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We have established the generality of using detergents for facilitating the reactivation of 6 M guanidinium chloride-denatured rhodanese that was recently described for the nonionic detergent lauryl maltoside (LM) (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). We report here that not only LM but other nonionic as well as ionic and zwitterionic detergents also have favorable effects in reactivating the denatured enzyme. Not all detergents are useful, and the favorable effects occur over a limited concentration range. Above and below that range there is little or no effect. Zwittergents, which represent a homologous series with varying critical micelle concentrations (CMCs) are effective only above their CMCs. Induction phases occur in the progress curves of rhodanese refolded in the presence of the effective detergents, suggesting the presence of refolding intermediates that are apparently stabilized by detergent interactions. Gel filtration chromatography of rhodanese with and without LM suggests that even though the renaturation of the denatured enzyme requires detergent at concentrations above its CMC, the enzyme does not bind an amount of detergent equivalent to a micelle. It is suggested that renaturation of other proteins might also be assisted by inclusion of "nondenaturing" detergents, although the optimal conditions will have to be determined for each individual case.
Rat pineal hydroxyindole-O-methyltransferase activity in crude homogenates is reduced by treatment with disulfides. Cystamine (IC50 = 128 microM) and selenocystamine (IC50 = 13 microM) are the most potent compounds tested. Reduced cystamine (cysteamine) and diaminohexane are inactive. N,N'-Diacetylcystamine, penicillamine disulfide, and glutathione disulfide are less potent or inactive; but several peptides (oxytocin, vasopressin, and arginine vasotocin) are active. Inactivation by cystamine is time- and temperature-dependent and is accelerated at higher pH. Disulfide treatment of intact pinealocytes also inactivates the enzyme. Addition of dithiothreitol during the enzyme assay completely reactivates inactivated enzyme formed by disulfide treatment of homogenates or intact cells. Rat hydroxyindole-O-methyltransferase is also inactivated in the absence of added disulfides and dissolved O2. This spontaneous inactivation is time-, temperature-, and pH-dependent and can be completely prevented, but not reversed, by dithiothreitol. In contrast to the inhibitory effects of cystamine on the rat enzyme, cystamine does not alter bovine hydroxyindole-O-methyltransferase and increases ovine hydroxyindole-O-methyltransferase activity. The bovine and ovine enzymes do not become inactive in the absence of added disulfides. Together these observations indicate that rat pineal hydroxyindole-O-methyltransferase can be inactivated by a protein thiol:disulfide exchange mechanism. This mechanism may contribute to the physiological regulation of this enzyme in the rat pineal gland but does not appear to be a common feature of pineal hydroxyindole-O-methyltransferase regulation in all species.
In Escherichia coli K12 the biosynthetic pathway of lysine, methionine and threonine is characterized by three isofunctional aspartokinases and two homoserine dehydrogenases. A single polypeptide chain carries the threonine-sensitive aspartokinase and homoserine dehydrogenase (AK I-HDH I), and a different polypeptide chain carries the methionine-repressible aspartokinase and homoserine dehydrogenase (AK II-HDH II). Immuno-adsorbants prepared with rabbit antibodies against AK I-HDH I bind the lysine-sensitive aspartokinase (AK III), the AK II-HDH II, and the homoserine kinase (HSK), an enzyme of the threonine biosynthetic pathway. Saturation of the immunoadsorbant with AK I-HDH I results in a decreased binding capacity for the other enzymes. Displacement of bound AK III or HSK can be obtained with pure AK I-HDH I, showing that the affinity of the antibodies to homologous antigens is higher than to heterologous ones. Immunoadsorbants prepared with anti-HSK antibodies show the same type of recognition: binding of the three aspartkinases and a capacity to displace the heterologous antigens bound. Accordingly, the same antibodies, implicated in the binding of the homologous antigen, bind the other enzymes. None of the other enzymes of the pathway, or the other kinases tested are recognized by the two immunoadsorbants. It can be postulated that in E. coli K12, duplication of a common ancestor gene gave rise to the three aspartokinases and to the homoserine kinase; two of the genes coding for the aspartokinases fused with those coding for the homoserine dehydrogenases. Indicating that only few epitopes are shared by these enzymes, by conventional immuno-diffusion techniques no precipitation lines appeared with antibodies against AK I-HDH I and the other proteins.
