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At least 703 records · Page 39Linked to original sources
PubMed1998

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism in human hepatic microsomes by ipomeanol analogs--an exploratory study.

The tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen in mice, rats and Syrian golden hamsters and a suspected human lung carcinogen. We have reported earlier that structural analogs of the naturally occurring pulmonary toxin 4-ipomeanol (IPO) were non toxic up to 50 micromol/mouse. Because these analogs are in part structurally similar to NNK, they are expected to compete for the same enzymes and/or reactive sites within DNA. Both NNK and IPO are primarily metabolized by cytochrome P450 enzymes in the Clara cells of the lung but also in the liver. We describe here the optimal conditions for the study of NNK metabolism in human liver microsomes and our investigation of four non-toxic IPO analogs as potential inhibitors of NNK activation. The IPO analogs studied were 4-hydroxy-1-phenyl-1-octanone (4-HPO), 1,4-diphenyl-4-hydroxy-1-butanone (DPHB), 4-hydroxy-1-phenylpentane (HPPentane) and amyl benzene (AB). When added to microsomal incubations of human liver cells at a concentration of 100 microM, all of these compounds were strong inhibitors of NNK activation, decreasing the total alpha-hydroxylation of NNK, which is the main pathway of activation, by 60-70% and preventing N-oxidation by 78-86%.

Antineoplastic Agents↗
PubMed1999

Thermally triggered metal binding by recombinant Thermus thermophilus manganese superoxide dismutase, expressed as the apo-enzyme.

Manganese superoxide dismutase from the extremely thermophilic eubacterium Thermus thermophilus has been cloned and expressed at high levels in a mesophilic host (Escherichia coli) as a soluble tetrameric protein mainly present as the metal-free apo-enzyme. Incubation of the purified apo-enzyme with manganese salts at ambient temperature did not restore superoxide dismutase activity, but reactivation could be achieved by heating the protein with Mn(II) at higher temperatures, approaching the physiological growth temperature for T. thermophilus. Heat annealing followed by incubation with manganese at lower temperature fails to reactivate the enzyme, demonstrating that a simple misfolding of the protein is not responsible for the observed behavior. The in vitro metal uptake is nonspecific, and manganese, iron, and vanadium all bind, but only manganese restores catalytic activity. Bound metal ions do not exchange during heat treatment, indicating that the formation of the metal complex is effectively irreversible under these conditions. The metallation process is strongly temperature-dependent, suggesting that substantial activation barriers to metal uptake at ambient temperature are overcome by a thermal transition in the apo-protein structure. A mechanism for SOD metallation is proposed, focusing on interactions at the domain interface.

Apoenzymes↗
PubMed1982

Inactivation and reactivation of rat liver 3-hydroxy-3-methylglutaryl-CoA-reductase phosphatases: effect of phosphate, pyrophosphate and divalent cations.

Incubation of four purified rat liver HMG-CoA-reductase phosphatases (Gil, G., Sitges, M. and Hegardt, F.G. (1981) Biochim. Biophys. Acta 663, 211-221) with Mn2 or Mg2 caused a concentration-dependent activation of enzyme activities. The maximum effect for Mn2 was at 5 mM for all phosphatases. Fe2 caused inactivation only in reductase phosphatases IIa and IIb. Ca2 10 mM showed a slight effect of inactivation. Phosphate, pyrophosphate and adenine nucleotides inhibited the four reductase phosphatases, this process being concentration-dependent. cAMP did not inhibit the four phosphatases at all in the range of 0.01-8 mM. Preincubation of reductase phosphatases with PPi and subsequent dilution did not diminish the inactivation effect, showing that this ion inhibits the enzyme prior to the binding to the substrate. Phosphorylated sugars, but not free sugar, inactivated the four reductase phosphatases. PPi-inactivated enzymes were reactivated by Mg2 or Mn2, this process being time-dependent. The four phosphatases had different patterns of reactivation. Phosphatases Ib and IIb (low-molecular mass forms) were shown to be different enzymes as judged by: their divergent behaviour when inhibited with Fe2; their PPi response; kinetics of reactivation by Mg2 or Mn2 or PPi-inactivated enzymes; and thermal stability. A metalloenzyme character is suggested for reductase phosphatases.

