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PubMed1986

The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences.

It has been proposed that recognition of specific DNA sequences by proteins is accomplished by hydrogen bond formation between the protein and particular groups that are accessible in the major and minor grooves of the DNA. We have examined the DNA-protein interactions involved in the recognition of the hexameric DNA sequence, GAATTC, by the EcoRI restriction endonuclease by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues hypoxanthine, 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, 5-bromouracil, uracil, 5-bromocytosine, and 5-methylcytosine were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the interactions between the EcoRI endonuclease and its recognition sequence were monitored by determining the steady state kinetic values of the hydrolysis reaction. The substitutions resulted in effects that varied from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those substrates that were reactive, whereas octanucleotide analogues containing N6-methyladenine at either adenine position, uracil at the second thymine position, or 5-bromocytosine or 5-methylcytosine at the cytosine position were unreactive. The results are discussed in terms of possible effects on interactions between the enzyme and its recognition site during the reaction. An accompanying paper presents the results of a similar study using these oligonucleotides with the EcoRI modification methylase.

Adenine↗

PubMed1982

Detection of group and interspecies reactivities of mammalian C-type virus p30 proteins by an enzyme-linked immunosorbent assay (ELISA): enhancement of interspecies reactivity by denaturation.

The applicability of the enzyme immunoassay technique to the detection of group and interspecies determinants of C-type retroviral proteins was tested. For this purpose four groups of C-type retroviruses (MuLV, FeLV, SiSV/GaLV, and BaEV/RD114) were assayed for group-specific and interspecies reactivities of their p30 proteins by an enzyme-linked immunosorbent assay (ELISA). We found that the ELISA can detect group-specific as well as interspecies determinants with sensitivity and reproducibility in purified p30 proteins, disrupted viruses, and cell extracts if an anti-p30 interspecies antiserum is used. If monospecific antisera against MuLV p30, SiSV p30, or BaEV p30 were used, only group-specific reactivities were detected reproducibly, whereas the detectability of interspecies determinants depended on the antisera used and varied even with the same antisera. In assays in which the reactivity of native and denatured p30 proteins was compared the detectability of sodium dodecyl sulphate-denatured MuLV p30 was better than that of native MuLV p30 suggesting that some of the most broadly cross-reactive sequences are localized inside the protein molecule and are freed by the denaturation process. Antisera raised against native and denatured p30 proteins showed identical spectra of reactivity.

Antigens, Viral↗

PubMed1985

SOS-type functions in mammalian cells. Enhanced reactivation of UV-irradiated SV 40 in UV-irradiated CV-1 cells.

The reactivation of UV-irradiated SV 40 was measured in UV-irradiated CV-1 cells as a function of time between irradiation of the cells and infection. To avoid the possible bias of multiplicity reactivation and/or virus propagation, infection was quantitated in terms of V-antigen-positive nuclei at the end of the first lytic cycle. In irradiated cell cultures enhanced reactivation of SV 40 was observed, which indicates induction of DNA repair enzymes. Maximal reactivation was obtained when the time interval between irradiation and infection was 72 h. The UV-inducible DNA repair enzymes might represent elements of an SOS-type response in mammalian cells.

Animals↗

PubMed1997

Focal cerebral ischemia enhances glial expression of ecto-5'-nucleotidase.