Explants of normal skin fail to react in direct immunofluorescence tests with stratum corneum antibodies. However, upon stripping with cellophane tape, the horny layer of such explants react in tissue culture. Swabbing of skin explants with ether and chloroform converts stratum corneum antigen (SCAg) from a nonreactive to a reactive form. Treatment with methanol, acetone or phosphate-buffered saline failed to bring about such a conversion. Treatment of skin explants with hyaluronidase and phosphilipase A converst SCAg of at least some skin explants to a reactive form. Treatment with trypsin, chymotrypsin and plasmin abolished the reactivity of SCAg upon prolonged incubation. However, upon short incubation with plasmin, SCAg was converted to a reactive form.
The kinetics of inactivation and reactivation of rabbit skeletal muscle phosphofructokinase have been studied as a function of pH and enzyme concentration at constant temperature in phosphate buffer. From the enzyme concentration dependence, we conclude that the minimal mechanism for inactivation involves a protonation step followed by isomerization to an inactive form and then dissociation to a species of one-half the molecular weight. Other data indicate a subsequent isomerization of the dissociated form. The pH and temperature dependence of the inactivation process shows that it is controlled by ionizable groups, and that the apparent pK for these groups is temperature-dependent in such a way as to make the enzyme show the characteristic of cold lability below pH 7. Reactivation of the inactive enzyme occurs by a kinetically different pathway involving deprotonation of an inactive, dissociated form to a form which may either isomerize to another inactive form, or dimerize to the active enzyme. A general mechanism is postulated in which the inactivation and reactivation processes are different aspects of the same mechanism. This mechanism assumes four species (two containing four subunits and two containing two subunits) each of which can exist in a protonated and unprotonated form. Inactivation or reactivation induced by changes in pH or temperature reflect the kinetic establishment of a new steady state between these forms. How the apparent pK values which control the distribution of the enzyme between protonated and unprotonated forms describe the pH-dependent characteristics of the enzyme is discussed in terms of the proposed mechanism.
Regional distributions of thiobarbituric acid-reactive products, activities of enzymes regulating metabolism of oxygen free radicals, and some of the related enzymes were studied in 10 areas of adult and aged rat brains. Thiobarbituric acid-reactive products were lower in cerebral cortex, septal area, hippocampus, caudate-putamen, and substantia nigra compared with other areas studied in adult rats; however, they increased significantly in the former areas with aging. A slight but significant reduction in superoxide dismutase activity was noted in frontal cortex, septal area, caudate-putamen, and substantia nigra with aging. Glutathione peroxidase and reductase activities were highest in caudate-putamen and in substantia nigra. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were lowest in cortical areas. Phosphofructokinase activity was lowest in septal area and hippocampus in aged rats. Glyceraldehyde-3-phosphate dehydrogenase activity showed only small regional and evolutional changes. Lactate dehydrogenase activity declined with age in most of the areas studied. sn-Glycerol-3-phosphate dehydrogenase activity showed small changes with aging except in hippocampus, where 40% reduction was noted. Generally, cerebral cortical areas, hippocampus, and septal areas were not particularly enriched in enzymes regulating the metabolism of oxygen free radicals. The results were discussed in relation to the role of free radicals in aging.
Reactive oxygen species produced by neutrophils contribute to the pathogenesis of focal cerebral ischemia/reperfusion injury and signal the inflammatory response. We have previously shown that honokiol, an active principle extracted from Magnolia officinalis, has a protective effect against focal cerebral ischemia/reperfusion injury in rats that paralleled a reduction in reactive oxygen species production by neutrophils. To elucidate the underlying mechanism(s) of the antioxidative effect of honokiol, peripheral neutrophils isolated from rats were activated with phorbol-12-myristate-13-acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in the presence or absence of honokiol. In this study, we found that honokiol inhibited PMA- or fMLP-induced reactive oxygen species production by neutrophils by three distinct mechanisms: (1) honokiol diminished the activity of assembled-NADPH oxidase, a major reactive oxygen species producing enzyme in neutrophils by 40% without interfering with its protein kinase C (PKC)-dependent assembly; (2) two other important enzymes for reactive oxygen species generation in neutrophils, i.e., myeloperoxidase and cyclooxygenase, were also inhibited by honokiol by 20% and 70%, respectively; and (3) honokiol enhanced glutathione (GSH) peroxidase activity by 30%, an enzyme that triggers the metabolism of hydrogen peroxide (H2O2). These data suggested that honokiol, acting as a potent reactive oxygen species inhibitor/scavenger, could achieve its focal cerebral ischemia/reperfusion injury protective effect by modulating enzyme systems related to reactive oxygen species production or metabolism, including NADPH oxidase, myeloperoxidase, cyclooxygenase, and GSH peroxidase in neutrophils.
