SH-group Inhibitors Research Articles

Redox-dependent mechanisms of regulation of breast epithelial cell proliferation

Activation of free radical oxidation in different cell types, including breast epithelial cells, may result in damage to macromolecules, in particular, proteins taking part in regulation of cell proliferation and apoptosis. The glutathione, glutaredoxin and thioredoxin systems play an essential role in maintaining intracellular redox homeostasis. Due to this fact, modulation of cellular redox status under the effect of an SH group inhibitor and an SH group protector may be used as a model for studying the role of redox proteins and glutathione in regulating cell proliferation in different pathological processes. In this study we have evaluated the state of the thioredoxin, glutaredoxin and glutathione systems as well as their role in regulating proliferation of HBL-100 breast epithelial cells under redox status modulation with N-ethylmaleimide (NEM) and 1,4-dithioerythriol (DTE). Modulating the redox status of breast epithelial cells under the effect of NEM and DTE influences the functional activity of glutathione-dependent enzymes, glutaredoxin, thioredoxin, and thioredoxin reductase through changes in the GSH and GSSG concentrations. In HBL-100 cells under redox-status modulation, we have found an increase in the number of cells in the S-phase of the cell cycle and a decrease in the number of cells in the G0/G1 and G2/М phases, as opposed to the values in the intact culture. The proposed model of proliferative activity of cells under redox status modulation may be used for development of new therapeutic approaches for treatment of diseases accompanied by oxidative stress generation.

THE ROLE OF PROTEIN OXIDATIVE MODIFICATION IN REDOX-REGULATION OF CASPASE-3 ACTIVITY IN BLOOD LYMPHOCYTES DURING OXIDATIVE STRESS IN VITRO

The formation of oxidative stress lies at the heart of many frequent and socially-important diseases. Blood lymphocytes are the cells which provide immunological control of our organism. As a result of their function implementation blood lymphocytes contact with different endogenic and exogenic factors, which can lead to active oxygen species production activation, macromolecules oxidative modification and to cell survival alteration. At the present time it is essential to expand and deepen the fundamental knowledge of blood lymphocytes apoptosis regulation peculiarities. The research objective was to establish the interaction among alterations of glutathione system condition, carbonylation level, protein glutathionylation and caspase-3 activity in blood lymphocytes during oxidative stress in vitro. Material and Methods. The material for research was blood lymphocytes cultivated with addition of hydrogen peroxide in final concentration of 0,5 mmol and/or protein SH-group inhibitor N- ethylmaleimide – 5 mmol, protector – 5 mmol – 1,4-dithioerythritol. Reduced, oxidized and protein-bound glutathione concentration was measured by method of spectropho-tometry, additionally, the ratio size of reduced to oxidized thiol fraction was estimated. With help of enzymoimmunoassay the level of protein carbonyl derivatives was evaluated; caspase-3 activity was registered by spectrofluorometric method. Results . Protein SH-group blocking in blood lymphocytes during oxidative stress in vitro was accompanied by protein-bound glutathione concentration rapid decrease in connection with increase of protein carbonyl derivatives content and caspase-3 activity. Protein SH-group protection in blood lymphocytes during oxidative stress in vitro was accompanied by concentration increase of protein-bound glutathione and protein carbonyl derivatives under comparable values of enzyme activity under study. Conclusion . The carried out research shows that caspase-3 and protein-bound glutathione are the molecular targets of selective control over programmed cell death. The received indices of caspase-3 activity change and protein-bound glutathione concentration alteration in blood lymphocytes during oxidative stress in vitro can be used when elaborating target therapy approaches to diseases accompanied by apoptosis disregulation.

