DTNB Treatment Research Articles

De-polymerisation and hydrolysis of seed storage protein is involved in the freezing tolerance of hydrated Brassica oleracea seeds

Hydrolysis of globulins might be involved in the freezing tolerance of hydrated lettuce seeds. The mechanism was further explored in hydrated brassica (Brassica oleracea) seeds, by application of 5,5'-dithiobis(2-nitro- benzoicacid) (DTNB), the Ellman's reagent, which can bind to the free sulphydryl side chain of cysteine. DTNB increased the viability of hydrated brassica seeds after slow cooling (-3°C hour-1 ) to -17°C. Two-dimensional electrophoresis and mass spectra analysis indicated the accumulation of small peptides derived from 12S globulins (cruciferin) in DTNB-treated seeds after slow cooling. The seed storage peptides with different molecular weight were cooled and the ice formation during cooling observed by cryomicroscopy, which proved that peptides of smaller molecular size produced smaller and rounder crystals. The smallest peptide in DTNB- treated seeds produced the smallest and roundest crystals during cooling. The levels of ASA, DHA and GSH were specifically up-regulated after DTNB treatment and following slow cooling, which indicates that DTNB enhanced the circulation of ASA and GSH to resist the oxidative stress. Consistent with this finding, RNA-seq and real time PCR indicated that glutathione reductase (GR) was up-regulated after DTNB treatment.

Thioredoxin (Trx1) regulates CD4 membrane domain localization and is required for efficient CD4-dependent HIV-1 entry

BackgroundCD4 is a glycoprotein expressed on the surfaces of certain immune cells. On lymphocytes, an important function of CD4 is to co-engage Major Histocompatibility Complex (MHC) molecules with the T Cell Receptor (TCR), a process that is essential for antigen-specific activation of T cells. CD4 localizes dynamically into distinct membrane microdomains, an important feature of its immunoregulatory function that has also been shown to influence the efficiency of HIV replication. However, the mechanism by which CD4 localization is regulated and the biological significance of this is incompletely understood. MethodsIn this study, we used confocal microscopy, density-gradient centrifugation and flow cytometry to analyze dynamic redox-dependent effects on CD4 membrane domain localization. ResultsBlocking cell surface redox exchanges with both a membrane-impermeable sulfhydryl blocker (DTNB) and specific antibody inhibitors of Thioredoxin-1 (Trx1) induces translocation of CD4 into detergent-resistant membrane domains (DRM). In contrast, Trx1 inactivation does not change the localization of the chemokine receptor CCR5, suggesting that this effect is targeted. Moreover, DTNB treatment and Trx1 depletion coincide with strong inhibition of CD4-dependent HIV entry, but only moderate reductions in the infectivity of a CD4-independent HIV pseudovirion. ConclusionsChanges in the extracellular redox environment, potentially mediated by allosteric consequences of functional disulfide bond oxidoreduction, may represent a signal for translocation of CD4 into DRM clusters, and this sequestration, another potential mechanism by which the anti-HIV effects of cell surface oxidoreductase inhibition are exerted. General significanceExtracellular redox conditions may regulate CD4 function by potentiating changes in its membrane domain localization.

Neutralization by metal ions of the toxicity of sodium selenide.

Inert metal-selenide colloids are found in animals. They are believed to afford cross-protection against the toxicities of both metals and selenocompounds. Here, the toxicities of metal salt and sodium selenide mixtures were systematically studied using the death rate of Saccharomyces cerevisiae cells as an indicator. In parallel, the abilities of these mixtures to produce colloids were assessed. Studied metal cations could be classified in three groups: (i) metal ions that protect cells against selenium toxicity and form insoluble colloids with selenide (Ag+, Cd2+, Cu2+, Hg2+, Pb2+ and Zn2+), (ii) metal ions which protect cells by producing insoluble metal-selenide complexes and by catalyzing hydrogen selenide oxidation in the presence of dioxygen (Co2+ and Ni2+) and, finally, (iii) metal ions which do not afford protection and do not interact (Ca2+, Mg2+, Mn2+) or weakly interact (Fe2+) with selenide under the assayed conditions. When occurring, the insoluble complexes formed from divalent metal ions and selenide contained equimolar amounts of metal and selenium atoms. With the monovalent silver ion, the complex contained two silver atoms per selenium atom. Next, because selenides are compounds prone to oxidation, the stabilities of the above colloids were evaluated under oxidizing conditions. 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the reduction of which can be optically followed, was used to promote selenide oxidation. Complexes with cadmium, copper, lead, mercury or silver resisted dissolution by DTNB treatment over several hours. With nickel and cobalt, partial oxidation by DTNB occurred. On the other hand, when starting from ZnSe or FeSe complexes, full decompositions were obtained within a few tens of minutes. The above properties possibly explain why ZnSe and FeSe nanoparticles were not detected in animals exposed to selenocompounds.

