Dihydropyridine binding to the L-type Ca2+ channel in rabbit heart sarcolemma and skeletal muscle transverse-tubules: role of disulfide, sulfhydryl and phosphate groups

The dihydropyridine receptor is associated with the L-type Ca2+ channel in the cell membrane. In this study we have examined the effects of group-specific modification on dihydropyridine binding in heart sarcolemmal membranes isolated from the rabbit. Specifically, dithiothreitol and glutathione were employed to assess the possible role of disulfide (-SS-) bonds in the binding of [3H]dihydropyridines. NEM, PCMS and iodoacetamide were employed to examine the effect of blocking free sulfhydryl groups (-SH) on the binding of [3H]dihydropyridines to their receptor in heart sarcolemma. Glutathione inhibited [3H]PN200-110 binding to sarcolemmal membranes 100%, with an IC50 value of 50 microM, while DTT inhibited maximally by 75% with an IC50 value in the millimolar range. Alkylation of free sulfhydryl groups by NEM or iodoacetamide inhibited binding of [3H]PN200-110 binding in cardiac sarcolemma approx. 40-60%. Blocking of free sulfhydryl groups by PCMS completely inhibited [3H]PN200-110 binding to their receptor in sarcolemmal membranes in a dose-dependent manner with an IC50 value of 20 microM. These results suggest the involvement of disulfide bonds and free sulfhydryl groups in DHP binding to the L-type Ca2+ channel in heart muscle. We also examined the effect of membrane phosphorylation on the specific binding of the dihydropyridine [3H]nitrendipine to its receptor. Phosphorylation was studied in cardiac sarcolemmal as well as skeletal muscle transverse-tubule membranes. Phosphorylation due to endogenous protein kinase and cAMP-dependent protein kinase was without effect on [3H]nitrendipine binding in both cardiac sarcolemmal and skeletal muscle membranes. Addition of exogenous calmodulin under conditions known to promote Ca2+/calmodulin-dependent phosphorylation increased [3H]nitrendipine binding 20% with no alteration in KD in both types of membrane preparation. These results suggest a role for calmodylin in dihydropyridine binding to L-type Ca2+ channels.     

Effects of cyclic nucleotides on the conformational states of the alpha core of the cyclic AMP receptor protein.

The alpha core gragment produced by limited proteolysis contains the cyclic AMP binding domain and the two buried sulfhydryl groups of the cyclic AMP receptor protein. The buried sulfhydryl groups of the alpha core react with 5,5'-dithio-bis(2-nitrobenzoic acid) after denaturation by 3 M urea or digestion with subtilisin. The rate of sulfhydryl modification in the presence of 3 M urea or subtilisin is markedly decreased in the presence of cyclic nucleotides which are proposed to tighten the conformation of the alpha core. Incubation of the alpha core in 3 M urea or dithionitrobenzoic acid does not affect cyclic AMP binding while dithionitrobenzoic acid plus 3 M urea inhibits cyclic AMP binding suggesting a role for the buried sulfhydryls in cyclic AMP binding or their proximity to the cyclic AMP binding domain of the alpha core. The data are consistent with a ligand-induced conformational change in the alpha region of the native cyclic AMP receptor protein that is required for DNA binding.     

Sulfhydryls of tubulin. A probe to detect conformational changes of tubulin.

The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 degrees C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 degrees C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects.     

Comparative inhibition studies of the phosphotransferase and glycerophosphate acylation systems in membrane vesicles of Escherichia coli

Purified membrane vesicles were treated with various reagents specific for different amino acid side-chains. Titration of sulfhydryl groups with specific reagents shows that the sulfhydryl content of membrane vesicles as estimated directly is similar to that found by treating spheroplasts or cells and then isolating the membrane vesicles. The blocking of sulfhydryl groups specifically inhibits the α-methylglucoside transport system (phosphotransferase system), whereas the glycerophosphate acylation system is not affected. The kinetics of inhibition of the first system show that a high reactivity of the sulfhydryl groups is involved. Inhibition of the acyltransferase activity by sulfhydryl reagents occurs only on partial denaturation of the membranes induced by mild sonication, heat or toluene treatment. The Inhibition is at the level of the glycerol 3-phosphate:acyl thioester acyltransferase.The effects of sonication and/or sulfhydryl reagents were measured by sulfhydryl titration, by assays of NADH oxidase and d-lactate dehydrogenase activities, as well as by 1-anilino-8-naphthalene sulfonate binding. The results support the hypothesis that the acyltransferase system is embedded within the membrane and that the readily accessible permease system is closer to (or at) the surface of the membrane.     

Domain structure of fibronectin and its relation to function. Disulfides and sulfhydryl groups.