The effect of ischemia on the reactive expression of ecto-5'-nucleotidase in rat brain was studied 6 h and 1, 2 and 7 days after permanent middle cerebral artery occlusion (MCAO). The distribution of 5'-nucleotidase in the infarcted brain was compared to markers for astrocytes (glial fibrillary acidic protein (GFAP)) and microglia (complement receptor type 3, antibody OX42) using histological staining or immunohistochemistry. 5'-Nucleotidase could be associated with reactive astrocytes by immunohistochemistry and with reactive microglia by enzyme histochemistry. In the untreated control 5'-nucleotidase was associated with astrocytes only in the hippocampus and the submeningeal space. After ischemia the enzyme was expressed on reactive astrocytes in the tissue surrounding the volume of infarction. Individual reactive astrocytes were observed 6 h after MCAO and the astrocytic expression became continuously enhanced during the following days. An enzyme histochemical analysis of 5'-nucleotidase activity revealed a postischemic increase in reaction product around the infarcted tissue. Seven days after MCAO a discrete band (0.2-0.4 mm) of reaction product characterized the rim of the infarcted area. This band of activity of 5'-nucleotidase colocalized with a band of immunoreactivity for OX42, indicative of an intense accumulation of 5'-nucleotidase expressing microglia. Our results suggest that ischemia following permanent MCAO results in an upregulation of the capacity for the hydrolysis of nucleotides within the tissue adjacent to the infarcted volume. Nucleotides released from the damaged cells can be hydrolyzed and the adenosine eventually formed may exert neuroprotective functions limiting the extent of damage.

5'-Nucleotidase↗

PubMedDate not supplied

Effects of mercury vapor inhalation on reactive oxygen species and antioxidant enzymes in rat brain and kidney are minimal.

Metals are known to induce the formation of reactive oxygen species (ROS) that initiate oxidative stress, an important mechanism of cell injury. The brain is particularly sensitive to oxidative attack because of its high level of unsaturated lipids and high rate of oxidative metabolism. The objective of this study was to determine if elemental mercury (Hg(0)) vapor inhalation increases ROS production and affects activities or levels of antioxidant-related biomolecules in the rat brain and kidney. Adult female Sprague-Dawley rats were exposed for 2 h per day for 11 consecutive days to Hg(0) vapor (1, 2, and 4 mg Hg(0) m(-3)). Brain regions (frontal cortex, cerebellum, brain stem) and kidney were assayed for total Hg, ROS and glutathione (GSH) levels, and for enzyme activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD). Marked exposure-related increases (2500-5600-fold) in total Hg were detected in the brain regions and in kidney. A statistically significant increase in ROS production (ca. 30% above controls) was observed only in the cortex of rats exposed to 1 mg m(-3) Hg vapor, but no significant changes were apparent at other exposures. Although a trend towards increasing ROS production was observed in the kidney, these effects were not statistically significant. Mercury vapor exposure had no significant effects on GSH levels or GPx activity in the three brain regions, however, statistically significant decreases in GSH and GPx activity were detected in the kidneys of rats exposed to 2 mg m(-3). Mercury exposure did not cause significant effects on SOD activity in the brain or kidney. The data indicate that oxidative stress and changes in GSH and activities of antioxidant enzymes do not play a major role in Hg(0) vapor toxicity in brain and kidney.

Administration, Inhalation↗

PubMed2002

Specificity of antioxidant enzyme inhibition in skeletal muscle to reactive nitrogen species donors.

Nitric oxide (*NO) and its by-products modulate many physiological functions of skeletal muscle including blood flow, metabolism, glucose uptake, and contractile function. However, growing evidence suggests that an overproduction of nitric oxide contributes to muscle wasting in a number of pathologies including chronic heart failure, sepsis, COPD, muscular dystrophy, and extreme disuse. Limited data point to the potential of inhibition various enzymes by reactive nitrogen species (RNS), including (.)NO and its downstream products such as peroxynitrite, primarily in purified systems. We hypothesized that exposure of skeletal muscle to RNS donors would reduce or downregulate activities of the crucial antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). Diaphragm muscle fiber bundles were extracted from 4-month-old Fischer-344 rats and, in a series of experiments, exposed to either (a) 0 (control), 1, or 5 mM diethylamine NONOate (DEANO: *NO donor); (b) 0, 100, 500 microM, or 1 mM sodium nitroprusside (SNP: *NO donor); (c) 0 or 2 mM S-nitroso-acetylpenicillamine (SNAP: *NO donor); or (d) 0 or 500 microM SIN-1 (peroxynitrite donor) for 60 min. DEANO resulted in a 50% reduction in CAT, GPX, and a dose-dependent inhibition of Cu, Zn-SOD. SNP resulted in significantly lower activities for total SOD, Mn-SOD isoform, Cu, Zn-SOD isoform, CAT, and GPX in a dose-dependent fashion. Two millimolar SNAP and 500 microM SIN-1 also resulted in a large and significant inhibition of total SOD and CAT. These data indicate that reactive nitrogen species impair antioxidant enzyme function in an RNS donor-specific and dose-dependent manner and are consistent with the hypothesis that excess RNS production contributes to skeletal muscle oxidative stress and muscle dysfunction.