THE THIOREDOXIN SYSTEM IN REGULATING MCF-7 CELL PROLIFERATION UNDER REDOX STATUS MODULATION

Introduction. Despite the available data on tumor cell functioning under the conditions of free radical-mediated oxidation, the mechanisms of redox regulation, cell proliferation management and apoptosis avoidance remain understudied. The objective of the study was to identify the role of the thioredoxin system in regulating MCF-7 breast cancer cell proliferation under redox status modulation with 1.4-dithioerythritol. Material and methods. The studies were conducted on the MCF-7 breast cancer cell line, grown in adherent cell culture. Cell redox status was modulated with5 mM N-ethylmaleimide – an SH group and peptide inhibitor and5 mM 1.4-dithioerythritol – a thiol group protector. The cell cycle was evaluated by flow cytometry, the same technique was used to measure the reactive oxygen species concentration. The levels of reduced and oxidized glutathione and the activity of thioredoxin reductase were identified by spectrophotometry. The intracellular concentrations of thioredoxin, cyclin E and cyclin-dependent kinase 2 were determined by Western blot analysis. Results and discussion. The essential role of the thioredoxin system in regulating MCF-7 breast cancer cell proliferation was exhibited. S-phase arrest under the effect of N-ethylmaleimide and G0/G1-phase arrest under the effect of 1.4-dithioerythritol are associated with the changes in the activity of redox-sensitive protein complexes (cyclins and cyclin-dependent kinases) that regulate cell proliferation. Conclusion. Redoxdependent modulation of proliferation regulating intracellular protein activity occurs due to the thioredoxin system. This is a promising research area for seeking molecular targets of breast cell malignization.

THE ROLE OF PROTEIN OXIDATIVE MODIFICATION IN REDOX-REGULATION OF CASPASE-3 ACTIVITY IN BLOOD LYMPHOCYTES DURING OXIDATIVE STRESS IN VITRO

The formation of oxidative stress lies at the heart of many frequent and socially-important diseases. Blood lymphocytes are the cells which provide immunological control of our organism. As a result of their function implementation blood lymphocytes contact with different endogenic and exogenic factors, which can lead to active oxygen species production activation, macromolecules oxidative modification and to cell survival alteration. At the present time it is essential to expand and deepen the fundamental knowledge of blood lymphocytes apoptosis regulation peculiarities. The research objective was to establish the interaction among alterations of glutathione system condition, carbonylation level, protein glutathionylation and caspase-3 activity in blood lymphocytes during oxidative stress in vitro.Material and Methods. The material for research was blood lymphocytes cultivated with addition of hydrogen peroxide in final concentration of 0,5 mmol and/or protein SH-group inhibitor N-ethylmaleimide – 5 mmol, protector – 5 mmol – 1,4-dithioerythritol. Reduced, oxidized and protein-bound glutathione concentration was measured by method of spectropho-tometry, additionally, the ratio size of reduced to oxidized thiol fraction was estimated. With help of enzymoimmunoassay the level of protein carbonyl derivatives was evaluated; caspase-3 activity was registered by spectrofluorometric method.Results. Protein SH-group blocking in blood lymphocytes during oxidative stress in vitro was accompanied by protein-bound glutathione concentration rapid decrease in connection with increase of protein carbonyl derivatives content and caspase-3 activity. Protein SH-group protection in blood lymphocytes during oxidative stress in vitro was accompanied by concentration increase of protein-bound glutathione and protein carbonyl derivatives under comparable values of enzyme activity under study.Conclusion. The carried out research shows that caspase-3 and protein-bound glutathione are the molecular targets of selective control over programmed cell death. The received indices of caspase-3 activity change and protein-bound glutathione concentration alteration in blood lymphocytes during oxidative stress in vitro can be used when elaborating target therapy approaches to diseases accompanied by apoptosis disregulation.

Healing Effects of Wheat Grass (Triticum aestivum L) Extracts on RBC Membrane Damage