Hamster cell fibronectin is a glycoprotein consisting of two 230,000-dalton subunits in a disulfide-bonded dimer. The molecule is composed of domains which can be separated by partial proteolytic cleavage. The carbohydrates, disulfide bonds, and a single free sulfhydryl group per chain are distributed nonuniformly among these regions. All the interchain disulfides are within 10,000 daltons of the end of the molecule and are removed by mild proteolysis which also generates 200,000- and 25,000-dalton fragments which do not contain interchain disulfides. The 200,000-dalton fragment contains all or most of the carbohydrate side chains, and the free sulfhydryl group, but is relatively poor in cystine. The 25,000-dalton fragment is carbohydrate-free and cystine-rich but has no free sulfhydryl groups. There is heterogeneity in carbohydrate content among the monomeric chains of intact fibronectin and the 200,000-dalton fragments. The gelatin binding site of fibronectin is in the 200,000 fragment. Intact disulfide bonds are required for binding of fibronectin to cells and to gelatin and blockage of the free sulfhydryl groups prevents binding of fibronectin to cells, suggesting that intermolecular disulfide bonding may be important.     

Influence of free thiol group(s) on autoantibody-defined epitope of proliferating cell nuclear antigen.

Proliferating cell nuclear Ag (PCNA) is an intranuclear protein involved in DNA replication directed by DNA polymerase delta and is the target Ag of autoantibodies in some patients with SLE. There is evidence that the epitope on PCNA recognized by human autoantibodies is conformation-dependent and is not a continuous peptide sequence. Thimerosal, a mercury-containing sulfhydryl blocking compound, markedly reduced or abolished the reactivity of this autoantibody-defined PCNA epitope. The thimerosal effect was observed in various Ag-detecting systems including indirect immunofluorescence, immunodiffusion, immunoprecipitation, and flow cytometry. The mechanism of the thimerosal effect appeared to be mediated through free but not readily accessible sulfhydryl group or groups. The sulfhydryl-modification by thimerosal could be reversed by competition with thiol-containing compounds. Experimentally induced mAb to PCNA generated by immunization with purified PCNA have been shown to recognize epitopes that are continuous peptide sequences and these epitopes were not affected by thimerosal. It has been shown that the human autoantibody-defined epitope is related to the function of PCNA because autoantibodies are able to inhibit DNA polymerase delta directed DNA replication, whereas experimentally induced antibodies are not. These studies show that certain sulfhydryl groups in PCNA have a role in determining the antigenicity of the epitope recognized by autoantibody and raise the possibility that certain sulfhydryl groups might also be associated with its function.     

Sulfhydryl Groups Modulate the Allosteric Interaction Between Glycine Binding Sites at the Inhibitory Glycine Receptor

We have investigated the effect of chemical reagents that modify sulfhydryl groups on the ligand binding properties of the glycine receptor (GlyR). The Hill coefficient (nH) for the displacement of [3H]strychnine binding by glycine was increased from approximately 0.8 to values significantly above 1 (approximately 1.2-1.4) in membranes pretreated with the disulfide-reducing agent dithiothreitol or glutathione. However, the affinity of strychnine or glycine for the GlyR was not affected by these treatments. This indicates that several glycine binding sites interact cooperatively for displacing bound strychnine under such experimental circumstances. A similar increase in the nH for glycine has been observed when the temperature of the binding assay was increased to 37 degrees C. Combination of dithiothreitol pretreatment and increased binding temperature led to nH variations similar to those observed with either of these treatments alone, a finding suggesting that their mechanisms of action are not independent. Conversely, modification of rat spinal cord membranes or of purified and reconstituted GlyR preparations with the sulfhydryl-alkylating agent N-ethylmaleimide or fluorescein-maleimide decreased nH values to approximately 0.5, without affecting glycine or strychnine affinities. This effect may be caused by an increased heterogeneity of GlyR populations. It is interesting that occupancy of the receptor by glycine or beta-alanine (but not by antagonists) specifically protects from the effects of the different sulfhydryl reagents. Moreover, the presence of some of the Eccles' anions, i.e., anions that permeate through the channels associated with GlyRs and gamma-aminobutyric acidA receptors, seems to be required for the action of both dithiothreitol and N-ethylmaleimide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Essential sulfhydryl groups in diamine oxidase from Euphorbia characias latex

Diamine oxidase from Euphorbia characias latex contains two sulfhydryl groups per mole of dimeric enzyme. The sulfhydryl groups are unreactive in the native enzyme but can be readily titrated by 4,4′-dithiodipyridine after protein denaturation, or anaerobically in the presence of the amine substrate. In the presence of both substrates (diamine and oxygen) they react sluggishly. The sulfhydryl groups show different reactivity toward various reagents, but in every case their modification inhibits catalytic activity. The insensitivity of the native enzyme to specific reagents suggests that the sulfhydryl groups are positioned in the interior of the protein and shielded from the solvent. Their reactivity in the presence of the amine substrate could be attributed to a conformational change occurring upon substrate binding or after substrate oxidation.     

