SH Reagents Research Articles

Energy transduction in vesicles of the methanogenic strain Gö1

Everted vesicles of the methanogenic strain Gö1 synthesized ATP in response to methanogenesis from methyl-coenzyme M and H 2. Simultaneously, a transmembrane pH gradient (ΔpH) was generated as evident from fluorescence quenching of acridine orange. Protonophorous uncouplers prevented ΔpH generation and ATP synthesis, but did not affect methanogenesis. The ATP synthase inhibitor diethylstilbestrol (DES) inhibited ATP synthesis but had no effect on methanogenesis and on ΔpH formation, indicating the essential role of the transmembrane proton potential in ATP synthesis. Progress has also been made in assigning specific functions to membrane components in methanogenesis from methyl-CoM and H 2. Separation of cell extracts into cytoplasmic and membrane fraction revealed an essential role of membrane-bound components in electron transfer: methanogenesis catalyzed by the cytoplasmic fraction from strain Gö1 was stimulated several fold by membranes from various methanogens. This stimulation was prevented if the membranes had been treated with oxidants (O 2, K 3[Fe(CN) 6]) or SH reagents (Ag +, p-chloromercuribenzoate, iodoacetamide) pointing to the involvement of functional SH groups in methanogenesis from methyl-CoM and H 2.

Energy transduction in vesicles of the methanogenic strain Gö1

Everted vesicles of the methanogenic strain Go1 synthesized ATP in response to methanogenesis from methyl-coenzyme M and H2. Simultaneously, a transmembrane pH gradient (ΔpH) was generated as evident from fluorescence quenching of acridine orange. Protonophorous uncouplers prevented ΔpH generation and ATP synthesis, but did not affect methanogenesis. The ATP synthase inhibitor diethylstilbestrol (DES) inhibited ATP synthesis but had no effect on methanogenesis and on ΔpH formation, indicating the essential role of the transmembrane proton potential in ATP synthesis. Progress has also been made in assigning specific functions to membrane components in methanogenesis from methyl-CoM and H2. Separation of cell extracts into cytoplasmic and membrane fraction revealed an essential role of membrane-bound components in electron transfer: methanogenesis catalyzed by the cytoplasmic fraction from strain Go1 was stimulated several fold by membranes from various methanogens. This stimulation was prevented if the membranes had been treated with oxidants (O2, K3[Fe(CN)6]) or SH reagents (Ag+, p-chloromercuribenzoate, iodoacetamide) pointing to the involvement of functional SH groups in methanogenesis from methyl-CoM and H2.

Purification and characterization of D-2-haloacid dehalogenase from Pseudomonas putida strain AJ1/23

A D-2-haloacid dehalogenase was isolated and purified to homogeneity from Pseudomonas putida strain AJ1/23. The enzyme catalysed the stereospecific dehalogenation of the D-isomer of 2-chloropropionate. Using a new ion-chromatograph assay, the enzyme was found to catalyse the dehalogenation of short-chain 2-halocarboxylic acids. Maximum enzyme activity occurred at pH 9.5 and 50 degrees C and the enzyme was insensitive to most -SH reagents. The enzyme has an Mr of about 135,000 and appears to be composed of four subunits of identical Mr.

Identification of the Reactive Cysteinyl Residue and ATP Binding Site in Bacillus cereus Glutamine Synthetase by Chemical Modification

Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.

Dependence on membrane components of methanogenesis from methyl‐CoM with formaldehyde or molecular hydrogen as electron donors

Methane formation from 2-(methylthio)-ethanesulfonate (methyl-CoM) and H2 by the soluble fraction from the methanogenic bacterium strain Gö1 was stimulated up to tenfold by the addition of the membrane fraction. This stimulation was observed with membranes from various methanogenic species belonging to different phylogenetic families, but not with membranes from Escherichia coli or Acetobacterium woodii. Treatment of the membranes with strong oxidants, i.e. O2 and K3[Fe(CN)6], or with SH reagents, i.e. Ag+, p-chloromercuribenzoate or iodoacetamide, caused an irreversible decrease or loss in stimulatory activity, as did heat treatment at temperatures above 78 degrees C. Methanogenesis from methyl-CoM with formaldehyde instead of H2 as electron donor depended similarly on the membrane fraction. With membranes, 1 mol HCHO was oxidized to 1 mol CO2 and allowed the formation of 2 mol CH4 from 2 mol CH3-CoM. Without membranes, per mol of HCHO oxidized 1 mol H2 was formed and 1 mol CH4 was produced from CH3-CoM; the rate was 10-20% of that in the presence of membranes. When methyl-CoM was replaced by an artificial electron acceptor system consisting of methylviologen and metronidazole, the formaldehyde-oxidizing activity was no longer stimulated by the membrane fraction. These results demonstrate for the first time an essential function of membrane components in methanogenic electron transfer.