The study was mainly designed to validate the healing effects of wheat grass on RBC membrane damage. RBC membrane damage can be due to several factors, of which the free radical damage takes a major part. Many medicinal herbs have been used in study to find out their potential role in healing effects and at the same time in scavenging the free radicals. Several works were proved of the possible role of the flavonoids and phenols in the healing effects. A detailed study was done on the extracts of wheat grass in relation to its healing effects on the RBC membrane damage. The fractions (Chloroform & Methanol) were initially screened for the Phenolics and Flavonoids and then estimated for the total polyphenols. Further, the fractions were used for tested for the invitro experiments on the rat blood sample. % inhibition of Haemolysis, Chelating activity and SH group measurement are tested with the evaluated fractions. The plant responded positive for both the phytochemicals screened. The results of Folin –Ciocalteu total phenols photometric assay showed that the Methanol fraction was shown to contain more polyphenols (41%), than the chloroform fraction. Both the chloroform and methanol extracts showed the inhibition of haemolysis. Percent inhibition of haemolysis was dose dependent in both the fractions. The IC50 values of chloroform and methanol are 1.689 mg/ml and 2.481 mg/ml respectively. The IC50 values were calculated from the equation of slope (Y=mx+c), where Y is taken as 50. The SH group content showed an abrupt decrease in the presence of the SH group inhibitor Tetrathionate. The control showed a high value normally. When the samples containing Tetrathionate were preincubated with the chloroform and methanol extracts, the values showed an abrupt increase in respect to the Tetrathionate treated samples. This increase in the values from 195 to about 326 and 389 showed the healing effects of the extracts. Methanol extract showed a high healing effect when compared to the chloroform extract. This healing effect might be due to the presence of phenolic in high content. Past studies have been done on Wheat grass in relation to their total phenolics and flavonoids. At the same phenolics were used in studying against the RBC membrane damage. Our study suggests that the wheat grass can be used as a better and novel therapeutic agent in healing the RBC membrane damage induced by free radicals.

Environmental and Synthetic Sulphydryl Group Inhibitors: Effects on Bioluminescence and Respiration in Vibrio fischeri

Elemental sulphur (as S0 and S8) is abundant in anaerobic sediments and soil, and is highly toxic in the Vibrio fischeri bioluminescence test. This mode of S0 action remains uncertain. The objective of this research was the analysis of the toxic effects of S0 on bioluminescence and respiration in V. fischeri, in joint action with N-ethylmaleimide (NEM) or 2,4-dithio-DL-threitol (DTT), which are -SH group inhibiting and maintaining synthetic agents, respectively. Non-toxic DTT immediately protected cell bioluminescence against S0 inhibition at low (5.5ppb) and high (55ppb) concentrations of S0, whilst restoration of the inhibitory effect of S0 took up to 30 minutes. NEM (62.5ppb) diminished cell bioluminescence by up to 50% after 5 minutes, but after 60 minutes, the inhibition reached 100%. DTT restored the bioluminescence function inhibited in vivo and in vitro by S0 and NEM. Enhancement of cell respiration by up to 20% and 33% was observed at 2.2ppm of S0 and 36.8ppm of 2,4-dinitrophenol (2,4-DNP; an uncoupler of oxidative phosphorylation), respectively; whilst NEM (3.1ppm) caused a reduction of up to 40%. This comparative analysis confirmed that S0 has multiple modes of action--it acts as both an -SH group inhibitor and an uncoupler of oxidative phosphorylation in V. fischeri cells.

Plant sucrose-H+ symporters mediate the transport of vitamin H.

A cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake-deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH-group inhibitor p-chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP), suggesting an energy-dependent biotin-H+ symport mechanism. The transport activity could contribute to the so-far uncharacterized plant sucrose-H+ symporter AtSUC5 which mediates the energy-dependent transport of biotin and sucrose, and restores growth of the biotin transport-deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin-H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.

Reaction mechanism of thioredoxin: 3'-phospho-adenylylsulfate reductase investigated by site-directed mutagenesis.

