Effect of adrenergically reactive drugs on peroxisomal enzymes in Tetrahymena.
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Aminophylline↗
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Results for “Enzyme Reactivators”
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[Reactivation with polyvinylpyrrolidone of enzymes inhibiting tannin].
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Glucosephosphate Dehydrogenase↗
Detection of conformational changes in rat kidney gamma-glutamyl transpeptidase by an antibody against a synthetic peptide belonging to part of the reactive centre of the enzyme.
Polyclonal antibodies were produced against rat kidney gamma-glutamyl transpeptidase (GGT) and against a synthetic peptide corresponding to residues 512-534 in rat GGT. The anti-peptide antibody bound denatured GGT, acivicin-treated GGT and GGT absorbed to microtiter plates, but not GGT in solution, and did not inhibit GGT. The antibody against native GGT inhibited its activity in solution and did not bind efficiently adsorbed GGT in direct ELISA. It was active in direct and competition ELISA using kidney brush border membranes as the adsorbed antigen. The results indicate that these antibodies recognize conformational rather than sequence epitopes in GGT, and that marked changes occur in the conformation of GGT upon its absorption to plates or its reaction with acivicin.
Amino Acid Sequence↗
Reactivities of feline calicivirus field isolates with monoclonal antibodies detected by enzyme-linked immunosorbent assay.
Reactivities of feline calicivirus (FCV) field isolates with monoclonal antibodies (MAbs) were examined by enzyme-linked immunosorbent assay (ELISA). The reactivities of the viruses in ELISA were different from our previous results using the neutralization tests (NT). Many isolates were positive in ELISA with MAbs which recognized neutralizing epitope 3B and/or 4. However, most were negative in NT in our previous study. After absorption of two FCV strains with host cells, the non-infectious virus fluid still reacted with MAb, which recognized epitope 3B and/or 4 in ELISA. These results indicated the possibility that neutralizing epitopes are expressed on non-infectious virus particles or exist as proteinaceous molecules in virus fluid.
Animals↗
Environmental contaminants in pathogenesis of breast cancer.
This review is an attempt to comprehend the diverse groups of environmental chemical contaminants with a potential for pathogenesis of breast cancer, their probable sources and the possible mechanisms by which these environmental contaminants act and interplay with other risk factors. Estrogens are closely related to the pathogenesis of breast cancer. Oxidative catabolism of estrogen, mediated by various cytochrome P450 enzymes, generates reactive free radicals that can cause oxidative damage. The same enzymes of estrogenic metabolic pathways catalyze biological activation of several environmental (xenobiotic) chemicals. Xenobiotic chemicals may exert their pathological effects through generation of reactive free radicals. Breast tissue can be a target of several xenobiotic agents. DNA-reactive metabolites of different xenobiotic compounds have been detected in breast tissue. Many phase I and II xenobiotic metabolizing enzymes are expressed in both normal and cancerous breast tissues. These enzymes play a significant role in the activation/detoxification of xenobiotic and endogenous compounds including estrogens. More than 30 carcinogenic chemicals are present in tobacco smoke; many of them are fat-soluble, resistant to metabolism and can be stored in breast adipose tissue. Similarly, pesticides are also known to cause oxidative stress; while some act as endocrine disruptor, some are shown to suppress apoptosis in estrogen sensitive cell lines. Reports have shown an association of smoking (both active and passive) and pesticides with breast cancer risk. However, the issues have remained controversial. Different mutagenic substances that are generated in the cooking process e.g., heterocyclic amines and polycyclic aromatic hydrocarbons (PAHs) can be a threat to breast tissue. PAHs and dioxins exert their adverse effects through the aryl hydrocarbon receptor (AhR), which activates several genes involved in the metabolisms of xenobiotic compounds and endogenous estrogens. These chemicals also induce AhR-dependent mitochondrial dysfunction. Many of the environmental pollutants suppress the immune system, which are implicated to risk. A better understanding about the biological effects of different environmental carcinogenic compounds and determination of their impact on rising incidence of breast cancer will be beneficial in improving preventive policy against breast cancer.