Purification and Characterization of a Growth Factor from Guinea Pig Harderian Gland

A polypeptide growth factor, Harderian gland-derived growth factor (HGDGF), has been purified approximately 43,000-fold from guinea pig Harderian gland by column chromatography on TSK gel DEAE-5PW, blue-Sepharose CL-6B, and Superose 12. The yield was approximately 10%. The Superose 12 fraction was further purified by Aquapore BU-300 reversed-phase chromatography to apparent homogeneity. HGDGF was eluted from TSK gel DEAE-5PW at 0.20-0.35 M NaCl, with a linear gradient of 0.15-0.80 M NaCl and at 2.20 M NaCl from blue-Sepharose CL-6B. The activity of HGDGF toward human embryonic cells (TIG-3) was quantitated, [3H]thymidine incorporation for 48 h being stimulated in a linear and dose-dependent manner. Purified HGDGF has a molecular weight of approximately 13,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve column chromatography. HGDGF is labile to treatment with SH reagents or acetic acid. Both trypsin digestion and boiling decrease the activity of HGDGF. Its pI is 5.1. HGDGF stimulates the multiplication of TIG-3 cells but has no effect on human endothelial cells K2T1 or A2T2 which require fibroblast growth factor for growth. HGDGF appears to differ from other growth factors, suggesting that it is a previously undescribed growth factor.

Cross binding and stiffening of the red cell membrane

Cross binding and stiffening of the human red cell membrane was studied using treatments with SH, amino, and carboxyl reagents, oxidizing and denaturing treatments and acidification. Membrane cross binding was initiated when, after red cell treatment, opposite areas of the cytoplasmic face of the red cell membrane were brought into contact by cell shrinking. Membrane cross binding was detected by light microscopy when this contact persisted upon swelling the cells in a hypotonic medium. Membrane stiffening was recorded as a decrease in elongation of red cells in the shear field of a viscous dextran solution. No correlation was found between membrane cross binding and membrane stiffening. The results are explained by the existence of two modifications of spectrin, type I causing solely membrane stiffening, type II causing membrane cross bonding as well as membrane stiffening. The amino and carboxyl reagents caused only type I modification. The other treatments caused both types of modification although with varying proportions. The results support the previously suggested mechanism of membrane cross bonding which involves a rearrangement of spectrin similar to denaturation by heat or urea, a decrease in associations within the membrane skeletal network, and a lateral aggregation of membrane proteins. These changes are proposed to occur by the type II modification. The data further substantiate the membrane stiffening effect of inter- and intra-molecular cross linking of spectrin which is identified with the type I modification. Finally, hypotheses are presented concerning the mechanism of membrane stiffening due to type II modifications of spectrin.

Analysis of free sulfhydryl groups and disulfide bonds in Na +,K +-ATPase

The content of free SH groups and disulfide bonds in the purified pig kidney Na +,K +-ATPase was determined by ammetric titration with silver nitrate. In the native enzyme, most of the free SH groups are masked due to their location in the polypeptide chain regions poorly accessible to SH reagents. Denaturation with 5% SDS and 8 M urea makes these regions accessible thus revealing 22 free SH groups/mol of the protein. After complete blocking of free SH groups with silver ions, 8 SH groups/mol of the protein are being released upon sulfitolysis which indicates the presence of four disulfide bonds in the enzyme. At least one disulfide bridge is located in the α-subunit whereas the β-subunit contains three disulfide bonds.

