Modification of membrane sulfhydryl groups in bacteriostatic action of nitrite.

The mechanism by which nitrite inhibits outgrowing spores of Bacillus cereus T was examined by using techniques developed earlier for nitrite analogs. The morphological stage of inhibition, cooperativity effects, effect of pH on inhibition, kinetics of protection against iodoacetate incorporation into membrane sulfhydryl groups, and protection against the bacteriocidal effect of carboxymethylation by iodoacetate indicate that nitrite acts as a membrane-directed sulfhydryl agent. The mechanism by which nitrite modifies the chemical reactivity of the sulfhydryl group could be either direct covalent modification or inactivation through communication with another modified membrane component. Profiles of pH effects suggest that the active agent is the protonated form of nitrite. The nitrite concentrations which modify membrane sulfhydryl activity coincide with those which have a bacteriostatic effect. These results are consistent with membrane sulfhydryl modification as a component of the mechanism of nitrite-induced bacteriostasis in this aerobic sporeformer.     

Identification and characterization of some bacterial membrane sulfhydryl groups which are targets of bacteriostatic and antibiotic action

Covalent modification of sulfhydryl groups which become sensitive toward sulfhydryl agents during germination of Bacillus cereus spores exerts a profound bacteriostatic effect, resulting in outgrowth inhibition. The modified spore components are membrane species of 13,000, 28,000, and 29,000 daltons. Detergent disruption of the membrane inactivated the sulfhydryl groups. A highly sigmoid inhibition curve (n = 11.8) with diamide suggested the participation of closely neighboring sulfhydryl groups. Substate and substrate analogs of the lactose and dicarboxylic acid permeases protected the sulfhydryl groups against modification. Nisin, a 34-residue peptide antibiotic, inhibited spore outgrowth and sulfhydryl modification at a concentration of about 0.1 microM. Since these sulfhydryl groups have been implicated as involved with the bacteriostatic action of nitrite, substances directed toward them may be a useful new class of bacteriostatic agents and antibiotics.     

Erythrocyte membrane sulfhydryl groups and cation permeability

Reaction of the slowly penetrating organic mercurial compound parachloromercuribenzene sulfonate (PCMBS) with intact erythrocytes has been characterized. Addition of concentrations of PCMBS which result in binding within the interior of the membrane of more than 1.9 × 10^?18 moles/cell produces alterations in Na⁺ and K⁺ permeability, but does not affect choline permeability. However, the increased cation permeability is observed only after a lag period of over two hours. After ten hours, a spontaneous slow “recovery” to normal rates of K⁺ leakage occurs at 25°C but not at 2°C. Subsequent to the effects on cation balance, increasing degrees of hemolysis occur, interpreted as colloid osmotic lysis. The relationships between the binding of the agent and its effects are as follows: a small, rapid initial uptake does not affect cation permeability; the subsequent slower uptake is associated with increased leakage of K⁺ and Na⁺; and the recovery at 25°C is associated with desorption of about half of the PCMBS due to competition by soluble thiol substances released into the medium from the cells. Desorption and “recovery” can be mimicked at any time by addition of small amounts of protein in the medium. The half of the PCMBS that cannot be desorbed is assumed to be bound by the hemoglobin inside the cell. The sulfhydryl groups involved in control of cation permeability constitute only a fraction of the total within the membrane (4–18%). They are located within the interior of the membrane separated from the medium and from the interior of the cell by diffusion barriers to PCMBS.     

Modification of sulfhydryl groups in ribosomal proteins by a dinitrophenyl hapten

A dinitrophenyl hapten capable of protein SH-group modification was synthesized and the specificity of its reaction with SH-groups of the E. coli ribosomal proteins was studied. The possibility of incorporation of the Dnp-modified protein into ribosomal subunits by in vitro reconstitution was demonstrated with the ribosomal protein S12. The Dnp-hapten attached to the protein S12 was found to be accessible for interaction with the Dnp-specific antibodies and therefore to be exposed on the surface of the reconstituted 30S subunit. Thus, the approach for incorporation of the antigenic groups into the protein components of supramolecular structures was proposed.     