Properties of purified recombinant adenosine 3'-phosphate 5'-phosphosulfate (PAdoPS) reductase from Escherichia coli were investigated. The Michaelis constants for reduced thioredoxin and PAdoPS are 23 microM and 10 microM, respectively; the enzyme has a Vmax of 94-99 mumol min-1 mg-1 and a molecular activity/catalytically active dimer of 95 s-1. Adenosine 3',5'-bisphosphate (PAdoP) inhibits competitively (Ki 4 microM) with respect to PAdoPS; adenosine 2',5'-bisphosphate and sulfite are not inhibitory. Alkylation by SH-group inhibitors irreversibly inactivates the enzyme. The structural gene (cysH) encodes for a small polypeptide with a single Cys residue located in a conserved cluster (KXECGI/LH) of amino acids. Involvement of the only Cys and of Tyr209 in the reduction of PAdoPS to sulfite was investigated by site-specific mutagenesis: cysH was mutated by single-strand-overlay extension PCR; the mutated genes were cloned in pBTac1 and expressed in E. coli RL 22 (delta cysHIJ). Homogenous Cys239Ser and Tyr209Phe mutant PAdoPS reductases were investigated for altered catalytic properties. Mutation of the single Cys reduced Vmax by a factor of 4.5 x 10(3) (Vmax = 0.02-0.013 mumol min-1 mg-1) with marginal effects on Km for PAdoPS (19 microM) and reduced thioredoxin (14 microM). Mutation of Tyr209 drastically affected saturation with thioredoxin (Km 1.5 microM) and decreased Vmax (0.22-0.25 mumol min-1 mg-1) in addition to a small increase in Km for PAdoPS (31 microM). Chromophores as prosthetic groups were absent from recombinant PAdoPS reductase. Difference absorption spectra between reduced and oxidized forms of wild-type and mutated proteins indicated that, in addition to Cys239 and Tyr209, an unidentified Trp (delta lambda max 292 nm) appears to be involved in the reduction. The data suggest a special ping-pong mechanism with PAdoPS reacting with the reduced enzyme isomer in a Theorell-Chance type mechanism.

A phloem‐specific sucrose‐H+ symporter from Plantago major L. supports the model of apoplastic phloem loading

In this paper the cloning of a full-length cDNA clone encoding the PmSUC2 sucrose-H+ symporter from Plantago major is described. This plant allows the simple preparation of vascular bundles from the basal regions of fully developed source leaves and thus a separation of vascular and non-vascular tissue. A cDNA library was constructed from poly(A)+ RNA isolated from vascular bundles and used for the subsequent cloning of cDNAs. The respective mRNA is specifically expressed in the vascular bundles as shown on Northern blots of total RNA from vascular and non-vascular tissues. The PmSUC2 protein has 12 putative transmembrane helices and is highly homologous to other plant sucrose transporters. Substrate specificity and energy dependence of the transporter encoded by this cDNA were determined by expression in baker's yeast Saccharomyces cerevisiae. The PmSUC2 protein catalyses the transport of sucrose into transgenic yeast cells. Invertase null mutants of yeast expressing PmSUC2 accumulate sucrose more than 200-fold. This transport was sensitive to uncouplers or SH-group inhibitors. Plasma membranes from yeast cells expressing the PmSUC2 protein were purified and fused to proteoliposomes containing cytochrome-c-oxidase. In this system sucrose is accumulated only when proton motive force is generated, indicating that PmSUC2 is a sucrose-H+ symporter. The apparent molecular weight of the PmSUC2 protein is 35 kDa on 10% SDS-polyacrylamide gels. The presented data strongly support the theory of phloem loading from the apoplastic space by a sucrose-H+ symporter.

Threonine is present instead of cysteine at the active site of urease from Staphylococcus xylosus.

DNA sequence analysis of the structural urease genes from Staphylococcus xylosus revealed that three enzyme subunits are encoded in the order of 11,000, 15,400 and 61,000 (mol. mass), which correspond to the single polypeptide chain of jack bean urease (90,800). Comparing the deduced amino acid sequence of S. xylosus urease with the amino acid sequence of jack bean urease an overall portion of 56% identical residues was found. For S. xylosus urease a subunit structure of (alpha beta gamma)4 was proposed, based on the comparison of the deduced amino acid content of the enzyme subunits with the total amino acid content of the purified enzyme. The staphylococcal enzyme contained no cysteine, as deduced from DNA sequence and confirmed by the determination of the total amino acid content in the purified enzyme. Instead of cysteine, known to be catalytically essential in the plant enzyme, and conserved among all bacterial ureases analyzed so far, threonine was found in S. xylosus. This amino acid-exchange was located within a highly conserved domain of 17 amino acids, supposed to be part of the active site. Sequence analysis of the respective region of Staphylococcus saprophyticus urease showed that it also contains threonine instead of cysteine. In contrast to jack bean urease S. xylosus urease was not affected by the SH-group inhibitor dipyridyl disulfide but was completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The presented results indicate that in these staphylococcal strains urea hydrolysis might function in a manner similar to the peptide bond cleavage by chymotrypsin.