Animals↗
Immunochemical studies of human placental-type variants of alkaline phosphatase. Structural differences between the "Nagao isoenzyme" and the placental "D-variant".
Hyperimmune antisera raised against the rare FD and SD phenotypes of human placental alkaline phosphatase and the related cancer-associated Nagao isoenzyme were exhaustively absorbed with the common SS and FF phenotype enzymes conjugated to Sepharose. The antibodies so obtained had one of three specificities, determined by testing all the common phenotypes of placental alkaline phosphatase, the rare FD, SD and S-17 phenotypes, and five samples of purified cancer patient enzymes: (I) antibodies reactive with the S, D and I variant enzymes, but not with the F, 17 or Nagao enzymes; (II) antibodies reactive with the F, 17 and Nagao enzymes, but not with the S, D, and I variant enzymes; and (III) antibodies reactive with the D, 17 and Nagao enzymes and not with the S, F or I variant enzymes. Based on these data, and on the information obtained from biochemical characterization, it is argued that the Nagao isoenzyme and the D-variant are closely related but distinct enzymes. We propose a hypothetical scheme for the divergence of these two enzymes from a common ancestral gene product. In this scheme, an ancestral gene duplication resulted in two isoenzymes, one of which is completely specific to the placenta, and one of which (the "Nagao isoenzyme") has an unknown normal tissue localization, but is found in some cases of cancer. We use the term "syn-placental" to describe this latter isoenzyme, reflecting its close structural relationship to the placental isoenzyme.
Alkaline Phosphatase↗
The presence of reactive SH groups in the enzymatically active dicyano derivative of creatine kinase.
It has been shown that the active dicyano derivative of creatine kinase (ATP:creatine N-phosphotransferase) obtained by cyanolysis of the 5,5'-dithiobis(2-nitrobenzoic acid)-modified and inactivated enzyme contains, as does the native enzyme, two reactive SH groups. Modification of these two SH groups leads to complete inactivation of the dicyano enzyme. Reaction with 4-iodoacetamido-1-naphthol introduces fluorescent labels at these reactive SH groups of the native and the dicyano enzymes. Following tryptic digestion, the respective fluorescent-labelled peptides have been separated by HPLC and the amino acid composition analysis of these peptides has shown that they are consistent with the sequence of the peptide segment containing the active-site SH of Cys-282 of creatine kinase for both the native and the dicyano enzymes, showing that the active SH groups are free in the dicyano enzyme. Upon mild denaturation in 3 M urea, it can be shown that two of the SH groups partially buried in the native enzyme have been cyanylated in the dicyano enzyme. The two reactive SH groups are therefore essential for the activity of creatine kinase and the two cyanylated SH groups are internal groups which probably contributes partially to the stabilization of an active conformation of the enzyme molecule.
Animals↗
Binding of beta-lactam antibiotics to the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R39.
Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic-enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their beta-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its beta-lactam ring. In Tris-NaCl-MgCl(2) buffer at pH7.7 and 37 degrees C, the rate constants for the dissociation of the antibiotic-enzyme complexes were 2.8x10(-6), 1.5x10(-6) and 0.63x10(-6)s(-1) (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [(14)C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a beta-lactam-antibiotic-destroying enzyme but did not function as a beta-lactamase. Incubation at 37 degrees C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [(14)C]benzylpenicillin-enzyme complex. The rate constants were 1.6x10(-5)s(-1) and 0.8x10(-4)s(-1) respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.
Binding, Competitive↗
Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646.
An aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646 was purified 196-fold by a combination of Mono-Q, Reactive Green 19 agarose affinity, and hydroxyapatite chromatographies. The purified enzyme runs as a single band of 140 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 163 +/- 3.8 kDa by gel filtration, indicating that this enzyme is a monomeric protein. The binding of the enzyme to Reactive Green 19 agarose was Mg2+ dependent. The binding capacity was estimated to be about 0.2 mg of Reactive Green agarose per ml in the presence of 10 mM MgCl2. This enzyme can catalyze the reduction of a wide range of aryl carboxylic acids, including substituted benzoic acids, phenyl-substituted aliphatic acids, heterocyclic carboxylic acids, and polyaromatic ring carboxylic acids, to produce the corresponding aldehydes. The Km values for benzoate, ATP, and NADPH were determined to be 645 +/- 75, 29.3 +/- 3.1, and 57.3 +/- 12.5 microM, respectively. The Vmax was determined to be 0.902 +/- 0.04 micromol/min/mg of protein. Km values for (S)-(+)-alpha-methyl-4-(2-methylpropyl)-benzeneacetic acid (ibuprofen) and its (R)-(-) isomer were determined to be 155 +/- 18 and 34.5 +/- 2.5 microM, respectively. The Vmax for the (S)-(+) and (R)-(-) isomers were 1.33 and 0.15 micromol/min/mg of protein, respectively. Anthranilic acid is a competitive inhibitor with benzoic acid as a substrate, with a Ki of 261 +/- 30 microM. The N-terminal and internal amino acid sequences of a 76-kDa peptide from limited alpha-chymotrypsin digestion were determined.
Aldehyde Oxidoreductases↗
Erythrocyte delta-aminolevulinate dehydratase measurements in Balkan endemic nephropathy.
Balkan endemic nephropathy (BEN) is a chronic progressive kidney disease leading to renal insufficiency. The possible etiological role of some toxic factors was considered in this study by investigating the activity of erythrocyte delta-aminolevulinate dehydratase (ALA-D), an enzyme influenced by various environmental factors. We observed that ALA-D activity was markedly decreased in patients with BEN and in 32% of their healthy family members. Glutathione concentration was found normal in all the groups studied, however, it was possible to reactivate the enzyme in vitro by addition of exogenous glutathione. Percent activation was significantly higher in the groups with decreased ALA-D activity. Blood lead levels were within normal range. The results suggest a normal synthesis of ALA-D apoenzyme, and also the presence of some factors, environmental, metabolic, or genetic, which may affect the enzyme activity through binding to the reactive groups in the active center of this enzyme or by oxidation of the reactive groups. Additional studies are necessary to further elucidate the significance of decreased ALA-D activity in BEN and their healthy relatives.
Balkan Nephropathy↗
Snapshots of tRNA sulphuration via an adenylated intermediate.
Uridine at the first anticodon position (U34) of glutamate, lysine and glutamine transfer RNAs is universally modified by thiouridylase into 2-thiouridine (s2U34), which is crucial for precise translation by restricting codon-anticodon wobble during protein synthesis on the ribosome. However, it remains unclear how the enzyme incorporates reactive sulphur into the correct position of the uridine base. Here we present the crystal structures of the MnmA thiouridylase-tRNA complex in three discrete forms, which provide snapshots of the sequential chemical reactions during RNA sulphuration. On enzyme activation, an alpha-helix overhanging the active site is restructured into an idiosyncratic beta-hairpin-containing loop, which packs the flipped-out U34 deeply into the catalytic pocket and triggers the activation of the catalytic cysteine residues. The adenylated RNA intermediate is trapped. Thus, the active closed-conformation of the complex ensures accurate sulphur incorporation into the activated uridine carbon by forming a catalytic chamber to prevent solvent from accessing the catalytic site. The structures of the complex with glutamate tRNA further reveal how MnmA specifically recognizes its three different tRNA substrates. These findings provide the structural basis for a general mechanism whereby an enzyme incorporates a reactive atom at a precise position in a biological molecule.
Anticodon↗
Isomerase-independent chaperone function of cyclophilin ensures aggregation prevention of adenosine kinase both in vitro and under in vivo conditions.