Modification of the erythrocyte membrane by sulfhydryl group reagents

Summary In this study, the consequences of modification of human erythrocyte membrane sulfhydryl groups by N-ethyl maleimide (NEM), 5,5dithiobis-(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuriphenyl sulfonate (PHMPS) were investigated. These reagents differ in chemical reactivity, membrane penetrability and charge characteristics.Results of sulfhydryl modification were analyzed in terms of inhibitory effects on activities of five membrane enzymes; Mg⁺⁺- and Na⁺, K⁺-ATPase, K⁺-dependent and independentp-nitrophenyl phosphatase (NPPase) and DPNase. Structural considerations involved in the sulfhydryl-mediated inhibition were evaluated by studying the changes in susceptibility to sulfhydryl alteration produced by shearing membranes into microvesicles and by the addition of the membrane modifiers, Mg⁺⁺ and ATP.Conclusions from the data suggest that the effects of NEM appeared to result from modification of a single class of sulfhydryls; DTNB interacted with two different sulfhydryl classes. Increasing concentrations of PHMPS resulted in the sequential modification of many types of sulfhydryls, presumably as a result of increasing membrane structural disruption. DTNB and PHMPS caused solubilization of about 15% of membrane protein at concentrations giving maximal enzyme inhibition.In contrast to the usually observed parallels between Na⁺, K⁺-ATPase and K⁺-dependent NPPase, activities of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase and K⁺-dependent NPPase varied independently as a result of sulfhydryl modification. We suggest complex structural and functional relationships exist among these components of the membrane ATP-hydrolyzing system.Our studies indicate that the effects of sulfhydryl group reagents on these membrane systems should not be ascribed to sulfhydryl modificationper se, but rather to the resulting structural perturbations. These effects depend upon the structural characteristics of the particular membrane preparation studied and on the chemical characteristics of the sulfhydryl group reagent used.     

The diversity of sulfhydryl groups in the human erythrocyte membrane

Human bank blood erythrocytes were exposed to the mercurials p-chloromercuribenzoate (PCMB), chlormerodrin (CM), p-chloromercuribenzenesulfonate (PCMBS), and 1-bromomercuri-2-hydroxypropane (BMHP) for different time intervals, at different concentrations and in combination with n-ethylmaleimide (NEM) added before, and 2-mercaptoethylguanidine (MEG) and reduced glutathione (GSH) added after the mercurial. Binding patterns of the mercurials to the cells and effects on permeability of the cells were measured. The results indicate that the erythrocyte membrane contains multiple classes of sulfhydryl groups, alteration of which has a variety of effects on cell permeability. PCMB, chlormerodrin and PCMBS react with at least three classes of sulfhydryls, two of which are associated with the sodium-potassium barrier and, when altered, result in potassium loss, sodium accumulation and hemolysis. BMHP reacts with at least two classes of sulfhydryls, one of which is associated with permeability, and, when altered, results in hemolysis in isotonic solutions of choline chloride or lactose. The results provide additional insight into the structure and function of the erythrocyte membrane.     

Intramembranous particles on freeze-fractured membrane replica and sulfhydryl groups

The correlated distribution of intramembranous particles and sulfhydryl groups was examined in normal adult rabbit erythrocyte ghosts. Ghosts were treated to exhibit characteristic patterns of particle distribution and sulfhydryl groups were stained with Fast Blue BBN. It was shown that particles and sulfhydryl groups were located in corresponding patterns. The results indicate that the intramembranous particles in the erythrocyte membrane consist predominantly of protein.     

Intramembranous particles on freeze-fractured membrane replica and sulfhydryl groups

Summary The correlated distribution of intramembranous particles and sulfhydryl groups was examined in normal adult rabbit erythrocyte ghosts. Ghosts were treated to exhibit characteristic patterns of particle distribution and sulfhydryl groups were stained with Fast Blue BBN. It was shown that particles and sulfhydryl groups were located in corresponding patterns. The results indicate that the intramembranous particles in the erythrocyte membrane consist predominantly of protein.Supported by grants from the Japanese Government, Nos. 244016 and 337001     

Control of the mitochondrial inner membrane permeability by sulfhydryl groups

The effect of some thiol alkylating agents (N-substituted maleimide derivatives) on the permeability of the mitochondrial inner membrane was investigated. Several experimental approaches were used to study the modifications of the permeability properties. Alkylation of sulfhydryl groups led to an increase in the nonspecific permeability as judged by (i) the augmentation of the rate of osmotic shrinkage of mitochondria induced by polyethylene glycol, (ii) the sensitization of succinate dehydrogenase toward oxaloacetate, (iii) the enhancement of the oxidation rate of exogenous NADH, and (iv) the increase of the sucrose permeable space. The sulfhydryl groups involved in the maintenance of the selective permeability were shown to be located in the hydrophobic core of the membrane. Energization of mitochondria provoked an unmasking of these sulfhydryl groups. When magnesium ions were present in the incubation medium, N-substituted maleimide derivatives promoted gross modifications of the intramitochondrial ionic contents. Effluxes of endogenous calcium ions, inorganic phosphate, adenine nucleotides, and NAD(P)H were established. It was concluded that sulfhydryl groups probably play a crucial role in the maintenance of the membrane integrity and thus control the mitochondrial inner membrane permeability.     