Animals↗

PubMed1983

Studies on the mechanisms of ornithine decarboxylase in vitro inactivation.

Hydrocortisone-induced rat liver ornithine decarboxylase appears quite stable in the soluble fraction of the homogenate incubated at 37 degrees C. In contrast, the incubation of the whole homogenate causes a rapid loss of activity. The ornithine decarboxylase-inactivating capacity appears mainly bound to microsomes. Lysosomes seem to play a role only after the microsome-induced inactivation. Different reducing agents (dithiothreitol, NADPH, NADH, GSH) are effective both in preventing and in reversing ornithine decarboxylase inactivation. NADPH is peculiar in that it can reactivate the enzyme at very low concentrations. Oxidized glutathione potentiates the inactivating effect of microsomes. On the basis of present results it is suggested that ornithine decarboxylase may be reversibly inactivated through microsome-catalyzed formation of mixed or enzyme-enzyme disulfides and that NADPH plays a crucial role in ornithine decarboxylase reactivation, probably by cytosolic reductase(s).

Aging↗

PubMedDate not supplied

[Reactivation of phosphorylated cholinesterase immobilized in a gelatin membrane].

The soluble and immobilized cholinesterases (acetyl cholinesterase of human blood erythrocytes (EC 3.1.1.7) and butyryl cholinesterase of equine blood serum, (EC 3.1.1.8] were inactivated by such irreversible inhibitors as diisopropyl fluorophosphate (DFP), O,O-dimethyl-O-(2,2)-dichlorovinyl) phosphate (DDVP), paraoxone, armine. The inactivated enzymes were reactivated under the effect of TMB-4 (1,1'-trimethylene-bis)-4-formyl-pyridine bromide (dioxime). The values of the reactivation rate constants proved to be equal both for the soluble and immobilized cholinesterases inactivated by the same irreversible inhibitor. The immobilized enzyme is simpler and more correct to study the reactivating action than the soluble one.

Acetylcholinesterase↗

PubMed1987

Oxygen sensitivity of sugar metabolism and interconversion of pyruvate formate-lyase in intact cells of Streptococcus mutans and Streptococcus sanguis.

Pyruvate formate-lyase (PFL) (formate acetyltransferase; EC 2.3.1.54) of oral streptococci is essential for metabolizing sugar into volatile compounds (formate, acetate, and ethanol). This enzyme is extremely sensitive to oxygen, and its activity is irreversibly inactivated by oxygen. When Streptococcus sanguis was anaerobically starved, a part of the active form of PFL was converted into a reversible inactive form that was tolerant of oxygen. This reversible inactive enzyme could be reactivated to the active enzyme by anaerobic sugar metabolism, with the recovery of volatile compound production. The PFL in Streptococcus mutans was not converted into an oxygen-tolerant inactive form by anaerobic starvation, and after exposure of the cells to oxygen the PFL could not be reactivated. These findings suggest that S. mutans can produce acids rapidly under anaerobic conditions because of its capacity to keep PFL active and that S. sanguis can protect its sugar metabolism from oxygen impairment because of its interconversion of PFL.

Acetyltransferases↗

PubMed1969

Purification and properties of alpha-D-mannosidase from rat epididymis.