Sulfhydryl oxidation: A potential strategy to achieve neuroprotection during severe hypoxia?

Previously we reported that sulfhydryl (SH) modulation affects the susceptibility of rat hippocampal slices to severe hypoxia. SH-oxidation by DTNB (5,5′-dithiobis 2-nitrobenzoic acid) or H2O2 postponed the onset of hypoxia-induced spreading depression (HSD), thereby delaying the loss of neuronal function, whereas SH-reduction by DTT (1,4-dithio-dl-threitol) hastened HSD onset. To judge the neuroprotective merit that might arise from a postponement of HSD by oxidants, we have extended our earlier observations by multiparametric recordings and screened for changes in the extracellular K+ accumulation, HSD propagation velocity, and its maximum spread. As parameters for neuronal network function, the failure of synapses during hypoxia and their posthypoxic recovery were analyzed. DTNB (2 mM) or H2O2 (5 mM) postponed HSD but did not attenuate the rise in extracellular K+ concentration ([K+]o), HSD propagation velocity or its maximum spread. H2O2 slightly postponed the synaptic failure during hypoxia; the posthypoxic recovery of synapses was, however, incomplete. DTNB slowed the synaptic recovery upon reoxygenation. DTT (2 mM) hastened HSD onset, but HSD propagation velocity and tissue invasion were not affected. Upon reoxygenation, however, normalization of [K+]o was disturbed and synaptic recovery failed. Therefore, SH-reducing conditions at the onset of HSD proved to be devastating for the hippocampal network. In conclusion, the only merit of DTNB or H2O2 treatment is a delayed HSD onset, i.e. some extra time before neuronal function is lost during severe hypoxia. Attenuation of the severe changes during HSD or an improved outcome was not observed. Nevertheless, combination of SH-oxidants with established neuroprotectants might be a potential therapeutic approach.

Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

Kinesin-1 motor proteins step along microtubules by a mechanism in which the heads cycle through microtubule-bound and unbound states in an interlaced fashion. An important contribution to head-head coordination arises from the action of the neck-linker that docks onto the core motor domain upon ATP binding. We show here that the docked neck-linker not only guides the microtubule-unbound head to the next microtubule binding site but also signals its position to the head to which it is attached. Cross-linking studies on mutated kinesin constructs reveal that residues at the interface motor core/docked neck-linker, among them most importantly a conserved tyrosine, are involved in this feedback. The primary effect of the docked neck-linker is a reduced microtubule binding affinity in the ADP state.

Characterization of Specific Nucleosomal States by Use of Selective Substitution Reagents in Model Octamer and Tetramer Structures

Packaging of the DNA in nucleosomes restricts its access to regulatory factors and enzymatic complexes, making a local remodeling of the nucleosome structure a prerequisite to the establishment of protein–DNA interactions. The use of an experimental system in which one nucleosome is reconstituted on a topologically constrained DNA minicircle allows the visualization of different conformations of the nucleoprotein particle. The single cysteine located at position 110 of histone H3 can be titrated with thiol reagents, such as iodoacetamide (IAM), N-ethylmaleimide (NEM), and dithiobisnitrobenzoic acid (DTNB), in both histone octamers and histone (H3–H4)2 tetramers. Treatment of histone H3 with IAM, NEM, and DTNB allows the trapping of different conformations of the (H3–H4)2 tetramer within the nucleoprotein particle. When H3 cysteines are titrated within the histone octamer, IAM, NEM, and DTNB block the tetramer in the left-handed conformation, the conformation it adopts within the nucleosome. The left-handed conformation is initially dictated by H2A–H2B dimers and then frozen by the thiol reagents. When cysteines are titrated within the histone tetramer, the flexibility of the particle becomes apparent. NEM and IAM behave differently from DTNB. The first two reagents block the particle in its left-handed conformation while DTNB treatment favors the right-handed conformation. These thiol reagents that block the nucleoprotein particles in a given conformation should allow their structural analysis. They may also help the investigation of the role of the (H3–H4)2 nucleoprotein particle structural transition in biological processes involving nucleosome dynamics, such as DNA transcription, replication, and repair.