Role of cysteine residues in tryptophanase for monovalent cation-induced activation

We cloned and sequenced the tryptophanase structural gene of Escherichia coli B/1t7-A strain. The results indicate that tryptophanase proteins of E. coli B/1t7-A and K-12 are identical. When cysteine residues in tryptophanase were chemically modified with 5,5′-dithio bis (2-nitrobenzoic acid) (DTNB), the stabilizing effect of the active cations such as K + and NH 4 + was abolished. In consideration of our previous results that Cys-298 was selectively modified by SH reagents [Honda T. et al. (1986) J. Chromatogr. 371, 353–360], Cys-298 seems to have a close relation to the expression of the effect of monovalent cations. Fluorescence decay measurement of the holoenzyme revealed that the fluorescence lifetime derived from the coenzyme, pyridoxal 5′-phosphate (PLP), was dependent on coexisting monovalent cations, whereas that of the tryptophyl residue was not, in either the apo- or the holoenzyme preparation. The results of the synchrotrom small-angle X-ray scattering measurements showed that radii of gyration which reflect the size and shape of the enzyme were constant at around 38 Å irrespective of the presence or absence of the K + ion. These results suggest that the monovalent cations interact specifically with the PLP-binding site, and that the conformational change of enzyme protein caused by the monovalent-cation binding is limited to a small range. The above results are compatible with the possibility that Cys-298 is involved in the formation of “monovalent cation binding site” in the holoenzyme.

Characterization of soluble and membrane-bound forms of a vanadate-sensitive ATPase from plasma membranes of the halotolerant alga Dunaliella salina

A plasma-membrane preparation, obtained by an osmotic lysis of cells of the halotolerant alga Dunaliella salina ( Sheffer and Avron, 1986 ) was further purified on glycerol gradients. Two fractions of a vanadate-sensitive ATPase activity were resolved on the gradients: a soluble and a membrane-bound fraction. The enzymes exhibit identical sensitivities to vanadate, dicyclohexylcarbodiimide, diethylstilbestrol, SH reagents and phloridzin but are insensitive to molybdate, nitrate, cyanide, azide and quercetin. The two ATPase activities also have an identical K m value for Mg-ATP (0.9 mM), optimal activity at pH 7, are stimulated by 200 mM KCl or NaCl but inhibited by high salt concentrations. Antibodies against yeast plasma membrane H + -ATPase cross-react with 92 kDa or with 60 kDa polypeptides in the plasma membrane and soluble ATPase fractions, respectively. Treatment of the purified plasma membranes with trypsin releases a soluble vanadate-sensitive ATPase from the membranes, and the release is protectable by ATP. It is suggested that the soluble ATPase, which is resolved on the glycerol gradients, is a proteolytic product of the plasma membrane ATPase which is a vanadate-sensitive H + -ATPase.

The uncoupling protein dimer can form a disulfide cross‐link between the mobile C‐terminal SH groups

Isolated uncoupling protein (UCP) can be cross-linked, by various disulfide-forming reagents, to dimers. The best cross-linking is achieved with Cu2+-phenanthroline oxidation. Because cross-linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross-linking is prevented by SH reagent and reversed by SH-reducing reagents. In mitochondria, cross-linking of UCP with disulfide-forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide-bridge formation does not inhibit GTP-binding to UCP. Cross-linked UCP re-incorporated in proteoliposomes either before or after cross-linking fully retains the H1-transport function. Rapid cross-linking by membrane impermeant reagents indicates a surface localization of the C-terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide-bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross-linked UCP was cleaved by BrCN. The CB-7 C-terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys-292, removed cross-linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C-terminal peptide of 11 residues contains only cystein-304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S-S bridge formation. Obviously, the C-terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross-linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane.

Phosphate transport protein of rat heart mitochondria: location of its SH-groups and exploration of their environment

(1) The properties of the SH groups of the phosphate transport protein of rat heart mitochondria were investigated on the basis of inhibition caused by SH reagents under different conditions. (2) The essential thiol groups are located near the external surface, as they are accessible to impermeable reagents from the external space. (3) The environment of the sulfhydryl groups influences their reactivity, as alteration of the external pH affects adversely their reactions with ionizable and non-ionizable SH reagents. (4) Intramitochondrial pH exerts a transmembrane effect: alkalinization augments and acidification diminishes the reaction rate of the sulfhydryl groups on the opposite surface of the membrane. (5) Changes of the concentration of the transported substrate occurring exclusively in the extramitochondrial space do not influence the reactivity of the essential SH groups. (6) It is concluded that in transport studies the phosphate transport protein of heart and liver mitochondria show basic similarity. It is suggested that the amino-acid sequence around the NEM-reactive cysteine (i.e., Lys-41 - Cys-42 - Arg-43) does not participate in substrate binding.