1. alpha-d-Mannosidase from rat epididymis was purified 300-fold. beta-N-Acetyl-glucosaminidase and beta-galactosidase were removed from the preparation by treatment with pyridine. Zn(2+) was added during the purification to stabilize the alpha-mannosidase. 2. Mammalian alpha-mannosidase is most stable at pH6. At lower pH values it undergoes reversible spontaneous inactivation. The enzyme is also subject to irreversible inactivation, which is delayed by the addition of albumin. 3. Reversible inactivation of alpha-mannosidase is accelerated by EDTA and reversed or prevented by Zn(2+). Other cations, such as Co(2+), Cd(2+) and Cu(2+), accelerate inactivation and the action of a toxic cation can be prevented by Zn(2+) or by EDTA in suitable concentration. 4. The enzyme is stabilized by substrate and neither Zn(2+), EDTA nor a toxic cation has more than a small effect in the assay of an untreated preparation. The addition of Zn(2+) is necessary, however, for a constant rate of hydrolysis during prolonged incubation of the enzyme with substrate. In an EDTA-treated preparation, Zn(2+) reactivates the enzyme during the assay. 5. Evidence is presented that alpha-mannosidase is a dissociable Zn(2+)-protein complex, in which Zn(2+) is essential for enzyme activity.

Acetone↗

PubMed2000

Comparison of oxime-initiated reactivation of organophosphorous-inhibited acetylcholinesterase in brains of avian embryos.

Organophosphorous (OP) insecticide-induced inhibition and oxime reactivation of acetylcholinesterase (AChE) was determined in whole-brain homogenates prepared from 15-d-old chick embryos. Doses of chlorpyrifos, parathion, acephate, and trichlorfon that inhibited AChE >70% were administered to the embryos. Following insecticide exposure, an in vitro system compared the capability of the oximes pralidoxime (2-PAM), obidoxime, TMB-4, and HI-6 to reactivate the OP-inhibited AChE. Concentration-related increases in AChE activities were noted in embryo brains reactivated with 2-PAM, TMB, and HI-6. 2-PAM was the most effective reactivator of trichlorfon-inhibited AChE; 2-PAM and obidoxime were relatively similar in effectiveness for reactivation of AChE inhibited with the other OP insecticides used as test agents. All oximes were similarly effective against acephate, but HI-6 was the least effective reactivator of AChE in chick embryo brain homogenates inhibited by the other OP insecticides. These results suggest that both the OP insecticide inhibiting AChE and the oxime reactivating this enzyme can contribute to the effectiveness of the avian brain AChE reactivation.

Acetylcholinesterase↗

PubMed2005

2-Hydroxyisocaproyl-CoA dehydratase and its activator from Clostridium difficile.

The hadBC and hadI genes from Clostridium difficile were functionally expressed in Escherichia coli and shown to encode the novel 2-hydroxyisocaproyl-CoA dehydratase HadBC and its activator HadI. The activated enzyme catalyses the dehydration of (R)-2-hydroxyisocaproyl-CoA to isocaprenoyl-CoA in the pathway of leucine fermentation. The extremely oxygen-sensitive homodimeric activator as well as the heterodimeric dehydratase, contain iron and inorganic sulfur; besides varying amounts of zinc, other metal ions, particularly molybdenum, were not detected in the dehydratase. The reduced activator transfers one electron to the dehydratase concomitant with hydrolysis of ATP, a process similar to that observed with the unrelated nitrogenase. The thus activated dehydratase was separated from the activator and ATP; it catalyzed about 10(4) dehydration turnovers until the enzyme became inactive. Adding activator, ATP, MgCl(2), dithionite and dithioerythritol reactivated the enzyme. This is the first demonstration with a 2-hydroxyacyl-CoA dehydratase that the catalytic electron is recycled after each turnover. In agreement with this observation, only substoichiometric amounts of activator (dehydratase/activator = 10 mol/mol) were required to generate full activity.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1990

Studies of recombinant plasminogen activator inhibitor-1 in rabbits. Pharmacokinetics and evidence for reactivation of latent plasminogen activator inhibitor-1 in vivo.