Preferential distribution of amphotericin B lipid complex into human HDL3 is a consequence of high density lipoprotein coat lipid content

The purpose of this study was to determine the plasma lipoprotein (LP) distribution of amphotericin B (AmpB) and amphotericin B lipid complex [ABLC; Abelcet composed of dimyristoyl phosphati-dylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG)] and define the relationship between LP lipid concentration and composition and the distribution of AmpB and ABLC in human plasma with varying total and lipoprotein cholesterol and triglycerides. AmpB and ABLC at a concentration of 20 μg amphotericin B/mL were incubated in plasma obtained from different human subjects (n = 7) for 60 min at 37 °C. Following these incubations plasma samples were separated into their high-density lipoprotein (HDL), triglyceride-rich lipoprotein (TRL; which contains very low-density lipoproteins and chylomicrons), low-density lipoprotein (LDL), and lipoprotein-deficient (LPDP) fractions by density-gradient ultracentrifugation (UC) and each fraction was assayed for AmpB using high-pressure liquid chromatography (HPLC). The HDL fraction was further separated into its HDL3 and HDL2 subclasses by UC and assayed for AmpB using HPLC. Separation of HDL into its subclasses was confirmed by gel electrophoresis. To assess the influence of modified lipoprotein concentrations and lipid composition on the plasma distribution of AmpB and ABLC, these compounds were incubated in plasmas from human subjects with varying total and lipoprotein lipid concentrations. In addition, to demonstrate that alterations in HDL lipid composition influence the plasma distribution of ABLC, ABLC (20 μg amphotericin B/mL) was incubated in plasma pretreated with dithionitrobenzoate (DTNB, a compound which inhibits lecithin:cholesterol acyltransferase conversion of HDL3 free cholesterol to esterified cholesterol) 18 h prior to the experiment or in untreated plasma for 60 min at 37 °C. Total plasma and lipoprotein cholesterol (TC), free cholesterol (fC), esterified cholesterol (CE), triglyceride (TG), phospholipid (PL), and protein (TP) concentrations in each human sample were determined by enzymatic assays. When AmpB was incubated in human plasmas of varying lipid concentrations, the majority of the drug was recovered in the LPDP fraction. However, the majority of AmpB was recovered in the HDL3 fraction following the incubation of ABLC. Differences in lipid coat content (fC and PL) carried by HDL influenced the distribution of ABLC within plasma of different human subjects. These findings were confirmed by the DTNB treatment experiments. These findings suggest that the association of AmpB with DMPC and DMPG to form drug–lipid complexes modifies the plasma distribution of the AmpB. In addition, the distribution of ABLC among plasma lipoproteins of different human subjects is defined by the HDL lipid coat content and is possibly an important consideration when evaluating the pharmacokinetics, toxicity, and activity of these compounds following administration to humans with differing plasma lipid concentrations.

Epileptiform activity but not synaptic plasticity is blocked by oxidation of NMDA receptors in a chronic model of temporal lobe epilepsy.