Novel Ca2+-activated neutral protease from an aquatic fungus, Allomyces arbuscula.

A Ca2+-activated neutral protease was purified to homogeneity from an aquatic Phycomycete fungus, Allomyces arbuscula. It requires millimolar concentrations of Ca2+ for activation (1.8 to 2 mM for 50% activation). Sr2+ can replace Ca2+ but at higher concentrations (4 mM for 50% activation). The enzyme is a dimer of 40-kilodalton subunits and contains six cysteine residues, three of which are revealed only after the addition of micromolar concentrations of Ca2+; the other three are free. Enzyme activity is strongly inhibited by SH-group inhibitors and some trypsin inhibitors (leupeptin and alpha-N-tosyl-L-lysine chloromethyl ketone). The enzyme lacks general trypsinlike specificity, since substrates containing tryptic cleavage sites are not cleaved nor is enzyme activity inhibited by other trypsin inhibitors. The enzyme has many functional similarities to the extensively characterized mammalian and avian Ca2+-activated neutral proteases but differs in its substrate specificity, inhibition by alpha-N-tosyl-L-phenylalanine chloromethyl ketone, and subunit structure. It is, nevertheless, presumed that this enzyme has a similar high order of specificity and is involved in the regulation of a specific growth function.

Uptake of Lupanine by Alkaloid-Storing Epidermal Cells ofLupinus polyphyllus

Epidermis of steins and petioles of LUPINUS POLYPHYLLUS accumulates quinolizidine alkaloids at a concentration of about 30 mM. Since lupanine is synthesized mainly in green mesophyll tissue and not in the epidermis, the alkaloids have to be transported into the epidermal cells. Uptake of [ (3)H]-lupanine into isolated epidermis was 3 to 20 times higher in epidermal cells as compared to the corresponding mesophyll cells. Uptake of lupanine is time dependent and proceeds against a concentration gradient. The uptake depends on temperature and can be characterized by an activation energy of 34 kJ/mol. The process shows multiphasic uptake kinetics and is reduced by SH-group inhibitors (NEM, PHMB) and inhibitors of the energy metabolism (cyanide, antimycine, DNP, CCCP). All these data provide first evidence that simple diffusion cannot be the mechanism for the uptake of lupanine into epidermal cells. The uptake is probably catalyzed by transport proteins.

Purification and immunochemical characterization of a dihydrofolate reductase-thymidylate synthase enzyme complex from wild-carrot cells

An enzyme complex with dihydrofolate reductase (DHFR, E.C.1.5.1.3.) activity was purified to apparent homogenity from wild-carrot cells. The complex has a mol. wt of 286 kd and contains five polypeptide chains of 95, 70, 50, 45 and 26 kd. The DHFR enzyme activity and methotrexate-binding site are on the 45-kd subunit. Folate analogs (methotrexate, aminopterin and formylaminopterin) as well as SH-group inhibitors [p-hydroxymercuribenzoate, 5,5' -dithiobis(2-nitrobenzoic acid), or N-ethylmaleimide] inhibit DHFR. Thymidylate synthase (TS, E.C.2.1.1.45) activity co-purified with the enzyme complex through each of seven steps and co-eluted from gel filtration columns with the DHFR activity at the mol. wt of the enzyme complex. Further identification of TS within the complex was achieved using a Leishmania DHFR-TS antisera which specifically inhibited the carrot TS, although it immunoprecipitated both TS and DHFR. Polyclonal antisera, raised against and specific for the complex as judged by Ouchterlony double diffusion tests and Western blot analysis, inhibited and immunoprecipitated both DHFR and TS. The Leishmania antisera also identified the 70-kd polypeptide within the purified complex as TS in a Western blot experiment. The functions of the other three polypeptides have not yet been established.