Tetrameric 20 beta-hydroxysteroid dehydrogenase (17,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) from Streptomyces hydrogenans was reactivated after inactivation, dissociation and denaturation with urea. The effect of several factors such as NAD+, NADH, substrate, sulphydryl reducing agents, extraneous proteins, pH and enzyme concentration on reactivation was investigated. The coenzymes were found to be essential for obtaining a high reactivation yield (about 90%), since in their absence the reactivation was less than 10%. NADH was effective at lower concentrations than NAD+. The reactivated enzyme, after the removal of inactive aggregates, showed physical and catalytic properties coincident with those of the native enzyme. The mechanism by which NADH affects the reconstitution of 20 beta-hydroxysteroid dehydrogenase was investigated using both soluble enzyme and enzyme immobilized on Sepharose 4B. The immobilization demonstrates that isolated subunits are inactive and incapable of binding NADH and suggests that subunit association to the tetramer is essential for enzymatic activity. NADH appears to act, after subunit assembly has taken place, by stabilizing tetramers and preventing their aggregation with monomers that would give rise to inactive polymers.
The glutathione inhibitor drugs, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and buthionine sulfoximine (BSO), were tested in vitro in order to assess their cytotoxic effectiveness when combined with an enzyme immunotoxin (eIT) composed of a T-cell reactive monoclonal antibody (mAb) 097 coupled to the reactive oxygen-generating enzyme, glucose oxidase (GO) (EC 1.1.3.4). As targets of this eIT we used mature human T-cells or leukemia cells that expressed the 097 epitope. We found that treatment of the cells with subtoxic amounts of mixtures of both a drug and the 097 eIT markedly potentiated cytotoxicity compared to either drug or eIT alone.
Reactivity and chemical properties of calf thymus topoisomerase I have been investigated with respect to enzyme ability to relax supercoiled DNA. The relaxation rate has been analyzed at optimum and relatively high salt concentration. Catalysis is processive at optimum salt concentration and distributive at a higher one; camptothecin decreases the initial rate of reaction in both salt conditions, but more so at the higher one. We conclude that: 1. calf thymus topoisomerase I requires, for its maximum reactivity, specific and characteristic reaction conditions; 2. salt concentration affects DNA processing, indeed influencing the initial rate of DNA relaxation and directly reflecting the salt-dependence for the enzyme-duplex DNA binding; 3. topoisomerase I, from various sources, maybe individually responds to alteration of assay parameters such as pH, Mg++ and NaCl concentrations, indicating that individual criteria could be responsible for the catalytic activity optimum.
Using high-resolution immuno-electron microscopy the steady-state subcellular distribution of tyrosine-nitrated proteins in different cells and tissues was evaluated. In quiescent eosinophils and neutrophils in the bone marrow intracellular nitrated proteins were mainly restricted to the peroxidase-containing secretory granules. The inducible nitric oxide synthase (iNOS) was expressed in the same granules. Proteins nitrated on tyrosine residues were also abundant in the cytosol of circulating erythrocytes. In the vasculature, nitrated proteins were mainly located in mitochondria and endoplasmic reticulum of the endothelial cells, fibroblasts, and smooth muscle cells. Endogenous nitrated proteins were also found in chondrocytes in cartilage, where it was typically associated with the cytoplasmic interface of the endoplasmic reticulum membrane. Nitrated proteins were also prominent in the peroxisomes of liver hepatocytes and of secretory cells in the lacrimal gland. Challenge of mouse dendritic cells with lipopolysaccharide induced iNOS protein expression in cytosol and peroxisomes and was associated with an increased 3-nitrotyrosine formation in cytosol, mitochondria, and peroxisomes. These data indicate that nitric oxide-dependent protein tyrosine nitration is a physiologically relevant process localized within specific subcellular compartments in close proximity to iNOS and to enzymes capable of peroxidative chemistry and reactive oxygen species production.