Adenine Nucleotides↗
PubMed1993

Immunohistochemical identification of muscle fiber types in normal and degenerated muscles.

Myosin ATPase reactions are generally used to differentiate between muscle fibers. However, those reactions have disadvantages including the need for delicate pH control and preincubation under non-physiological pH. Furthermore enzyme activity is only an indirect reflection of myofilament characteristics. In this study, an immunohistochemical method with anti-slow and anti-fast myosin heavy chain antibodies was used to observe: 1) whether muscle fiber types could be distinguished in degenerated muscles, 2) immunoreactivity of type 2C fibers in denervated muscles, and 3) discrepancies in structural disorders. Thirty one muscle biopsies which included neurogenic, myogenic, and control muscles were examined. Muscle fiber types were recognized in normal and in severely degenerated muscles. Type 2C muscle fibers were not necessarily constantly immunoreactive to both anti-slow and anti-fast myosin antibodies. In some targetoid fibers sites with absence of myosin ATPase activity had the same or rather higher immunoreactivity. Some muscle fibers undergoing fiber-type transformation showed discrepancies in reactivity between enzyme- and immuno-reactivity. This immunohistochemical method is capable of observing changes of muscle fibers during denervation, reinnervation, and also sports activity.

Adolescent↗
PubMed1994

Substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida and its relationship to cell growth.

Catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida consists of four identical subunits, each containing one ferrous ion. The enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatechol. Two mutants of catechol 2,3-dioxygenases (4ECR1 and 4ECR6) able to oxidize 4-ethylcatechol, one mutant (3MCS) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and 4-methylcatechol, and one phenotypic revertant of 3MCS (3MCR) which had regained the ability to oxidize 3-methylcatechol were characterized by determining their Km and partition ratio (the ratio of productive catalysis to suicide catalysis). The amino acid substitutions in the four mutant enzymes were also identified by sequencing their structural genes. Wild-type catechol 2,3-dioxygenase was inactivated during the catalysis of 4-ethylcatechol and thus had a low partition ratio for this substrate, whereas the two mutant enzymes, 4ECR1 and 4ECR6, had higher partition ratios for it. Similarly, mutant enzyme 3MCS had a lower partition ratio for 3-methylcatechol than that of 3MCR. Molecular oxygen was required for the inactivation of the wild-type enzyme by 4-ethylcatechol and of 3MCS by 3-methylcatechol, and the inactivated enzymes could be reactivated by incubation with FeSO4 plus ascorbic acid. The enzyme inactivation is thus most likely mechanism based and occurred principally by oxidation and/or removal of the ferrous ion in the catalytic center. In general, partition ratios for catechols lower than 18,000 did not support bacterial growth. A possible meaning of the critical value of the partition ratio is discussed.

Ascorbic Acid↗
PubMed1989

Zymogen/enzyme discrimination using peptide chloromethyl ketones.

Glutamylglycinylarginyl chloromethyl ketone, tyrosylglycinylarginyl chloromethyl ketone, and phenylalanylprolylarginyl chloromethyl ketone have been labeled at their amino termini using fluorescein, rhodamine-X, lissamine-rhodamine, pyrene, and the 1,5-, 2,5-, and 2,6-dimethylaminonaphthalene-1-sulfonyl moieties. These peptidyl chloromethyl ketones have also been modified by incorporation of biotin and epsilon-amino caproyl biotin. The ability of these various chloromethyl ketones to be incorporated into a collection of zymogen-enzyme pairs has been evaluated using a variety of coagulation and fibrinolytic proteins. All labeled chloromethyl ketones were efficiently incorporated into the proteases tested, with the exception of urokinase which was refractory to inhibition by phenylalanylprolylarginyl chloromethyl ketone derivatives. No modification of any zymogen species was observed even under conditions designed to detect minimal reactivity. When enzymes were modified using chloromethyl ketones labeled with epsilon-amino caproyl biotin, the modified proteins readily reacted with avidin under a variety of different conditions. The observed reactivity with avidin was used in enzyme "blotting" following electrophoretic resolution of polypeptide chains and to remove active enzyme present in enzyme-zymogen mixtures. These reagents have been used to evaluate the potential for active site expression by the single-chain human factor VII molecule. Studies conducted with tissue factor, phospholipids, and calcium using factor X as substrate demonstrate that no activity can be obtained without initial activation of either factor X to factor Xa or factor VII to factor VIIa by an external source. We thus conclude that factor VII is a true zymogen, inert in the blood clotting process prior to its cleavage to factor VIIa.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Chloride bound to oxidized cytochrome c oxidase controls the reaction with nitric oxide.