Crucial role of sulfhydryl groups in the mitochondrial inner membrane structure

The mitochondrial inner membrane lost its selectivity for the transport of solutes after reaction of hydrophobic sulfhydryl groups with alkylating agents (maleimide derivatives). The nature of the thiol reagent-induced membrane perturbations was investigated. Modifications of the interactions between membrane components after treatment with thiol reagents were assessed by measuring the binding parameters of 1-anilinonaphtalene-8-sulfonate. An enhancement (about 50%) of the fluorescence intensity, a weak increase of the number of binding sites, and a decrease of the apparent dissociation constant were observed. However, no significant modification of the net surface charge was detected. The osmotic behavior of mitochondria in hypotonic solutions of sucrose was altered after thiol modification. The outer membrane did not seem to influence the matricial volume expansion when thiols were alkylated. After swelling in an isotonic solution of permeant ions, N-butylmaleimide-treated mitochondrial lost one-half of their malate dehydrogenase content, whereas fumarase and glutamate dehydrogenase did not leave the matrix space. Addition of polyethylene glycol of molecular weight below 6000 to swollen mitochondria induced a rapid but transient shrinkage. In swollen mitochondria, the above results indicate a possible holes formation in the membrane structure. The size of these holes was estimated to be about 3 nm. This process which required the presence of the outer membrane, was favored by increasing the temperature and was antagonized by specific effectors of the adenine nucleotide translocator.     

Erythrocyte membrane sulfhydryl groups and the active transport of cations

RNA synthesis was studied by autoradiographic analysis using tritiated uridine incorporation in the Chinese hamster cell line Dede after a one-minute pulse labeling period. RNA synthesis continues during all stages of interphase and mitosis except during metaphase and anaphase. Cytoplasmic RNA was apparently synthesized in the nucleus, since no grains were observed above the background level in the sample immediately following the labeling. Nucleoli synthesize their own RNA and are not reservoirs for RNA synthesized elsewhere. Both actinomycin D and nogalamycin inhibited the RNA synthetic activity of chromatin and nucleoli. However, the nucleolar synthetic activity was more susceptible to these agents than that of chromatin. Furthermore, actinomycin D was a stronger inhibitor than nogalamycin.     

Modification of cellular protein sulfhydryl groups by activated soluble immune response suppressor

Soluble immune response suppressor (SIRS), a product of murine Ly-2+ T lymphocytes, is activated to SIRSox by H2O2 produced by macrophages: SIRSox directly inhibits cell division by normal and neoplastic cells and antibody secretion by B lymphocytes. To examine the mechanism of SIRSox-mediated inhibition, a variety of cellular functions were measured after treatment of cells with SIRSox. These included respiration, glucose transport, microtubule content, glutathione content, production of H2O2 or superoxide anion, and the activities of a variety of different enzymes. Several cellular activities or measurements were inhibited or lowered after SIRSox-treatment, including cell division, microtubule content, glutathione reductase activity, and thioredoxin reductase activity; inhibition was partially reversed by the sulfhydryl reducing agent dithiothreitol. Protein sulfhydryl content of P815 mastocytoma cells and several other cell types was lowered by 35 to 45% after exposure to SIRSox. Protein sulfhydryl loss was also partially restored after incubation with dithiothreitol. Sulfhydryl loss was not due to cell lysis. In addition, treatment of crude cellular particulate fractions with SIRSox resulted in protein sulfhydryl loss and formation of protein sulfenyl derivatives. A comparison of the amount of SIRS and H2O2 present to the number of protein sulfhydryls lost or sulfenyl derivatives formed suggests that SIRSox acts catalytically, serves as a co-factor in protein sulfhydryl oxidation, or that it activates a second pathway that is directly responsible for sulfhydryl oxidation.     

Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

Electron paramagnetic resonance was used to characterize the first use of a thiol-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-Δ³-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.