The pharmacokinetics of human recombinant plasminogen activator inhibitor-1 (rPAI-1) was studied in rabbits. Latent rPAI-1 (0-2 units of tissue-type plasminogen activator neutralizing activity per microgram protein); reactivated rPAI-1 (approximately 150 units/micrograms); and chloramine T-oxidized, nonreactivatable rPAI-1 (approximately 0.7 units/microgram) were studied. The pharmacokinetic parameters for the disposition of rPAI-1 antigen after an intravenous bolus injection of 1.0 or 2.5 mg/kg rPAI-1 were very similar for all three forms: the initial volume of distribution was approximately 60 ml/kg, the initial half-life in plasma was 6 minutes, and the plasma clearance was approximately 4 ml/kg/min. The disposition of PAI activity after injection of reactivated rPAI-1 was similar to that of rPAI-1 antigen. Injection of latent rPAI-1 was associated with a nearly threefold increase in the specific activity of circulating PAI-1 from 2 units/micrograms to 5.0 +/- 1.1 units/micrograms (p less than 0.01) within 1 minute, followed by a cumulative 25-fold increase in specific activity over 1 hour (p = 0.01). In contrast, the specific activity of oxidized or reactivated preparations of rPAI-1 did not increase in the first several minutes after injection. These findings support the existence of a fast-acting but low-capacity mechanism for the reactivation of rPAI-1 in vivo.

Analysis of Variance↗

PubMed1977

Homoserine dehydrogenase: spontaneous reactivation by dissociation of p-mercuribenzoate from an inactive enzyme--p-mercuribenzoate complex.

Incubation of Rhodospirillum rubrum homoserine dehydrogenase (L-homoserine:NAD+ oxidoreductase, EC 1.1.1.3) with p-mercuribenzoate (PMB) in the presence of 0.2 M KCl and 2 mM L-threonine resulted in complete loss of enzyme activity. Upon removal of excess PMB, KCl, and L-threonine, a time-dependent recovery of enzyme activity was observed in 25 mM phosphate/I mM EDTA buffer, pH 7.5. Circular dichroism studies indicated that the transition from inactive to reactivated form of the enzyme was accompanied by a conformational change in the protein. Experiments with [14C]PMB revealed loss of enzyme-bound radioactivity during reactivation. Increase in ionic strength of the phosphate buffer and/or addition of L-threonine, leading to enzyme aggregation, decreased the rate of enzyme reactivation, aggregated enzyme that remained inactive retained [14C]PMB on the enzyme. Sulfhydryl titration of various forms of the enzyme suggested a preferential release of PMB from a sulfhydryl group essential to enzymic activity. We conclude that reactivation of the inactive enzyme is due to dissociation of PMB from an "active-site" sulfhydryl group and that changes in the protein structure influence the rate of dissociation of the enzyme-PMB complex.

Alcohol Oxidoreductases↗

PubMed1987

Immuno cross-reactivity suggests that catecholamine biosynthesis enzymes and beta-adrenergic receptors may be related.

Turkey red blood cell, beta 1-adrenergic receptors (BARs) were prepared to electrophoretic homogeneity by affinity chromatography, size exclusion high performance liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to prepare rabbit polyclonal anti-BAR antibodies. Anti-BAR activity was confirmed by immunoadsorption of [125I]cyanopindolol-labeled BAR to a protein A affinity column using the anti-BAR antibodies. BAR was compared to the catecholamine biosynthetic enzyme dopamine B-hydroxylase (DBH) by anti-BAR antibody cross-reactivity. DBH was purified from bovine adrenal medullae chromaffin vesicles by ion exchange, size exclusion, and concanavalin A-Sepharose chromatography. Final DBH specific activities were 42 +/- 4 units/mg of protein. Homogeneity was confirmed by nondenaturing polyacrylamide gel electrophoresis. Both DBH and BAR were recognized by the anti-BAR antibodies on Western transfer and immunoblotting. No interactions were observed with preimmune controls. Similar results were obtained with glycosylated and deglycosylated DBH, suggesting that the anti-BAR antibodies recognize specific portions of DBH amino acid sequence and not associated carbohydrate. DBH-cross-reactive antibodies were also purified by affinity chromatography using immobilized DBH and shown to immunoadsorb [125I]cyanopindolol-labeled BAR by protein A affinity chromatography. These results suggest that the catecholamine biosynthetic enzyme DBH and BAR may be related in structure.