The reaction of nitric oxide (NO) with oxidized fast cytochrome c oxidase was investigated by stopped-flow, amperometry, and EPR, using the enzyme as prepared or after "pulsing." A rapid reduction of cytochrome a is observed with the pulsed, but not with the enzyme as prepared. The reactive species (lambdamax = 424 nm) reacts with NO at k = 2.2 x 10(5) M-1 s-1 at 20 degreesC and is stable for hours unless Cl- is added, in which case it decays slowly (t1/2 approximately 70 min) to an unreactive state (lambdamax = 423 nm) similar to the enzyme as prepared. Thus, Cl- binding prevents a rapid reaction of NO with the oxidized binuclear center. EPR experiments show no new signals within 15 s after addition of NO to the enzyme as prepared. Amperometric measurements show that the pulsed NO-reactive enzyme reacts with high affinity and a stoichiometry of 1 NO/aa3, whereas the enzyme as prepared reacts to a very small extent (<20%). In both cases, the reactivity is abolished by pre-incubation with cyanide. These experiments suggest that the effect of "pulsing" the enzyme, which leads to enhanced NO reactivity, arises from removing Cl- bound at the oxidized cytochrome a3-CuB site.

Animals↗
PubMed1986

Quantitation of apolipoprotein B by polyclonal and monoclonal antibodies.

A non-competitive sandwich enzyme linked immunosorbent assay for the quantitation of apolipoprotein B with polyclonal and monoclonal antibodies was developed. Polyclonal antibodies were used as 'coater'. In the assay with polyclonal antibodies, the same antibody was used as conjugate with alkaline phosphatase. For studies with monoclonal antibodies, enzyme conjugated anti-mouse immunoglobulin had to be used, since monoclonal antibodies lost their reactivity upon enzyme conjugation. Two murine monoclonal antibodies were employed: MAB B-1 with specificity for apolipoproteins (Apo) B-48 and B-100 and MAB B-5 with specificity for B-100 (Radioimmunoassay Inc.). In a reference group Apo B values of 0.82 +/- 0.20 g/l were measured with polyclonal antibodies, 0.68 +/- 0.19 g/l and 0.95 +/- 0.33 g/l with MAB B-1 and MAB B-5. In pure hypercholesterolemia, a similar increase was found with all three antibodies, while in combined hyperlipoproteinemia MAB B-5 gave greater than 40% lower values. Differences were also found with respect to the correlation between Apo B and cholesterol or triglycerides.

Antibodies↗
PubMed1986

Dirofilaria immitis: comparison of cytosolic and mitochondrial glutamate dehydrogenases.

Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8-8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and less than 100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 microM NADH, the Km for the substrate, alpha-ketoglutarate, was equivalent for the two enzymes, but the Km for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 microM NADH, 1 mM ADP approximately doubled and 1 mM ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 mM glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate Km of M-GDH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1968

A new hepatic protein inactivating glucose 6-phosphate dehydrogenase.

Two NADP-cleaving enzymes, namely NADP glycohydrolase and NADP pyrophosphatase, are present in a rat liver extract that inactivates G6PD (glucose 6-phosphate dehydrogenase). The following results suggest that a third G6PD-inactivating protein is present in this extract. (1) Nicotinamide, which selectively inhibits NADP glycohydrolase, enhances the G6PD inactivation under conditions where G6PD activity in control experiments is rather stable. (2) DEAE-cellulose adsorbs the bulk of both NADP glycohydrolase and NADP pyrophosphatase, whereas most of the G6PD-inactivating ability is unadsorbed. (3) Out of 37 liver extracts that were prepared, two were found to lack NADP pyrophosphatase. After removal of NADP glycohydrolase from these extracts by centrifugation, they were still found to inactivate G6PD. (4) Deproteinization of DEAE-cellulose supernatants results in a complete loss of G6PD-inactivating ability; moreover, kinetic experiments performed with the extracts lacking pyrophosphatase strongly support the view that the inactivating protein is an enzyme, although its mechanism is not clear. (5) NADP protects G6PD from inactivation and also reactivates the enzyme completely, thus supporting the view of some action of the inactivating protein on the G6PD-bound NADP.

