Defining the "fast-reacting" thiols of myosin by reaction with 1, 5 IAEDANS.

The specificity of the fluorescent reagent N-iodoacetyl-N-(5-sulfo-1-naphthyl)ethylenediamine (1,5 IAEDANS) for a specific thiol group of myosin has been characterized by a comparison with iodoacetamide (IAA) and by observing maximal enhancement of the Ca²⁺-ATPase activity and inhibition of the K⁺-EDTA-ATPase activity of myosin. The stoichiometry of the [³H]1,5 IAEDANS bound to myosin indicates the presence of two fast-reacting thiols which correspond to the “SH₁” groups responsible for the catalytic properties of myosin. Moreover, it has been unequivocally demonstrated by gel electrophoresis that the fast-reacting thiol is located on the myosin heavy chain. A single radioactivity-labeled thiol peptide obtained from tryptic digests of myosin labeled with [³H]1,5 IAEDANS or iodo[1-¹⁴C]acetamide indicates strongly that the identical thiol was labeled by both reagents.     

Reaction of thiol groups of gizzard myosin heavy chains with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Chicken gizzard myosin treated with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in a 65% inhibition of the K(+)-ATPase (myosin ATP phosphohydrolase (actin translocating), EC 3.6.1.32) activity and 3.5 mol of the reagent was bound per 4.7 x 10(5) g protein. The labeling was limited to the heavy chain region and none of the light chains were lost. MgATP had no effect on the inactivation or labeling pattern. Thiolysis of NBD-myosin with dithiothreitol restored the K(+)-ATPase activity and concurrently, 1 mol of the NBD group was removed from the heavy chain region. Cysteine residues were modified in NBD-myosin at sites other than the active site when the enzyme activity was inhibited. There was a difference in the extent of NBD-Cl modification of gizzard myosin at 0.6 M KCl (6 S elongated state) when compared to that at 0.15 M KCl (10 S folded state). This was also seen in the heavy meromyosin-like chymotryptic fragments and tryptic fragments of NBD-myosin. The reagent NBD-Cl can detect changes in the conformation of gizzard myosin by way of its reaction with thiol groups of the heavy chain region.     

Electron microscopic visualization of the SH1 thiol of myosin by the use of an avidin-biotin system

One of the reactive thiols in the myosin head, SH1, was covalently labeled with a biotin derivative, N-iodoacetyl-N'-biotinylhexylenediamine. When 50% of the SH1 thiol was modified with the biotin reagent as judged from measurements of ATPase activities, the biotinylated myosin bound one mole of avidin per mole of myosin at the saturating level. The avidin-myosin complex was readily formed in the presence of MgADP or MgATP. Peptide maps of the biotinylated myosin revealed that SH1 is actually the site of biotinylation with N-iodoacetyl-N'-biotinylhexylenediamine. Electron microscopic examination of the avidin-myosin complex showed that the attachment site of avidin on the myosin head is 130 A from the head-rod junction, indicating that the SH1 thiol is located there.     

Peroxidative crosslinking of myosins.

The effect of myoglobin, free hemin and H2O2 on myosins from heart and skeletal muscle was studied. SDS-gel electrophoresis revealed that each agent caused intermolecular thiol crosslinking of both myosins dissociable by excess of beta-mercaptoethanol. In the simultaneous presence of H2O2 and myoglobin or H2O2 and free hemin, myosin formed covalent aggregates undissociable by beta-mercaptoethanol and therefore assessed to formation of non S-S inter molecular covalent bonds. The latter aggregates are suggested to result from pairing of myosin radicals formed by the H2O2 induced ferryl iron state in myoglobin, free hemin or hemo-myosin.     

The reactivity of the thiol groups of calf thymus deoxyribonucleohistone

The reactivities of the two cysteine thiol groups of calf thymus F3 histone were investigated using 5,5'-dithiobis-[2- nitrobenzoic acid], (DTNB). In isolated histone, both thiol groups were available for reaction. However, analysis of reaction profiles of native deoxyribonucleohistone, (DNH), in various solvent conditions, together with gel electrophoresis studies of DNH modified with DTNB, showed that only one of the thiol groups is normally modified by the reagent. If NaCl is present (above 1.OM) the other thiol group can also be modified. The reactivities of both groups were largely independent of the degree of DNH supercoiling and of the binding of F3 to the DNA.     

