decreased. The possible involvement of lipid peroxidative process in the inhibition of 5′-D activity inColumba livia is suggested.     

Dual Role of Prostaglandins (PGE2) in Regulation of Channel Density and Open Probability of Epithelial Na+ Channels in Frog Skin (R. pipiens)

Prostaglandins are important in signaling pathways involved in modulating the rates of Na⁺ transport in a diverse group of tissues possessing apical membrane epithelial channels. PGE₂ is known to cause either stimulation, inhibition or transient stimulatory changes of Na⁺ transport. We have continued our studies of frog skins that are known to respond to forskolin and PGE₂ with large steady-state increases of transport and have used noninvasive methods of blocker-induced noise analysis of Na⁺ channels to determine their channel densities (N _T ) and open probabilities (P _o ). In the absence of exogenous hormones, baseline rates of Na⁺ transport are especially high in scraped skins (R. pipiens pipiens) studied in the fall of the year. Na⁺ transport was inhibited by indomethacin and by removal of the unstirred layers of the corium (isolated epithelia) alone suggesting that PGE₂ is responsible for the sustained and elevated rates of transport in scraped skins. Changes of transport caused by indomethacin, forskolin or PGE₂ were unquestionably mediated by considerably larger changes of N _T than compensatory changes of P _o . Since cAMP caused no change of P _o in tissues pretreated with indomethacin, PGE₂ appears in this tissue to serve a dual role, increasing the steady state N _T by way of cAMP and decreasing P _o by unknown mechanisms. Despite appreciable PGE₂-related decreases of P _o , the net stimulation of transport occurs by a considerably greater cAMP-mediated increase of N _T . Received: 28 February 1996/Revised: 22 August 1996     

Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens

Sulfite-oxidizing enzyme activities were analyzed in cell-free extracts of aerobically grown cells of Acidianus ambivalens, an extremely thermophilic and chemolithoautotrophic archaeon. In the membrane and cytoplasmic fractions, two distinct enzyme activities were found. In the membrane fraction, a sulfite:acceptor oxidoreductase activity was found [530 mU (mg protein)^–1; apparent K _m for sulfite, 3.6 mM]. In the cytoplasmic fraction the following enzyme activities were found and are indicative of an oxidative adenylylsulfate pathway: adenylylsulfate reductase [138 mU (mg protein)^–1], adenylylsulfate:phosphate adenyltransferase [“ADP sulfurylase”; 86 mU (mg protein)^–1], adenylate kinase [650 mU (mg protein)^–1], and rhodanese [thiosulfate sulfur transferase, 9.2 mU (mg protein)^–1]. In addition, 5′,5′′′-P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A) synthase and Ap₄A pyrophosphohydrolase activities were detected. Received: 17 August 1998 / Accepted: 29 April 1999     

Interaction of pyridoxal phosphate with the amino groups at the active site of 5- aminolevulinic acid dehydratase in maize

Pyridoxal 5′-phosphate strongly and reversibly inhibited maize leaf 5-amino levulinic acid dehydratase. The inhibition was linearly competitive with respect to the substrate 5-aminolevulinic acid at pH values between 7 to 9.0. Pyridoxal was also effective as an inhibitor of the enzyme but pyridoxamine phosphate was not inhibitory. The results suggest that pyridoxal 5′-phosphate may be interacting with the enzyme either close to or at the 5-aminolevulinic acid binding site. This conclusion was further corroborated by the detection of a Schiff base between the enzyme and the substrate, 5-aminolevulinic acid and by reduction of pyridoxal phosphate and substrate complexes with sodium borohydride     

Chemical ligation and recombination of DNA fragments through formation (exchange) of disulfide bonds located in the sugar-phosphate backbone

Effective methods of the directed introduction of diphosphoryl disulfide bridges into hairpin DNA duplexes in place of natural phosphodiester groups were developed using the H₂O₂-effected ligation of 3′- and 5′-thiophosphorylated oligonucleotides or by autoligation of a preactivated oligonucleotide derivative with a phosphorothioate-bearing oligomer. The postsynthetic recombination of the disulfide-linked oligonucleotide fragments was characterized. It was shown that, along with template-directed reactions, out-of-duplex formation and exchange of diphosphoryl disulfide bonds in the DNA sugar-phosphate backbone may occur. In modified hairpin DNA, a spontaneous exchange of disulfide-linked fragments virtually does not take place because of the intramolecular duplex formation.     

Differences in the cellular zinc content and 5′-nucleotidase activity of normal and acrodermatitis enteropathica (AE) fibroblasts

The acrodermatitis enteropathica (AE) mutation affects zinc (Zn) metabolism in human fibroblasts. We hypothesize that the mutation affects the cell Zn content, which subsequently affects the activity of various zinc-dependent enzymes, such as 5′-nucleotidase. Therefore, normal and AE fibroblasts were grown in normal medium containing physiological levels of Zn (16 Μmol/L) for ∼24 h. The medium was replaced by normal medium (16 Μmol/L Zn), Zn-depleted medium (1.5 Μmol/L Zn), or Zn-supplemented medium (200 Μmol/L Zn) for another 24 h. Regardless of the Zn concentration of the growth medium, the AE fibroblasts contained significantly less Zn than normal fibroblasts grown in comparable medium. Nevertheless, growth of the fibroblasts in 200 Μmol/L Zn medium significantly increased the cell Zn content fourfold of both normal and AE fibroblasts. The activity of 5′-nucleotidase in the AE fibroblasts grown in 16 Μmol/L Zn or 1.5 Μmol/L Zn medium was also significantly lower than in normal fibroblasts. Changing the growth medium from 16 Μmol/L Zn to 1.5 Μmol/L Zn medium did not affect the activity of the enzyme in either genotype. Cells grown in 200 Μmol/L Zn medium exhibited threefold greater 5′-nucleotidase activity in AE fibroblasts, but had no affect on enzyme activity in normal cells. In summary, altering the cell Zn content of normal fibroblasts did not result in a significant change in their 5′ -nucleotidase activity. However, AE fibroblasts grown in 200 Μmol/L Zn medium exhibited recovery of their 5′-nucleotidase activity to normal levels. These results support the hypothesis that the AE mutation affects the cellular Zn content. The lower cell Zn content subsequently affects the activity of 5′-nucleotidase.