Simultaneous extracellular recordings were performed in stratum radiatum and stratum pyramidale of hippocampal slices 7 days following unilateral intracerebroventricular injections of kainic acid. In this ex vivo experimental model of human temporal lobe epilepsy, stimulation of the surviving commissural fibres in stratum radiatum produced graded epileptiform activity in the CA1 area. The oxidizing reagent 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) acting at NMDA receptors redox sites decreases NMDA receptor-mediated responses by half and suppresses evoked epileptiform discharges. We have examined the effect of DTNB on NMDA-dependent bidirectional synaptic plasticity and EPSP/spike coupling. DTNB treatment did not prevent either long-term potentiation induced by tetanic stimulation or long-term depression induced by low frequency stimulation of field EPSPs. Application of DTNB alone did not induce EPSP/spike dissociation. However, both high and low frequency stimulations induced EPSP/spike potentiation indicating that neurons had a high probability to discharge in synchrony. These results suggest that oxidizing reagents may provide novel antiepileptic treatments since they decrease NMDA-dependent evoked epileptiform activity but do not interfere with either NMDA-dependent synaptic plasticity or the probability of synchronous discharge.

The Regulatory Light Chains of Myosin Modulate Cross-bridge Cycling in Skeletal Muscle

We investigated the kinetics of Ca2+ activation of skeletal muscle contraction elicited by the photolysis of caged Ca2+. Previously we showed that partial extraction of the 18-kDa regulatory light chains (RLCs) of myosin decreased the rate of force development and was subsequently increased by approximately 20% following reconstitution with RLCs (Potter, J. D., Zhao, J. and Pan, B. S. (1992) FASEB J. 6, A1240). We extend here the RLC-extraction study to the complete removal of the RLCs. The complete removal of RLCs was achieved by a combination of 5,5'-dithiobis-(2-nitrobenzoic acid) and EDTA treatment followed by reduction of oxidized sulfydryl groups by dithiothreitol. Under these conditions the complete extraction of RLCs was accompanied by the extraction of endogenous troponin C, resulting in the loss of isometric tension. Steady state force was restored to 65-75% following troponin C reconstitution and increased to 75-85% as a result of RLC reincorporation into the fibers. The rates of force transients generated by UV-flash photolysis of 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N' -tetrakis[(oxycarbonyl)methyl]-1,2-ethanediamine) or nitrophenyl-EGTA, photoliberating bound Ca2+, decreased 2-fold after RLC extraction and troponin C reconstitution and then increased to the values of intact fibers after RLC reconstitution. These results support our earlier findings that the regulatory light chains of myosin play an important role in the kinetics of cross-bridge cycling.

Anoxic LTP is mediated by the redox modulatory site of the NMDA receptor

1. The effects of redox reagents, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and tris(carboxyethyl)phosphine (TCEP), on anoxia-induced long-term potentiation (LTP) were investigated in CA1 hippocampal neurons using extracellular recording techniques. Experiments were performed in the presence of 0.1 mM MgCl2 and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to pharmacologically isolate N-methyl-D-aspartate (NMDA) receptor-mediated responses. 2. DTNB (200 microM), a thiol oxidizing reagent, reduces by 52 +/- 9% (mean +/- SE) (n = 9/9) NMDA-receptor field potentials evoked by electrical stimulation of Schaffer collaterals and this effect could not be reversed by extensive washing. Nearly the same reduction of the initial response was obtained with different concentrations of DTNB (100 and 500 microM), but the time required to reach the maximal inhibition was concentration-dependent. 3. In keeping with an earlier study oxygen and glucose deprivation for 2-3 min induced a long-term potentiation (LTP) of the NMDA receptor response (+65 +/- 16%, n = 4/6). This potentiation was reversed by DTNB (100-500 microM) (-47 +/- 18%; n = 4/4) and the initial LTP could not be restored upon extensive washing of the drug. 4. TCEP (200 microM), a reagent which reduces S-S bond, amplified the electrically evoked NMDA-receptor EPSP (+27 +/- 12%; n = 3). In addition, TCEP (200 microM), nearly completely reversed the effect of DTNB (200 microM) on anoxia-induced LTP (+56 +/- 19%; n = 3/3). Preliminary results also indicate that TCEP occlude anoxic-LTP (n = 3/4). 5. Following DTNB (200 microM) treatment, oxygen and glucose deprivation did not generate anoxic LTP and extensive washing did not restore a potentiated NMDA field potential. 6. These observations strongly suggest that the redox site of the NMDA receptor is involved in the induction and the maintenance of the anoxic LTP of the NMDA receptor-mediated response in CA1.