N-(3-Triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC): a heterobifunctional reagent for immobilization of oligonucleotides on glass surface

A new heterobifunctional reagent, namely, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC) was developed and its potentiality for fixing of thiol (-SH) modified oligonucleotides were tested. The covalent attachment of oligonucleotides with the reagent was achieved through its maleimide functionality at one end via stable thioether linkage while the other end bearing triethoxysilyl functionality has been utilized for coupling with the virgin glass surface with simplified methodologies. Immobilization of oligonucleotides was achieved by two alternating ways. The PATH-1 involves formation of conjugate of reagent and SH-modified oligonucleotides through thioether linkage and was subsequently immobilized on unmodified glass surface through triethoxysilyl group and alternatively, PATH-2 involves reaction of reagent first with unmodified glass surface to get maleimide functionality on the surface and then the SH-modified oligonucleotides were immobilized via thioether linkage. The specificity of immobilization was tested by hybridization study with complementary fluorescein labeled oligonucleotide strand.     

Thiol reactivity as a sensor of rotation of the converter in myosin

Smooth muscle myosin has two reactive thiols located near the C-terminal region of its motor domain, the “converter”, which rotates by ∼70° upon the transition from the “nucleotide-free” state to the “pre-power stroke” state. The incorporation rates of a thiol reagent, 5-(((2-iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), into these thiols were greatly altered by adding ATP or changing the myosin conformation. Comparisons of the myosin structures in the pre-power stroke state and the nucleotide-free state explained why the reactivity of both thiols is especially sensitive to a conformational change around the converter, and thus can be used as a sensor of the rotation of the converter. Modeling of the myosin structure in the pre-power stroke state, in which the most reactive thiol, “SH1”, was selectively modified with IAEDANS, revealed that this label becomes an obstacle when the converter completely rotates toward its position in the pre-power stroke state, thus resulting in incomplete rotation of the converter. Therefore, we suggest that the limitation of the converter rotation by modification causes the as-yet unexplained phenomena of SH1-modified myosin, including the inhibition of 10S myosin formation and the losses in phosphorylation-dependent regulation of the basic and actin-activated Mg-ATPase activities of myosin.     

Structure of the actin-myosin complex produced by crosslinking in the presence of ATP

The structure of the actin-myosin complex during ATP hydrolysis was studied by covalently crosslinking myosin subfragment 1 (S1) to F-actin in the presence of nucleotides (especially ATP) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The fluorescence energy transfer was measured between N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine and 6-(iodoacetamide)fluorescein bound to the SH1 thiol of S1 and the Cys374 thiol of actin. The covalent acto-S1, produced by crosslinking in the absence of nucleotide or in the presence of ADP, showed transfer efficiency of 0.50 to 0.52 and intersite distance of 4.5 to 4.7 nm, which were equal to those obtained with non-crosslinked acto-S1 in the absence of nucleotide. However, the covalent acto-S1, produced by crosslinking in the presence of either 5'-adenylyl imidodiphosphate (AMPPNP) at high ionic strength or ATP, showed a significant decrease in the efficiency to 0.26 to 0.34 and hence an increase in the distance to 5.2 to 5.5 nm. These results suggest that AM-ATP and/or AM-ADP-P (formed during ATP hydrolysis) and AM-AMPPNP have a very different conformation from AM and AM-ADP (in which A is actin and M is myosin).     

The effect of a cross-bridging thiol reagent on the catecholamine fluxes of adrenal medulla vesicles