Comparative electrophoresis and some properties of alkaline proteinases produced by Monascus spp.

Some strains in the genus Monascus secreted extracellular alkaline proteinases on a wheat bran medium, along with propagation of cells. Crude enzymes prepared from six strains of five species and one variety of Monascus were separated by polyacrylamide slab gel electrophoresis and their activities were visualized with a printing technique on a casein-containing gel. We found that their proteinase activities were based on at least three different proteins which occurred commonly in all the strains examined. These enzymes were completely inhibited by phenylmethylsulfonyl fluoride and were insensitive to chelating and SH reagents. They were characterized as serine proteinases different from chymotrypsin- or trypsin-type enzymes by their insensitivity to the affinity-labeling reagents, N-α-tosyl-L-lysyl chloromethyl ketone and N-tosyl-L-phenylalanyl chloromethyl ketone.

Characterization of nitrile hydratase and amidase, which are responsible for the conversion of dinitriles to mononitriles, from Corynebacterium sp.

Two enzymes, nitrile hydratase and amidase, which participate in the conversion of trans-1,4- dicyanocyclohexane (t-DCC) to frans-4-cyanocyclohexane-l-carboxylic acid (t-MCC), a tranexamic acid intermediate, were purified and characterized. Nitrile hydratase was obtained in a homogeneous state. The molecular weight of the native enzyme was 61,400 and that of the subunit 26.900, indicating a dimer structure. Valeronitrile and butyronitrile were good substrates for the enzyme. The enzyme could also hydrate benzonitrile, p-hydroxybenzonitrile and 4-cyanobenzoic acid. t-DCC was ex-clusively hydrated to fnzws-4-cyanocycIohexane-l-car boxy amide (t-MCMA), further hydration of the nitrile group of t-MCMA and t-MCC not being observed. The presence of pyrroloquinoline quinone in the enzyme was confirmed. The presence of iron was also confirmed. The amidase of the strain was also purified. The latter enzyme could hydrate t-MCMA, yielding t-MCC. The enzyme was highly resistant to SH reagents.

Zinc uptake in human seminal spermatozoa: characterization and effects on cell membranes.

Human ejaculated spermatozoa take up zinc in a time- and concentration-dependent manner. The kinetics of 65Zn2+ uptake is suggestive of an at least partly carrier-mediated transport. The lack of effect of the respiratory chain inhibitor antimycin A could mean that mitochondrial ATP is not required as an energy source for the uptake. The failure of nonpermeant SH reagent mersalyl to modify zinc uptake indicates that functional membrane sulfhydryl groups are not involved in the process. A dose-dependent inhibition of 65Zn2+ uptake was induced by the "anticalmodulin" drug trifluoperazine, suggesting that the calcium-binding protein calmodulin could have a role in zinc transport. In in vitro experiments this cation brought about a powerful effect in protecting the spermatozoa from being damaged by hypo-osmosis.

Calcium uptake in human spermatozoa: characterization and mechanisms.

Basal 45Ca2+ influx was analyzed in human seminal spermatozoa using a method that allows these highly reactive cells to be easily and safely handled. The uptake was a time-dependent process, with its maximum at 400 s. The kinetics of 45Ca2+ transport was saturating as a function of extracellular Ca2+ concentration with a Km of 429 microM and a Vmax of 1.6 nmol 45Ca2+/mg protein/2.5 min. Depolarizing conditions and the calcium channel blocker verapamil did not affect the uptake; based on this, the presence of operating calcium channels in seminal spermatozoa is excluded. The independence of 45Ca2+ uptake on external concentration of both Na+ and Ca2+ suggests that Na+/Ca2+ exchange does not occur in these cells. The anticalmodulin drug trifluoperazine, the mitochondrial inhibitor antimycin A, and the SH reagents N-ethylmaleimide and mersalyl all inhibited the ion transport. A calmodulin-regulated, energy-requiring, proteinaceous Ca2+ transporter seems to be the main operating mechanism of calcium uptake in human seminal gametes.