A cysteine in the C-terminal region of alanyl-tRNA synthetase is important for aminoacylation activity.

Alanyl-tRNA synthetase (AlaRS) from Escherichia coli is a multimeric enzyme that catalyzes the esterification of alanine to tRNA(Ala) in the ATP-dependent aminoacylation reaction. The functional binding of all three substrates follows Michaelis-Menten kinetics. The role of cysteines in this enzyme has been evaluated via modification of these residues with p-(hydroxymercuri)phenylsulfonic acid, monobromobimane, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The former two reagents induce nearly complete inactivation of AlaRS aminoacylation activity and the release of all tightly bound zinc. In the case of mild DTNB treatment, only two of the six cysteines in AlaRS are modified, with release of all zinc and partial loss of aminoacylation activity. These experiments indicate the importance of one or more cysteines, other than those thought to be coordinated with zinc, in the aminoacylation reaction. Substitution of each of the cysteine residues outside the zinc-binding motif with serine does not disrupt zinc binding. However, the cysteine most removed in primary sequence from the active site (Cys665) is identified as important in the aminoacylation step. Mutation of Cys665 to serine induces a 120-fold decrease in the catalytic efficiency of this enzyme, primarily through a kcat effect, and introduces sigmoidal kinetics (nH = 1.8) with respect to the RNA substrate. The results demonstrate that a simple manipulation in the C-terminal region can introduce positive cooperativity in this otherwise noncooperative enzyme.

The effects of thiol reduction and oxidation on the inhibition of NMDA-stimulated neurotransmitter release by ethanol

The N- methyl- D- aspartate (NMDA) receptor is sensitive to inhibition by pharmacologically relevant concentrations of ethanol and may be an important target for the actions of ethanol in the brain. The mechanisms responsible for ethanol's action are unknown but may involve perturbation of one of the multiple regulatory sites that have been identified on the receptor. Previous studies have shown that thiol reducing and oxidizing agents selectively alter the activity of the NMDA receptor via a redox modulatory site. In this study, these agents were tested for their ability to modify the inhibitory actions of ethanol on NMDA-stimulated neurotransmitter release from rat brain slices and the potentiation of NMDA-stimulated release by the glycine agonist d-serine. Treatment of hippocampal slices with the thiol reducing agent dithiothreitol (DTT) significantly potentiated (> 2-fold) the NMDA-stimulated release of tritiated norepinephrine ([ 3H]NE) from rat hippocampal slices. Slices treated with the thiol oxidizing agent dithionitrobenzoic acid (DTNB) had slightly lower (10–15%) levels of NMDA-stimulated neurotransmitter release. Ethanol (25–200 mM) dose-dependently inhibited the NMDA-stimulated release of [ 3H]NE from control slices with a calculated IC 50 value of 73.1 mM. The inhibitory potency for ethanol was increased following DTNB treatment (IC 50 of 40.9 mM) while DTT treatment slightly reduced ethanol's potency (IC 50 of 85.6 mM). The ability of d-serine to augment NMDA-stimulated neurotransmitter release was tested in the presence and absence of ethanol following treatment of slices with either DTT or DTNB to examine any potential interaction between these modulatory sites. d-serine (1–30 μM) potentiated the NMDA-stimulated release of neurotransmitter from both control and DTNB treated slices but did not appear to alter the inhibition produced by 50 mM ethanol in either case. In contrast, d-serine did not potentiate NMDA-stimulated release in DTT treated slices under control conditions nor did it significantly alter the inhibition of neurotransmitter release produced by 50 mM ethanol. These results suggest that changes in the redox state of the NMDA receptor may alter the sensitivity of the receptor to the inhibitory actions of ethanol. These results also suggest that the inhibitory effects of ethanol are not mediated via a selective interaction with the glycine site on the receptor.