The thiol groups of the vesicular protein of bovine adrenal medulla were allowed to react with the bifunctional thiol reagent bis-(N-maleimidomethyl) ether and with the monofunctional thiol reagent N-ethylmaleimide, and the ATP-dependent and -independent catecholamine fluxes of the modified preparations were studied. 1. During the initial phase of the reaction bis-(N-maleimidomethyl) ether blocks twice as many thiol groups as does N-ethylmaleimide at equimolar concentrations. 2. Labelling of the bis-(N-maleimidomethyl) ether-protein compound with [(14)C]-cysteine shows that 70-80% of the blocked thiol groups are interconnected by the bifunctional thiol reagent. 3. At a low extent of reaction (1.5mol of thiol groups/10(6)g of protein) the catecholamine efflux is diminished. If more than 2mol of thiol groups/10(6)g of protein are blocked, the efflux is enhanced whichever thiol reagent is applied. 4. If 2-4mol of thiol groups/10(6)g of protein are blocked the inhibition of the catecholamine influx increases linearly with the proportion of the thiol groups blocked. 5. ATP protects the catecholamine influx and the adenosine triphosphatase activity against bis-(N-maleimidomethyl) ether poisoning somewhat less effectively than against N-ethylmaleimide poisoning.     

The reaction of rabbit muscle creatine kinase with some derivatives of iodoacetamide.

The dimeric enzyme creatine kinase from rabbit muscle was treated with three derivatives of iodoacetamide that are capable of introducing fluorescent groups into the enzyme. All the three reagents (4-iodoacetamidosalicylate (IAS), 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulphonate (IAEDANS) and 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate (IAANS)) were shown to react at the same single thiol group on each enzyme subunit, leading to complete inactivation of the enzyme. The reaction with IAS was extremely rapid by comparison with the reaction with iodoacetamide or iodoacetate, but various lines of evidence suggest that IAS is not a true affinity label. However, kinetic and binding studies indicate that salicylate itself probably binds at the nucleotide-binding site on the enzyme. As the size of the modifying reagent increased, the first thiol group reacted more rapidly than the second; this trend was more pronounced at 0 degree C than at 25 degree C. With the largest modifying reagent used (IAANS), the pronounced biphasic nature of the modification reaction permitted the preparation of a hybrid enzyme in which only one subunit was modified, but a study of the thiol-group reactivity showed that this hybrid enzyme preparation underwent subunit rearrangement.     

Selective modification of myosin SH1 with 1,2,4-trinitrobenzene. VIII. Thiols of myosin

Myosin has 2 mol of the most reactive thiol, named SH1. 1,2,4-Trinitrobenzene (TNB), a novel dinitrophenyl(DNP)ating reagent [Takahashi et al. (1983) Chem. Lett. 1445-1448], was found to react only with SH1 without any other amino acid residues in myosin under the conditions used. Its reaction with myosin SH1 was about 30 times faster than that with N-acetylcysteine (NAC). The reaction rate of TNB with SH1 was about twice compared with that of NEM, the most reactive selective reagent for SH1 so far found, although its rate with NAC was only one sixtieth that of NEM. As to the lambda max of the absorption spectrum of SH1-DNP-myosin, a large red shift of as much as 20 nm was observed compared with low molecular S-DNP derivatives. This red shift disappeared in 8 M urea. This outstanding feature of SH1 modification with TNB was discussed in terms of affinity labeling by interaction with an aromatic amino acid near SH1.     

MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection

MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.     

Mapping of acyl carrier domain within the subunit of type I bacterial fatty acid synthetase

A fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, was used to label the acyl carrier site of the bacterial fatty acid synthetase from Brevibacterium ammoniagenes. The reagent bound preferentially to the 4'-phosphopantetheine thiol group of the acyl carrier domain and irreversively inactivated the enzyme. The modified enzyme was cleaved by proteinases for the mapping of the labeled site. The fluorescent fragment was readily detected on a polyacrylamide gel after electrophoresis. The region of 45 kDa containing the 4'-phosphopantetheine was located on the polypeptide at around two-thirds of the full length from the N-terminal.     

Cooperativity in F-actin: chemical modifications of actin monomers affect the functional interactions of myosin with unmodified monomers in the same actin filament.

We have chemically modified a fraction of the monomers in actin filaments, and then measured the effects on the functional interaction of myosin with unmodified monomers within the same filament. Two modifications were used: (a) covalent attachment of various amounts of myosin subfragment-1 (S1) with the bifunctional reagent disuccinimidyl suberate and (b) copolymerization of unmodified actin monomers with monomers cross-linked internally with 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. Each of these modifications abolished the interaction of the modified monomers with myosin, so the remaining interactions were exclusively with unmodified monomers. The two modifications had similar effects on the interaction of actin with myosin in solution: decreased affinity of myosin heads for unmodified actin monomers, without a change in the Vmax of actin-activated myosin ATPase activity. However, modification (b) produced much greater inhibition of actin sliding on a myosin-coated surface, as measured by an in vitro motility assay. These results provide insight into the functional consequences of cooperative interactions within the actin filament.     