Origin and fate of the lysophosphatidylethanolamine in a chain-forming mutant (envC) of Escherichia coli.

The role of phospholipid metabolism in the functioning of the bacterial envelope was investigated in the chain-forming Escherichia coli envC. Lysophosphatidylethanolamine (LPE) which accumulated in this strain during growth was identified as the product of phosphatidylethanolamine (PE) hydrolysis by a phospholipase A1, i.e. 2-acylLPE. Isotopically labelled LPE transferred into intact mutant and parent cells by liposome/bacteria interaction was rapidly reacylated to PE. However, in envC the final PE/LPE ratio was lower than that in the parent, thus showing that the fate of LPE is modified. Crude cell extracts degraded LPE to a lesser extent in envC than in the parent but were unable to promote reacylation activity under our experimental conditions. In both strains, the lysophospholipase activity was neither calcium-dependent nor inhibited by the SH-group inhibitors pHMB or pCMPS, and hydrolysed 1-acylLPE as well as 2-acylLPE. These results indicate the existence of a deacylation-reacylation cycle in E. coli and show that this cycle is perturbed in envC cells, especially at the lysophospholipase step.

Purification and properties of indole 2,3-dioxygenase from maize leaves

An indole 2,3-dioxygenase was purified ca 38-fold from maize leaves. The enzyme had an MW of about 98000, an optimum pH of 5.0 and the energy of activation was 9.1 kcal/mol. The K max for indole was 1.4 × 10 −4 M. The enzyme was inhibited by diethyldithiocarbamate, salicylaldoxime and sodium dithionite. The inhibition by diethyldithiocarbamate was specifically reversed by Cu 2+. The dialysed enzyme was stimulated by Cu 2+. Four atoms of oxygen were utilized in the disappearance of 1 mole of indole. Inhibition of the enzyme by -SH compounds and -SH group inhibitors, and their partial removal by Cu 2+ only, suggested the involvement of -SH groups in binding of Cu 2+ at the catalytic site.

A novel enzyme, tagatose kinase, from Mycobacterium butyricum

A novel enzyme catalyzing the phosphorylation of d-tagatose to d-tagatose 6-phosphate with ATP has been identified in extracts of dulcitol-grown Mycobacterium butyricum. The enzyme was purified 100-fold with 29% recovery. It required Mg 2+, Mn 2+ or Fe 2+ and showed maximum activity at pH 7.5. The molecular weight as determined by Sephadex G-100 filtration amounted to 63 000. The apparent Michaelis constants for d-tagatose and ATP were 0.8 and 1.0 mM, respectively. Apart from tagatose, none of 14 common sugars including fructose and sorbose served as a substrate. The enzyme preparations were not very sensitive to SH group inhibitors and heavy metals but rapidly lost activity on heating above 50°C.

Characterization of a lignin-specific O-Methyltransferase in aspen wood

O-Methyltransferases were extracted from the differentiating xylem of 10-yr-old Populus euramericana. The enzymes were partially purified by ammonium sulfate precipitation, and column chromatography on DEAE-cellulose, Sephadex G200 and hydroxyapatite. The enzymes were resolved into two peaks by DEAE-cellulose chromatography, and the MWs of the respective enzymes were estimated to be 72 000 and 75 000 by gel filtration chromatography. The enzyme corresponding to the latter peak was unstable and thus only the former peak enzyme was characterized completely. Magnesium ions had no effect, EDTA moderately stimulated and heavy metals and SH group inhibitors strongly inhibited enzyme activity. K m m values for caffeate and 5-hydroxyferulate were estimated to be 3.8 x 10 −4 and 3.1 x 10 −4 M, respectively. The ratio of V max/ K m for 5-hydroxyferulate was 5.4 times greater than that for caffeate. The enzyme(s) catalysing the formation of ferulate from caffeate and of sinapate from 5-hydroxyferulate were not separated during the purification or by the disc electrophoresis using polyacrylamide gel. Quercetin, cyanin and catechin were not methylated by the enzyme preparation. The O-methyltransferase of aspen wood, where the phenolic metabolism is almost exclusively directed to lignin biosynthesis, catalyses the methylation of both guaiacyl and syringyl lignin precursors, with preferential utilization of the latter substrate. These findings lead to the conclusion that the enzyme is a typical angiosperm-type O-methyltransferase related to guaiacyl and syringyl lignin biosynthesis in aspen wood.