Animals↗

PubMed1979

Irreversible inactivation of pyruvate decarboxylase in the presence of substrate and an oxidant. An example of paracatalytic enzyme inactivation.

Pyruvate decarboxylase from yeast is progressively inactivated in the presence of pyruvate and an extrinsic oxidant such as 2,6-dichloroindophenol or hexacyanoferrate(III). The inactivation is linked to the oxidation of the hydroxyethylthiamine diphosphate intermediate to acetate. Removal of low-molecular compounds by gel filtration does not reactivate the enzyme. The rate of inactivation obeys saturation kinetics with respect to substrate concentration and is independent of enzyme concentration. In analogy to the paracatalytic inactivation of other enzymes forming oxidizable carbanion intermediates [Christen, P. (1977) Methods Enzymol.46, 48--54], the oxidation of enzyme-bound hydroxyethylthiamine diphosphate is thought to generate a transiently reactive intermediate which, without being released from the enzyme, covalently modifies a group at or near the active site. Reconstitution experiments indicate that the protein rather than the coenzyme moiety is modified.

2,6-Dichloroindophenol↗

PubMed2000

Vascular oxidant stress: molecular mechanisms and pathophysiological implications.

The term oxidative stress refers to a situation in which cells are exposed to excessive levels of either molecular oxygen or chemical derivatives of oxygen (ie, reactive oxygen species). Three enzyme systems produce reactive oxygen species in the vascular wall: NADH/NADPH oxidase, xanthine oxidoreductase, and endothelial nitric oxide synthase. Among vascular reactive oxygen species superoxide anion plays a critical role in vascular biology because it is the source for many other reactive oxygen species and various vascular cell functions. It is currently thought that increases in oxidant stress, namely excessive production of superoxide anion, are involved in the pathophysiology of endothelial dysfunction that accompanies a number of cardiovascular risk factors including hypercholesterolemia, hypertension and cigarette smoking. On the other hand, vascular oxidant stress plays a pivotal role in the evolution of clinical conditions such as atherosclerosis, diabetes and heart failure.

Animals↗

PubMed1988

Purification and kinetic and structural properties of spinach leaf NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase.

NADP-dependent nonphosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from spinach leaves has been purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation, molecular sieving on Sephadex G-200, DEAE-cellulose, and 2',5'-ADP-Sepharose affinity chromatography. The purified enzyme exhibited a specific activity of 15 mumol (mg protein)-1 min-1 and was characterized as a homotetramer with a native molecular weight of 195,000. Preincubation of the purified enzyme with NADP+ resulted in an almost twofold increase in enzymatic activity. The rate of activation was slower than the rate of catalysis, indicating that the enzyme has hysteretic properties. This behavior results in a lag phase during activity measurement of the enzyme preincubated without NADP+. Substrate interaction and product inhibition studies suggest a rapid equilibrium random BiBi mechanism for the reaction. Thiol modifying reagents, iodoacetamide and diamide, completely inactivated the purified enzyme. Inactivation by iodoacetamide exhibited pseudo-first-order kinetics with a rate constant of 0.17 min-1. D-Glyceraldehyde 3-phosphate effectively protected the enzyme against inactivation by thiol reagents, suggesting that modification occurred at or near the substrate-binding site. Complete inactivation of the dehydrogenase was correlated with incorporation of 8 mol [1-14C]iodoacetamide/mol enzyme. Total protection afforded by D-glyceraldehyde 3-phosphate against enzyme inactivation by iodoacetamide was correlated with a protection of 4 mol reactive residues/mol enzyme. On the basis of these results it is suggested that one sulfhydryl group per enzyme subunit is essential for catalysis in spinach leaf nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase. A kinetic and molecular mechanism for the reaction is proposed.

Ammonium Sulfate↗