The location of the thiol groups of myosin that are protected by pyrophosphate against reaction with 2,4-dinitrophenyl β-hydroxyethyl disulphide

Myosin modified in the presence or in the absence of pyrophosphate by 2,4-dinitrophenyl beta-hydroxyethyl disulphide was treated with iodo[1-(14)C]acetamide. The residual Ca(2+)-stimulated adenosine triphosphatase (ATPase) activity of the modified myosin was different depending on the presence or absence of PP(i) during modification and the number of 2,4-dinitrophenyl beta-hydroxyethyl disulphide-modified thiol groups. The radioactivity incorporated into the light components of myosin correlated with the Ca(2+)-stimulated ATPase activity of the modified myosin and decreased with decreasing residual Ca(2+)-stimulated ATPase activity of the modified myosin. When native myosin was treated with low concentrations of iodo[1-(14)C]acetamide the residual Ca(2+)-stimulated ATPase activity of carboxyamidomethylated myosin was high and the radioactivity incorporated into the light components of myosin was negligible. The thiol groups of the light components of myosin are essential to preserve the ATPase activity of the protein and are close to the pyrophosphate-binding sites.     

Evidence for a thiol ester in duck ovostatin (ovomacroglobulin)

The structure and the mechanism for proteinase inhibition of the egg white protein ovostatin (ovomacroglobulin) are similar to those of plasma alpha 2-macroglobulin, but previous studies have shown that chicken ovostatin lacks a reactive thiol ester (Nagase, H., and Harris, E. D., Jr. (1983) J. Biol. Chem. 258, 7490-7498). Here we show that duck ovostatin has conserved such a thiol ester and is capable of inhibiting both metallo- and serine proteinases stoichiometrically. Evidence for thiol esters was established by the following results with duck ovostatin: 1) autolysis into fragments of Mr = 123,000 and 60,000 occurred by heating in sodium dodecyl sulfate, but was prevented by treatment with CH3NH2; 2) covalent linkages were formed with proteinases on complex formation; 3) reaction with CH3NH2 generated 3.6 SH groups/mol, and 3.9 mol of [14C]CH3NH2 were incorporated per mol of protein; and 4) saturation with a proteinase liberated 3.8 SH groups/mol of the inhibitor. Conformational rearrangement of duck ovostatin upon reacting with CH3NH2 or proteinases was demonstrated by an increased mobility of the protein in polyacrylamide gel electrophoresis. CH3NH2-treated duck ovostatin was able to bind and inhibit proteinases without forming covalent bonds, but, unlike unmodified ovostatin, its inhibitory activity was destroyed by freezing and thawing. Complexes formed between CH3NH2-treated duck ovostatin and a proteinase were not dissociable except under denaturing conditions. These results and other evidence indicate that covalent bond formation through reaction with a thiol ester is a separate process from the trapping and inhibition of proteinases by this family of proteins.     

Fluorescence properties of o-phthaldialdehyde derivatives of amino acids.