Biochemistry of terminal deoxynucleotidyltransferase: identification, characterization, requirements, and active-site involvement in the catalysis of associated pyrophosphate exchange and pyrophosphorolytic activity

Terminal deoxynucleotidyltransferase (TdT) has been found to catalyze both pyrophosphate exchange and pyrophosphorolysis reactions. Both reactions are strongly inhibited by antiserum to TdT. The reactions require the presence of a divalent cation, a single- or double-stranded oligomeric or polymeric DNA or RNA, and deoxyribonucleoside triphosphates (for PPi exchange only). Of the three divalent cations tested, Mg2+ and Co2+ are equally effective, while Mn2+ neither is used for catalysis nor inhibits the Mg2+-catalyzed reactions. Ribonucleoside triphosphates have been found to support the PPi exchange reaction to a minor extent and have no inhibitory effect on the catalysis mediated by dNTPs. Inhibition studies, using SH group inhibitors, Zn chelator, and a substrate binding site specific reagent, revealed that PPi exchange and pyrophosphorolysis reactions may be distinguished by differences in their sensitivity to inhibition by various reagents. While the PPi exchange reaction is strongly inhibited by sulfhydryl reagents, o-phenanthroline, and pyridoxal phosphate, the pyrophosphorolysis reaction is insensitive to these reagents. In addition, the pyrophosphorolysis reaction is also found not to require a free 3'-OH terminus of a primer. This difference in the susceptibility of the two reactions indicates that discrete active-site structures exist in TdT which catalyze PPi exchange and pyrophosphorolysis reactions.

Evidence for the Existence of meta and para Directing O-Methyltransferases in Tobacco Cell Cultures

The O-methyltransferase of tobacco cell culture was resolved to its meta and para directing forms by chromatography on DEAE-cellulose. Despite similarities in molecular weights and pI values of the two forms, however, evidence from pH optima, SH-group inhibitors, methylation ratios, SDS-acrylamide gels and mixed substrate experiments indicates the existence of two discrete enzymes acting at the meta and para positions of caffeic acid and quercetin, respectively; though the latter enzyme was less substrate specific than its meta counterpart.

Surface Sulphydryl Groups and Phagocytosis‐Associated Oxidative Metabolic Changes in Human Polymorphonuclear Leucocytes

The role of surface sulphydryl (-SH) groups of human polymorphonuclear leucocytes on phagocytosis-associated oxidative metabolic changes was studied. p-Chloromercurybenzene sulphonic acid, a surface -SH group inhibitor, had no effect on phagocytosis, superoxide production during phagocytosis, or killing of S. aureus by the leucocytes, while it inhibited phagocytosis-associated stimulation of hexose monophosphate pathway activity and H2O2 production. In contrast, N-ethylmaleimide, which inhibits both intracellular and surface -SH groups, inhibited phagocytosis-associated oxidative metabolic changes. p-Chloromercurybenzene sulphonic acid also inhibited the stimulation of hexose monophosphate pathway activity by exogenous H2O2, suggesting a regulatory role of plasma membrane on the pathway's activity in human polymorphonuclear leucocytes. The results also suggest that: (I) superoxide production is the primary event of oxidative metabolic changes during phagocytosis, (2) superoxide plays an important role in the killing of S. aureus, and (3) the hexose monophosphate pathway plays a primary role in producing NADPH for H2O2 production from superoxide.