Animals↗
PubMed1991

Comparative antigen analysis of different life stages of Schistosoma mansoni by crossed immunoelectrophoresis.

Two-dimensional (crossed) immunoelectrophoresis was used for analysis of soluble antigen extracts obtained from the three developmental stages, cercariae, adult worms and eggs, of Schistosoma mansoni by using homologous hyperimmune sera produced in sheep. The antigenic relationships between the three stages as well as the possible relationship to the intermediate snail host were studied. Seven antigen components were shown to be shared between all three life stages of S. mansoni. Furthermore, one antigen was common to adult worm and snail, and one other antigen was shared between cercaria and snail. By using an intermediate gel containing lectin in the antigen-antibody system or by enzyme staining of the immune precipitates it was possible to identify schistosome antigens possessing lectin reactivity or enzyme activity. Characterization of enzyme activities revealed three individual precipitating antigens in adult worm of S. mansoni possessing esterase, leucyl-glycyl-glycine peptidase and phenylalanyl-leucine peptidase activities, respectively. One further precipitinogen with malate dehydrogenase activity was identified for all three life stages.

Animals↗
PubMed2004

In vitro reactivation of acetylcholinesterase using the oxime K027.

The ability of a new bisquaternary oxime, K027 (1-[4-hydroxyiminomethylpyridinium]-3-[carbamoylpyridinium] propane dibromide), to reactivate the enzyme acetylcholinesterase (AChE) inhibited by the nerve agents Tabun, sarin and VX was evaluated. Its reactivation potency was compared to the AChE reactivators pralidoxime (2-PAM), obidoxime and HI-6; K027 seems a good reactivator of organophosphates-inhibited AChE. Its reactivation potency is lower compared to the other oximes for reactivation of sarin-inhibited AChE, but it is sufficient to significantly increase the activity of sarin-inhibited AChE. Its reactivation ability is comparable to obidoxime for reactivation of VX- and tabun-inhibited AChE and is higher than the reactivation potency of HI-6, for tabun-inhibited AChE. HI-6 is currently regarded the most promising reactivator of organophosphates-inhibited AChE.

Acetylcholinesterase↗
PubMedDate not supplied

Comparative studies on the ATPase-binding sites in Ca2+-ATPase and (Na+ + K+)-ATPase by the use of ATP-analogues.

The effects of ATP-analogues on Ca2+-ATPase and (Na++ K+)-ATPase have been studied. The participation of sulfhydryl groups in the recognition of ATP by both transport ATPases is indicated by the fact, that the disulfide of thioinosine triphosphate inactivates both enzymes. The reactivity of rapidly and slowly reacting sulfhydryl groups in the ATP binding sites of both enzymes is altered by the presence of transport substrates. At lease in (Na+ + K+)-ATPase Na+ and Mg2+ appear to alter the structure of the ATP binding site, which conclusion is fortified by the fact, that the photoinactivation of the enzyme by 3'-O-[3-(2-nitro-4-azidophenyl)-propionyl]-ATP needs Mg+. Chromium(III)ATP, a MgATP analogue, inactivated both transport ATPases by the formation of a stable chromo-phosphointermediate. In the case of Ca2+-ATPase this was concomited by the occlusion of Ca2+ in a stable form. No occlusion of Na+ was observable so far in the (Na+ + K+)-ATPase. Contrary to the expectation of the Albers-Post-scheme the hydrolysis of the phosphointermediate formed from chromium(III)ATP was protected by K+, but activated by high concentrations of Na+. Consequently, despite of the inhibition of (Na+ + K+)-ATPase activity, chromium(III)-ATP supported the Na+ - Na+-exchange reaction in everted red blood cells.

Adenosine Triphosphate↗
PubMed1984

A solid-phase enzyme immunoassay for C-reactive protein: clinical value and the effect of rheumatoid factor.