AMP-deaminase from human uterine smooth muscle: the effect of DTNB treatment on kinetic and regulatory properties of the enzyme

Reactivity of sulfhydryl groups of human uterine smooth muscle AMP-deaminase with DTNB, and the effect of their chemical modification on kinetic and regulatory properties of the enzyme were investigated. (1), Approx. 7 and 5 sulfhydryl groups per mol of the enzyme have been shown to be accessible for DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) titration in denaturated and native AMP-deaminase, respectively. (2), Titrated groups were not homogenous; some of them reacted with DTNB much faster than others. (3), The activity of the modified enzyme was very low, and the modified enzyme manifested unusual hyperbolic saturation kinetics with the substrate. (4), Exhaustive dialysis against a buffer containing 10 mM thioethanol reactivated the modified enzyme, and restored its original regulatory properties. Experimental results obtained indicate that modified sulfhydryl groups play a significant role in the maintenance of the proper, catalytically-efficient conformation of the enzyme.

Redox active sulfhydryls are required for fructose 2,6-bisphosphate activation of plant pyrophosphate fructose-6-phosphate 1-phosphotransferase

The classical, α β -subunit form (Q 2) of green tomato pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), a cytosolic enzyme functional in carbohydrate metabolism, was rapidly inactivated on incubation with the oxidant 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). Analysis of the DTNB-treated sample by a fluorescence procedure revealed that inactivation was accompanied by oxidation of sulfhydryl groups, primarily on the α-subunit. Phosphate metabolites—fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, P i, and PP i—protected against DTNB inactivation to varying degrees. The K m values for fructose 6-phosphate and PP i were not changed by DTNB treatment, but the capability for activation by fructose 2,6-bisphosphate was severely diminished. The oxidative inactivation of PFP was reversed by dithiothreitol, but not by monothiols (reduced glutathione or β-mercaptoethanol). Reactivation was accompanied by restoration of the ability to undergo activation by fructose 2,6-bisphosphate. The findings suggest that sulfhydryl groups are essential for the activation of PFP by fructose 2,6-bisphosphate and raise the possibility that a reversible change in their redox status can take place under certain conditions. Evidence that this is the case was obtained with a preparation from wheat flour which, in the absence of an added oxidant, required reduction by a dithiol for activation by fructose 2,6-bisphosphate (dithiothreitol and reduced thioredoxin h).

Water transport in human red cells: effects of ‘non-inhibitory’ sulfhydryl reagents

The water diffusional permeability of human red blood cells following exposure to various sulfhydryl group (SH) reagents have been studied using a nuclear magnetic resonance technique. Exposure of red blood cells up to 12 mM N-ethylmaleimide (NEM) or 10 mM 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNE) alone does not affect water diffusion. In contrast, when DTNB treatment follows a preincubation of the cells with NEM, a small (18% at 37°C) but significant inhibition of water permeability occurs. The NEM and DTNB treatment of the cells caused no change of the cell shape and volume or of the cell water volume. Consequently, the inhibition observed after NEM and DTNB treatment has a real significance.

Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase

Three amino acid residues (glycine-14, cysteine-135, and cysteine-218) previously speculated to be important for the structure and function of Drosophila melanogaster alcohol dehydrogenase have been investigated by using site-directed mutagenesis followed by kinetic analysis and chemical modification. Mutating glycine-14 to valine (G14V) virtually inactivates Drosophila ADH, and substitution of alanine at this position (G14A) causes a 31% decrease in activity. Thermal denaturation and kinetic and inhibition studies further demonstrate that replacing glycine-14 with either alanine or valine leads to structural changes in the NAD binding domain. These results provide direct evidence for the role played by glycine-14 in maintaining the correct conformation in the NAD binding domain. On the other hand, changing of cysteine-135, -218, or both to alanine (C135A, C218A, and C135A/C218A) causes no decrease in the catalytic activity of the enzyme, indicating that neither of the cysteinyl residues is essential for catalysis. C135A and wild-type enzyme are both inactivated by DTNB. In contrast, C218A and C135A/C218A are unaffected by DTNB treatment. DTNB modification of cysteine-218 can be prevented by the substrates NAD and 2-propanol, suggesting that cysteine-218 may be in the vicinity of the active site. Cysteine-135 which is normally insensitive to DTNB becomes accessible in the presence of 2-propanol and/or NAD, suggesting a conformational change induced by binding of these substrates.