Interaction of phenylisothiocyanates with the mitochondrial phosphate carrier. I. Covalent modification and inhibition of phosphate transport

The effects of phenylisothiocyanate (PITC) and of the polar analogue p-sulfophenylisothiocyanate ( p-sulfoPITC) on the phosphate carrier of bovine heart mitochondria have been investigated. Incubation of mitochondria with the two phenylisothiocyanates leads to inhibition of the phosphate carrier protein. The inhibition of phosphate transport by PITC is unaffected by the addition of dithioerythritol (DTE) or by variation of the pH. The inhibition by p-sulfoPITC is in part removed by DTE; the remaining inactivation of the phosphate carrier, which can be attributed to the reaction with NH 2 groups, is temperature and pH-dependent. Inhibition of phosphate transport by both p-sulfoPITC and PITC depends on the time of incubation and the concentration of the inhibitor. Preincubation with mersalyl protects the carrier protein against the inactivation by p-sulfoPITC but not against PITC. Other SH reagents tested do not show any protective effect. It can thus be concluded that two types of lysine residues are essential for the activity of the phosphate carrier. Lysine(s) of the former type are located at the surface of the membrane and are topologically related to the functional SH groups of the protein. Lysine residue(s) of the latter type are buried in the hydrophobic phase of the membrane.

Calcium release from isolated sarcoplasmic reticulum due to 4,4'-dithiodipyridine.

The effects of SH reagents on Ca2+ release from sarcoplasmic reticulum (SR) vesicles were examined by the tracer method using 45Ca2+. Among the various SH reagents tested, 4,4'-dithiodipyridine (PDS) was found to induce Ca2+ release most specifically from the heavy fraction of SR vesicles. Further, the following results were obtained. (i) PDS bound covalently to proteins in the SR membrane and induced Ca2+ release. (ii) The Ca2+ release was further enhanced by ATP and caffeine, but inhibited by procaine, ruthenium red and various divalent cations. (iii) PDS enhanced the Ca2+ release in the whole range of Ca2+ concentrations tested. (iv) Choline permeability was also enhanced by PDS. Further, the electrical conductance of the Ca2+-induced Ca2+ release channels was studied by incorporating them into lipid bilayers and it was found that PDS increased the probability of opening of the channels. These results suggest that PDS binds to certain SH groups of the Ca2+-induced Ca2+ release channels in the SR membrane and thus induces Ca2+ release.

Photocross-linking from dinitrophenylated SH1 in myosin head. II. Cross-linked site on 50-kDa fragment.

We reported in the preceding paper [Muno, D., et al. (1987) J. Biochem. 101, 661-669] that the dinitrophenyl group exclusively introduced to SH1 on the 20-kDa fragment of myosin subfragment 1 was cross-linked to the 50-kDa fragment by irradiation, and that limited trypsinolysis of the cross-linked S1 generated an 83-kDa peptide, a cross-linking product between the 20- and 50-kDa fragments. This paper will deal with the location of the cross-linked residue on the 50-kDa fragment. When the 83-kDa fragment labeled at SH2 with a fluorogenic SH reagent was subjected to bromocyanolysis, a main fluorescent band, which implied a cross-linked peptide, appeared in the position with an apparent molecular mass of 18.5-kDa on SDS-PAGE. On the other hand, another cross-linked peptide was obtained from a complete tryptic digest of a 83-kDa fragment rich fraction. Amino acid sequence analysis of the two cross-linked peptides revealed that the DNP moiety attached at SH1 was cross-linked with a residue in the segment of the heavy chain spanning the 485-493 region from the N-terminus of the heavy chain.

Calcium regulation by lens plasma membrane vesicles

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca 2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca 2+ ( K 1 2 = 0.08 μm Ca 2+ ) potassium (maximally at 50 mM K +), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC 50 = 5 μM) and the calmodulin inhibitor R24571 (IC 50 = 5 μM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K +, stimulated calcium uptake, suggesting that the calcium pump either countertransports K +, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na +, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca 2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca 2+ in this condition.