Physostigmine, aldicarb and carbaryl were potent inhibitors of acetylcholinesterase (AChE). The physostigmine-inhibited AChE fluoresced at 300 nm excitation and 500 nm emission wavelengths, but the aldicarb and carbaryl inhibited enzyme did not. This suggests that the carbamylated active center is not the fluorescing site in AChE. The fluorescence intensity of physostigmine-inhibited AChE decreased with increasing the substrate (acetylthiocholine) concentration, thus indicating that physostigmine binding to the active site is essential for the development of fluorescence. Thus, the physostigmine-inhibited AChE fluoresces due to the binding of trimethylpyrrolo[2,3-b]indol (TMPI) moiety, formed by the hydrolysis of physostigmine, to a peripheral site in AChE. The fluorescence intensity of the physostigmine-inhibited enzyme decreased when the inhibited-enzyme was dialyzed for either 30 min that poorly reactivated the enzyme or 180 min that fully reactivated the enzyme. This suggests that dialysis dissociates the AChE-TMPI complex much faster than it reactivates the carbamylated AChE. Ephedrine, propranolol and phenothiazines including trifluoparazine (TPZ) caused non-competitive inhibition, while hexamethonium caused an uncompetitive inhibition of AChE activity. TPZ, upon binding with AChE, formed a fluorescent TPZ-enzyme complex. The fluorescence intensity of TPZ-AChE complex was effectively decreased by ephedrine, but not by propranolol or hexamethonium. This indicates that TPZ and ephedrine bind to the same site in AChE which is different from the site/or sites to which propranolol or hexamethonium bind. Hexamethonium protected AChE from inhibition by carbamates and decreased the fluorescence intensity of the physostigmine-inhibited AChE. Phenothiazines and ephedrine did not modulate the enzyme inhibition or the fluorescence intensity of the physostigmine-inhibited AChE. Propranolol and TPZ potentiated the enzyme inhibition and increased the fluorescence intensity in the presence of physostigmine. These compounds, however, did not affect the inhibition of AChE by carbaryl or aldicarb. Ephedrine blocked the effects of TPZ, but did not alter the effects of propranolol on physostigmine-inhibited AChE. AChE, therefore, contains multiple peripheral binding sites which, upon binding to specific ligands, transduce differential signals to the active center.
Adenosine-5'-phosphosulfate (APS) kinase from Penicillium chrysogenum, loses catalytic activity at temperatures greater than approximately 40 degrees C. When the heat-inactivated enzyme is cooled to 30 degrees C or lower, activity is regained in a time-dependent process. At an intermediary temperature (e.g. 36 degrees C) an equilibrium between active and inactive forms can be demonstrated. APS kinase from P. chrysogenum is a dimer (Mr = 57,000-60,000) composed of two apparently identical subunits. Three lines of evidence suggest that the reversible inactivation is a result of subunit dissociation and reassociation. (a) Inactivation is a first-order process. The half-time for inactivation at a given temperature is independent of the original enzyme concentration. Reactivation follows second-order kinetics. The half-time for reactivation is inversely proportional to the original enzyme concentration. (b) The equilibrium active/inactive ratio at 36 degrees C increases as the total initial enzyme concentration is increased. However, Keq,app at 5 mM MgATP and 36 degrees C calculated as [inactive sites]2/0.5 [active sites] is near-constant at about 1.7 X 10(-8) M over a 10-fold concentration range of enzyme. (c) At 46 degrees C, the inactive P. chrysogenum enzyme (assayed after reactivation) elutes from a calibrated gel filtration column at a position corresponding to Mr = 33,000. Substrates and products of the APS kinase reaction had no detectable effect on the rate of inactivation. However, MgATP and MgADP markedly stimulated the reactivation process (kapp = 3 X 10(5) M-1 X s-1 at 30 degrees C and 10 mM MgATP). The kapp for reactivation was a nearly linear function of MgATP up to about 20 mM suggesting that the monomer has a very low affinity for the nucleotide compared to that of the native dimer. Keq,app at 36 degrees C increases as the MgATP concentration is increased. The inactivation rate constant increased as the pH was decreased but no pK alpha could be determined. The reactivation rate constant increased as the pH was increased. An apparent pK alpha of 6.4 was estimated.