The fluorescence properties of the products formed by reaction of o-phthaldialdehyde with amino acids and their derivatives, in the presence of thiol compounds, have been studied. The emission spectra, quantum yields, and lifetimes depend on the primary amine and thiol compound used; the observations confirm the report (Simsons, S.S., Jr. and Johnson, D.F. (1978) J. Org. Chem 43, 2886--2891) that the product incorporates molecules of all three types of compounds. The fluorescence quantum yields of o-phthaldialdehyde derivatives of the naturally occuring amino acids ranged from 0.33 to 0.47, using 2-mercaptoethanol as the thiol compound. The fluorescence lifetimes were about 18--20 ns. Lower quantum yields were obtained when mercaptoethanol was replaced by dithiothreitol or ethnethiol. Derivatives of amino acid amides and peptides had quantum yeilds as low as 0.03, due to quenching by the carboxamide group. The intramolecular quenching was relieved by the detergent, sodium dodecyl sulfate, and by dimethylsulfoxide. Monosubstituted lysine exhibited a normal fluorescence, but the di-substituted product was largely quenched, presumably due to interaction between the two isoindole fluorophors. Fluorscence stopped-flow experiments showed that the alpha- and epsilon-amino groups reacted at different rates, with the epsilon-amion group reacting 10 times faster, with a t 1/2 of about 6 s under pseudo first order conditions at pH 9.0 with 10(-3) M o-phthaldialdehyde. The amount of instability shown by the o-phthaldialdehyde derivatives depended on the thiol compound used, the primary amine involved, and the solvent. Cysteine and o-phthaldialdehyde reacted to give an unstable, weakly fluorescent product; but cysteine could be assayed normally if its sulfhydryl was blocked. The o-phthaldialdehyde reagent was discussed in relation to fluorescamine, another reagent for primary amines.     

Application of a fluorogenic reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate for detection of cystine-containing peptides

A simple, sensitive and reliable method for the detection of cystine-containing peptides has been developed. A peptide bridged with a disulfide bond was reduced and cleaved with tributylphosphine, and then coupled with a thiol specific reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, under alkaline conditions. After incubation at 60 degrees C for 1 h, the fluorescent derivative formed was measured with excitation at 385 nm and emission at 515 nm. The intensity of fluorescence labeled to the peptide was very stable and the peptide containing disulfide was quantitatively determined in the range of 100 pmol to 10 nmol, when oxidized glutathione was used as a standard. This method was very useful for specific detection of cystine-containing peptides in the column effluents on reverse phase high performance liquid chromatography.     

Reactive thiol groups in rat liver acid phosphatase

Dimeric rat liver acid phosphatase P1 of Mr 92,000 is inactivated by p-chloromercuribenzoate and fluorescein mercuriacetate (FMA). The enzyme is protected against the mercurials by the substrate analogue Pi. The reaction with FMA is accompanied by changes in absorbance at 495 nm and in fluorescence emission at 520 nm that are characteristic of reaction of this compound with thiol groups. Titration of P1 with FMA monitored by spectrophotometry or by fluorimetry indicated that equivalence is reached at an FMA/P1 ratio of 3. Since FMA can act as a bifunctional reagent, it is likely that P1 contains either 3 or 6 reactive thiol groups per molecule. Analysis of FMA inactivation/modification data by a statistical method suggests that of 6 reactive thiol groups, 2 are essential so that there are probably 3 thiol groups per subunit, one of which is located at the active site. If the total thiol number is 3, analysis suggests 1 essential thiol per subunit.     

MgADP-induced changes in the structure of myosin S1 near the ATPase-related thiol SH1 probed by cross-linking

The structural consequences of MgADP binding at the vicinity of the ATPase-related thiol SH1 (Cys-707) have been examined by subjecting myosin subfragment 1, premodified at SH2 (Cys-697) with N-ethylmaleimide (NEM), to reaction with the bifunctional reagent p-phenylenedimaleimide (pPDM) in the presence and absence of MgADP. By monitoring the changes in the Ca2(+)-ATPase activity as a function of reaction time, it appears that the reagent rapidly modifies SH1 irrespective of whether MgADP is present or not. In the absence of nucleotide, only extremely low levels of cross-linking to the 50-kDa middle segment of S1 can be detected, while in the presence of MgADP substantial cross-linking to this segment is observed. A similar cross-link is also formed if MgADP is added subsequent to the reaction of the SH2-NEM-pre-modified S1 with pPDM in the absence of nucleotide. Isolation of the labeled tryptic peptide from the cross-linked adduct formed with [14C]pPDM, and subsequent partial sequence analyses, indicates that the cross-link is made from SH1 to Cys-522. Moreover, it appears that this cross-link results in the trapping of MgADP in this S1 species. These data suggest that the binding of MgADP results in a change in the structure of S1 in the vicinity of the SH1 thiol relative to the 50-kDa "domain" which enables Cys-522 to adopt the appropriate configuration to enable it to be cross-linked to SH1 by pPDM.