Measurement of serum C-reactive protein (CRP) concentration is useful in monitoring the progress of chronic inflammatory diseases. Rheumatoid factor, by its interaction with the Fc portion of IgG, has the potential to interfere with solid-phase immunoassays for CRP and other serum proteins. To determine the effect of RF on a solid-phase enzyme immunoassay for CRP we compared assays employing whole antisera or F(ab')2 fragments. In 92 sera with RF latex titres ranging from 1/80 to 1/81,920, no correlation was found between RF concentrations and CRP measurements. CRP concentrations measured by use of whole antisera (mean +/- standard error of the mean, 59.7 +/- 5.7 mg/l, n = 92) were lower than those measured with F(ab')2 fragments (62.5 +/- 5 mg/l), indicating that exaggeration of CRP measurements did not occur in RF containing sera under the conditions of the assay. Our results show that in the CRP-ELISA, interference from RF was precluded by the high serum dilutions employed. At lower serum dilutions RF binding was detected. Consequently, in solid-phase enzyme immunoassays at lower serum dilutions the presence of RF may lead to false positive results and exaggerated measurements.

Arthritis, Rheumatoid↗
PubMed1993

Regulation of horse-liver glutathione reductase.

1. The enzyme was rapidly inactivated by NAD(P)H, GSH, dithionite or borohydride, while activity increased in the presence of NAD(P)+ or GSSG. NADH was more efficient for inactivation than NADPH. Redox inactivation required neutral or alkaline pH, was maximal at pH 8.5, and depended on the presence of metal cations. 2. GSSG and dithiothreitol fully protected the enzyme from inactivation at concentrations stoichiometric with NAD(P)H. Ten-fold higher ferricyanide or GSH concentrations were required to obtain partial protection. NAD+ or NADP+ were quite ineffective. 3. GSSG fully reactivated the inactive enzyme at 38 degrees C and neutral to acidic pH (5.5-7.5). Reactivation by dithiothreitol was accomplished in short periods of time at pH 8.5 although the activity was progressively lost afterwards. Ferricyanide and GSH also reactivated the enzyme to different extents.

Animals↗
PubMed2006

Mitochondria produce reactive nitrogen species via an arginine-independent pathway.

We measured the contribution of mitochondrial nitric oxide synthase (mtNOS) and respiratory chain enzymes to reactive nitrogen species (RNS) production. Diaminofluorescein (DAF) was applied for the assessment of RNS production in isolated mouse brain, heart and liver mitochondria and also in a cultured neuroblastoma cell line by confocal microscopy and flow cytometry. Mitochondria produced RNS, which was inhibited by catalysts of peroxynitrite decomposition but not by nitric oxide (NO) synthase inhibitors. Disrupting the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of complex I abolished the DAF signal, which was restored by complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone/ubiquinol cycle or dissipating the proton gradient had no effect on DAF fluorescence. We conclude that mitochondria from brain, heart and liver are capable of significant RNS production via the respiratory chain rather than through an arginine-dependent mtNOS.

Animals↗
PubMed2007

Structure-function analysis of the cuprous oxidase activity in Fet3p from Saccharomyces cerevisiae.

The Fet3 protein from Saccharomyces cerevisiae is a multicopper oxidase with specificity toward Fe(II) and Cu(I). Fet3p turnover of Fe(II) supports high affinity iron uptake across the yeast plasma membrane, whereas its turnover of Cu(I) contributes to copper resistance in yeast. The structure of Fet3p has been used to identify possible amino acid residues responsible for this protein's reactivity with Cu(I), and structure-function analyses have confirmed this assignment. Fet3p Met(345) is required for the enzyme's reactivity toward Cu(I). Although the Fet3pM345A mutant exhibits wild type spectral and electrochemical behavior, the kinetic constants for Cu(I) turnover and for single-turnover electron transfer from Cu(I) to the enzyme are significantly reduced. The specificity constant with Cu(I) as substrate is reduced by one-fifth, whereas the electron transfer rate from Cu(I) is reduced 50-fold. This mutation has little effect on the reactivity toward Fe(II), indicating that Met(345) contributes specifically to Fet3p reactivity with the cuprous ion. These kinetic defects render the Fet3pM345A unable to support wild type cellular copper resistance, suggesting that there is a finely tuned copper redox balance at the yeast plasma membrane.

Cell Membrane↗