Reactive oxygen species are known to increase in plant senescence. We investigated the participation of antioxidative enzymes in initiation of cotyledon senescence. Senescence of bean (Phaseolus vulgaris L.) cotyledons was modulated by UV C irradiation and by the decapitation of plant apices. Senescence was accompanied by a decrease of protein content and by a decrease of photochemical efficiency. A drop in activity of antioxidative enzymes preceded the onset of senescence in control plants. In cotyledons with prolonged life span, the decrease of antioxidant activities and the markers of senescence onset appeared at a similar age as in controls. Thus we presumed that the period from senescence initiation to cotyledon abscission was extended. On the other hand, in UV C irradiated plants we did not observe actual senescence initiation, and antioxidant enzymes although elevated, did not effectively play their role. The decrease of antioxidant enzymes activity and the markers of senescence appeared at a similar age both in control and in decapitated (D) plants, so we can presume that we prolonged mainly the period from senescence onset to cotyledon abscission in D plants. In UV C irradiated plants the antioxidative enzymes were probably destroyed before the process of senescence could begin.
Enzyme immunoassay (EIA) with sixty types of monoclonal antibodies (MAbs) was used to study cross-reactive epitopes on the attenuated and virulent strains of the Eastern equine encephalomyelitis (EEE) and Venezuelan equine encephalomyelitis (VEE) viruses. All three structural proteins of the EEE and VEE viruses were demonstrated to have both cross-reactive and specific antigenic determinants. The glycoprotein E1 of EEE and VEE viruses possesses three cross-reactive epitopes for binding to MAbs. The glycoprotein E2 has a cluster of epitopes for 20 cross-reacting MAbs produced to EEE and VEE viruses. Cross-reactive epitopes were localised within five different sites of glycoprotein E2 of VEE virus and within four sites of that of the EEE virus. There are no cross-neutralising MAbs to the VEE and EEE viruses. Only one type of the protective Mabs was able to cross-protect mice against lethal infection by the virulent strains of the VEE and EEE viruses. Eight MAbs blocked the hemagglutination activity (HA) of both viruses. Antigenic alterations of neutralising and protective sites were revealed for all attenuated strains of the VEE and EEE viruses. Comparative studies of the E2 proteins amino acid sequences show that the antigenic modifications observed with the attenuated strains of the VEE virus may be caused by multiple amino acid changes in positions 7, 62, 120, 192 and 209-213. The escape-variants of the VEE virus obtained with cross-reactive MAbs 7D1, 2D4 and 7A6 have mutations of the E2 protein at positions 59, 212-213 and 232, respectively. Amino acid sequences in these regions of the VEE and EEE viruses are not homologous. These observations indicate that cross-reactive MAbs are capable of recognising discontinuous epitopes on the E2 glycoprotein.
Nitrous oxide, or laughing gas, is an anaesthetic agent that inactivates cobalamin-dependent methionine synthase. This enzyme uses the highly reactive, enzyme-bound cob(I)alamin oxidation state of the prosthetic group to effect methyl group transfer from 5-methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. The cob(I)alamin is capable of reductively degrading nitrous oxide, and here we characterize the modifications that occur to the Escherichia coli enzyme following electrochemical inactivation. Methionine synthase was inactivated on a milligram scale by equilibrating enzyme containing bound cob(II)alamin with a reduced electrochemical mediator to give the reactive cob(I)alamin state under an anaerobic atmosphere of nitrous oxide. The primary damage occurs to a 37.2-kDa domain that binds S-adenosylmethionine (AdoMet), and inactive enzyme can no longer be reductively methylated using AdoMet. The damage is oxidative, and it includes the covalent addition of the mediator, triquat, to the enzyme selectively at valine 1177, as well as the formation of a covalent cross-link between peptides containing the only two cysteines within this domain. Spectrally, the prosthetic group bound to inactive enzyme resembles cob(II)alamin, although some loss in absorbance is apparent. When the enzyme was reconstituted with [57Co]cobalamin and the inactivation repeated, the cobalamin was recovered unmodified in approximately 75% yield, but two products derived from the cobalamin were also observed. We interpret the finding of oxidatively modified products as strong evidence that reductive degradation of nitrous oxide releases a potent oxidant, presumably hydroxyl radical or its equivalent, that is capable of modifying sites proximal to the cobalamin.