Light chains of eel myosin isolation and characterization

1. 1. The three light chains have been isolated from the white muscle myosin of eel ( Anguilla anguilla) by a method consisting of urea (LC 1 and LC 3) or DTNB (LC 2) treatment followed by DEAE-cellulose chromatography. 2. 2. They have been analysed for molecular weight, stoichiometry, isoelectric point, amino acid composition, ultraviolet spectrum and peptide maps. 3. 3. Eel myosin light chains are homologous to the corresponding light chains of other fishes so far reported except for small species-specific differences.

Modification of the catalytic and regulatory properties of beef heart AMP-deaminase by DTNB treatment

1. 1. In beef heart AMP-deaminase (EC 3.5.4.6.), 7 SH-groups out of 26 half-cysteine residues in the protein molecule have been shown to be accessible to alkylation by DTNB in the absence of ATP. The addition of ATP showed that only 6 SH-groups were accessible. 2. 2. DTNB-modified enzyme showed about 30% of the native catalytic activity but no sensitivity to the ATP-activating effect. 3. 3. Almost full reactivation of the modified enzyme and the restoration of the activatory effect of ATP could be achieved by exhaustive dialysis against mercaptoethanol.

Modification of the Cl −-activated arginine aminopeptidases from rat liver and human erythrocytes: A comparative study

The amino acid residues important for the catalytic activity of the Cl −-activated arginine aminopeptidases from human erythrocytes and rat liver were studied using enzyme modification. The general inhibition characteristics were similar with both enzymes. Inactivation with 5,5′-dithiobis-(2-nitrobenzoic acid) revealed one essential SH-group per active enzyme unit in both aminopeptidases. l-Arginyl- l-phenylalanine and N- l-arginyl-2-naphthylamide protected the enzymes against inactivation by DTNB, the former substrate being more effective. The rat liver enzyme was more sensitive to DTNB than the erythrocyte enzyme. Titration with DTNB revealed only fast reacting SH-groups in rat liver APB (mean 7.8). The erythrocyte enzyme, however, revealed SH-groups which reacted fast with low concentrations of DTNB, while high concentrations of DTNB or SDS treatment were needed to reveal all enzyme SH-groups (mean 8.0). The presence of at least one essential imidazole group in the erythrocyte enzyme was indicated by photooxidation in the presence of methylene blue, as previously found with the rat liver enzyme ( Mäkinen and Hopsu-Havu, 1967, Enzymologia 32, 333 ). The pH dependence curves of both enzymes also supported the presence of SH- and imidazole groups at or near the active site. Thus, the functional groups identified were the same for both enzymes. Neither enzyme had essential COOH or arginyl groups and they did not contain any zinc. The absence of Zn suggests that the reaction mechanism recently presented by other authors, based on the presence of Zn in the active center, does not apply to the Cl −-activated arginine aminopeptidases. Accordingly, this enzyme group cannot be classified to metallopeptidases.

The use of polyacrylamide gel electrophoresis to study relations between the chains of myosin

Polyacrylamide gel electrophoresis (PAGE) of native proteins shows that myosin subfragment-1 (S-1), prepared by α-chymotryptic digestion of myosin, can be separated into two well-spaced bands corresponding to two S-1 isozymes. One of these consists of a heavy chain fragment (HC) and light chain (LC1); the other to HC and light chain 3 (LC3). Addition of light chain 2 (LC2), lost during the digestion process, speeds up the migration of the LC1-bearing S-1 (HC · LC1) but leaves the LC3-bearing S-1 (HC · LC3) essentially unehanged. This suggests that LC2 has a stronger affinity for the former and forms with it the complex. HC · LC1 · LC2. 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) treatment of myosin is known to remove only about half of the LC2 (“DTNB light chain”). Although S-1 prepared from such myosin cannot be well-resolved by DEAE-cellulose chromatography into two peaks, the beginning of the peak is largely HC · LC1 · LC2 and the ending is largely HC · LC3, as revealed by sodium dodecyl sulfate (SDS)-PAGE. Thus, the loss of LC2 during DTNB treatment is mainly from S-1 bearing LC3.