Using inactive aggregates of adenosine kinase (AdK) from Leishmania donovani as the model substrate, we recently demonstrated that a cyclophilin (LdCyP) from the same source in an isomerase-independent fashion reactivated the enzyme in vitro by disaggregating its inactive oligomers [Chakraborty et al. (2002) J. Biol. Chem. 277, 47451-47460]. Besides disrupting preformed aggregates, LdCyP also prevents reaggregation of the newly formed active protein that is generated after productive refolding from its urea-denatured state. To investigate possible physiological implications of such phenomena, a unique expression system that simultaneously induces both AdK and LdCyP in naturally AdK-deficient Escherichia coli, was developed. Both in vitro and in vivo experiments revealed that oligomerization is an inherent property of this particular enzyme. In vivo protein cross-linking studies, activity determination analysis and Ado phosphorylation experiments carried out in cells coexpressing both the proteins unequivocally demonstrated that, similar to the phenomena observed in vitro, aggregates of the enzyme formed in vivo are able to interact with both LdCyP and its N-terminal truncated form (N(22-88)DEL LdCyP) in a crowded intracellular environment, resulting in aggregation prevention and reactivation of the enzyme. Our results indicate that the isomerase-independent chaperone function of LdCyP, detected in vitro, participates in vivo as well to keep aggregation-prone proteins in a monomeric state. Furthermore, analogous to yeast/bacterial two-hybrid systems, development of this simple coexpression system may help in the confirmation of interaction of two proteins under simulated in vivo conditions.
Adenosine Kinase↗
Radical catalysis of B12 enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases.
Enzymatic radical catalysis is defined as a mechanism of catalysis by which enzymes catalyze chemically difficult reactions by utilizing the high reactivity of free radicals. Adenosylcobalamin (coenzyme B12) serves as a cofactor for enzymatic radical reactions. The recent structural analysis of adenosylcobalamin-dependent diol dehydratase revealed that the substrate 1,2-propanediol and an essential potassium ion are located inside a (beta/alpha)8 barrel. Two hydroxyl groups of the substrate coordinate directly to the potassium ion which binds to the negatively charged inner part of the cavity. Cobalamin bound in the base-on mode covers the cavity to isolate the active site from solvent. Based on the three-dimensional structure and theoretical calculations, a new mechanism for diol dehydratase is proposed in which the potassium ion plays a direct role in the catalysis. The mechanisms for generation of a catalytic radical by homolysis of the coenzyme Co-C bond and for protection of radical intermediates from undesired side reactions during catalysis are discussed based on the structure. The reactivating factors for diol and glycerol dehydratases have been identified. These factors are a new type of molecular chaperone which participate in reactivation of the inactivated holoenzymes by mediating ATP-dependent exchange of the modified coenzyme for free intact coenzyme.
Bacteria↗
Unfolding kinetics of glutathione reductase from cyanobacterium Spirulina maxima.
The kinetics of the irreversible unfolding of glutathione reductase (NAD[P]H:GSSG oxidoreductase, EC 1.6.4.2.) from cyanobacterium Spirulina maxima was studied at pH 7.0 and room temperature. Denaturation was induced by guanidinium chloride and the changes in enzyme activity, aggregation state, and tertiary structure were monitored. No full reactivation of enzyme was obtained, even after very short incubation times in the presence of denaturant. Reactivation plots were complex, showing biphasic kinetics. A very fast early event in the denaturation pathway was the dissociation of tetrameric protein into reactivatable native-like dimers, followed by its conversion into a nonreactivatable intermediary, also dimeric. In the final step of the unfolding pathway the latter was dissociated into denatured monomers. Fluorescence measurements revealed that denaturation of S. maxima glutathione reductase is a slow process. Release of the prostethic group FAD was previous to the unfolding of the enzyme. No aggregated species were detected in the unfolding pathway, dismissing the aggregation of denatured polypeptide chains as the origin of irreversibility. Instead, the transition between the two dimeric intermediates is proposed as the cause of irreversibility in the denaturation of S. maxima glutathione reductase. A value of 106.6 +/- 3 kJ mol(-1) was obtained for the activation free energy of unfolding in the absence of denaturant. No evidence for the native monomer in the unfolding pathway was obtained which suggests that the dimeric nature of glutathione reductase is essential for the maintenance of the native subunit conformation.
Bacterial Proteins↗
Reactive oxygen species and antioxidant enzymes activity of Anabaena sp. PCC 7120 (Cyanobacterium) under simulated microgravity.
It was found that reactive oxygen species in Anabaena cells increased under simulated microgravity provided by clinostat. Activities of intracellular antioxidant enzymes, such as superoxide dismutase, catalase were higher than those in the controlled samples during the 7 days' experiment. However, the contents of glutathione [correction of gluathione], an intracellular antioxidant, decreased in comparison with the controlled samples. The results suggested that microgravity provided by clinostat might break the oxidative/antioxidative balance. It indicated a protective mechanism in algal cells, that the total antioxidant system activity increased, which might play an important role for algal cells to adapt the environmental stress of microgravity.
Anabaena↗
Cytochrome P-450-dependent covalent binding of carbon disulfide to rat liver microsomal protein in vitro and its prevention by reduced glutathione.
Carbon disulfide, a hepatotoxic solvent, is metabolized by liver microsomal enzymes to reactive sulfur atoms which get bound to the microsomal enzymes, causing inhibition of the enzyme system. These studies were carried out to examine whether glutathione can protect the liver enzymes from the sulfur binding and against carbon disulfide toxicity. When liver microsomes isolated from phenobarbital-pretreated rats were incubated with 35S-CS2, NADPH and glutathione, almost 60% decrease in sulfur binding to microsomal protein was observed under the experimental conditions. It was further observed that the addition of glutathione to microsomal incubations resulted in almost complete recovery of the activity of the enzyme system as measured by cytochrome P-450 concentration and benzphetamine metabolism. The data suggest that the presence of glutathione in sufficient amount in the liver of subject exposed to CS2 may significantly decrease the liver toxicity of this highly toxic compound.
Animals↗
Sulfhydryl groups of an extramitochondrial acetyl-CoA hydrolase from rat liver.
An extramitochondrial acetyl-CoA hydrolase (EC 3.1.2.1) purified from rat liver was inactivated by heavy metal cations (Hg2+, Cu2+, Cd2+ and Zn2+), which are known to be highly reactive with sulfhydryl groups. Their order of potency for enzyme inactivation was Hg2+ greater than Cu2+ greater than Cd2+ greater than Zn2+. This enzyme was also inactivated by various sulfhydryl-blocking reagents such as p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and iodoacetate (IAA). DL-Dithiothreitol (DTT) reversed the inactivation of this enzyme by DTNB markedly, and that by PHMB slightly, but did not reverse the inactivations by NEM, DTNB and IAA. Benzoyl-CoA (a substrate-like competitive inhibitor) and ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by PHMB, NEM, DTNB and IAA. These results suggest that the essential sulfhydryl groups are on or near the substrate binding site and nucleotide binding site. The enzyme contained about four sulfhydryl groups per mol of monomer, as estimated with DTNB. When the enzyme was denatured by 4 M guanidine-HCl, about seven sulfhydryl groups per mol of monomer reacted with DTNB. Two of the four sulfhydryl groups of the subunit of the native enzyme reacted with DTNB first without any significant inactivation of the enzyme, but its subsequent reaction with the other two sulfhydryl groups seemed to be involved in the inactivation process.
Acetyl-CoA Hydrolase↗
Purification and immunological characterization of a new form of gamma-glutamyltransferase of human semen.
A new form of gamma-glutamyltransferase was purified from human seminal plasma. The purified enzyme was composed of two non-identical subunits with apparent molecular masses of 150 and 95 kDa on polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), and showed a molecular mass of 500 and 250 kDa on gel filtration in the absence and presence of 1% Triton X-100, respectively. This enzyme was different from human renal gamma-glutamyltransferase not only in apparent molecular masses, but also in amino acid compositions of both the subunits to each other. Experiments with the antisera raised against the purified enzyme revealed that the enzyme was different from the renal, hepatic and testicular enzymes in reactivity to the antibody though partially related to those enzymes. Ouchterlony double diffusion analysis indicated that both human seminal plasma and prostatic extract contained two types of gamma-glutamyltransferase, one is that we purified and the other the renal type. Hence, it is most likely that gamma-glutamyltransferase accounting for most of the enzyme activity in semen results from prostata followed by secretion to seminal plasma.
Amino Acids↗