A spin label study of human erythrocyte ghost membranes damaged by methyl phenyldiazenecarboxylate (azoester)

The mechanism of methyl phenyldiazenecarboxylate (azoester) damage to human erythrocyte membranes has been investigated by means of spin labels. Azoester treatment exposed protein binding sites for non-covalently bound stearic acid and androstane spin labels that were occult in the untreated membrane. Experiments with iodoacetamide and N-alkylmaleimide spin labels suggested that azoester destroyed membrane sulfhydryl groups. No change in the structural integrity of membrane lipid components could be detected.     

Mitochondrial glutathione transferases. The alkylation of mitochondrial membrane yields a catalyst with glutathione-transferase-like properties

The pretreatment of rat liver mitochondria with alkylating agents (N-ethylmaleimide, iodoacetamide or vinyl pyridine) increased the activity of mitochondrial glutathione transferase (GST) by 100-250%. Further experiments provided evidence that mitochondrial membranes contain an enzymatically inactive protein which can be alkylated through its sulfhydryl groups. By alkylation, this protein achieves catalytic properties similar to those of native GST.     

Biochemical aspects of the visual process. XXIII. Sulfhydryl groups and rhodopsin photolysis

1. The number of exposed sulfhydryl groups in cattle rod photoreceptor membranes has been determined in suspension and after solubilization in various detergents both before and after illumination.2. In suspensions, two sulfhydryl groups are modified per mole of rhodopsin, both by Ellman's reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide, while no extra SH groups are uncovered upon illumination. Neither reagent affects the spectral integrity of rhodopsin at 500 nm and the recombination capacity is retained upon modification of both rhodopsin and opsin.3. However, in detergents (digitonin, Triton X-100 and cetyltrimethylammonium bromide (CTAB)) 2–3 additional sulfhydryl groups appear upon illumination, in agreement with earlier reports.4. A total number of six sulfhydryl groups and two disulfide bridges are found in rod photoreceptor membranes, expressed per mole of rhodopsin.5. DTNB reacts somewhat faster with membrane suspensions after than before illumination. The less reactive sulfhydryl modifying agents O-methylisourea and methyl-p-nitrobenzene sulfonate show a similar behavior.6. It is concluded that illumination of rhodopsin in vivo will not uncover additional SH groups, although the reactivity of one exposed SH group may increase somewhat. These findings also exclude a role of SH groups in the covalent binding of the chromophore.     

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.     

The role of sulfhydryl groups in the bleaching and synthesis of rhodopsin

The condensation of retinene₁ with opsin to form rhodopsin is optimal at pH about 6, a pH which favors the condensation of retinene₁ with sulfhydryl rather than with amino groups. The synthesis of rhodopsin, though unaffected by the less powerful sulfhydryl reagents, monoiodoacetic acid and its amide, is inhibited completely by p-chloromercuribenzoate (PCMB). This inhibition is reversed in part by the addition of glutathione. PCMB does not attack rhodopsin itself, nor does it react with retinene₁. Its action in this system is confined to the —SH groups of opsin. Under some conditions the synthesis of rhodopsin is aided by the presence of such a sulfhydryl compound as glutathione, which helps to keep the —SH groups of opsin free and reduced. By means of the amperometric silver titration of Kolthoff and Harris, it is shown that sulfhydryl groups are liberated in the bleaching of rhodopsin, two such groups for each retinene₁ molecule that appears. This is true equally of rhodopsin from the retinas of cattle, frogs) and squid. The exposure of new sulfhydryl groups adds an important element to the growing evidence that relates the bleaching of rhodopsin to protein denaturation. The place of sulfhydryl groups in the structure of rhodopsin is still uncertain. They may be concerned directly in binding the chromophore to opsin; or alternatively they may furnish hydrogen atoms for some reductive change by which the chromophore is formed from retinene₁. In the amperometric silver titration, the bleaching of rhodopsin yields directly an electrical variation. This phenomenon may have some fundamental connection with the role of rhodopsin in visual excitation, and may provide a model of the excitation process in general.     

Sulfhydryl groups of Mimosa pudica tubulin implicated in colchicine binding and polymerization in vitro.

The important characteristic of novel Mimosa pudica tubulin is its ability to bind colchicine only when dithiothreitol is included in the isolation buffer, indicating the involvement of sulfhydryl groups in colchicine binding. Modification of sulfhydryl groups by a sulfhydryl modifying agent also affects the normal assembly of tubulin into microtubules, as revealed by electron microscopic and spectrophotometric studies. The number of free sulfhydryl groups present in tubulin protein responsible for both colchicine binding and polymerization has been found to be 4, distributed in alpha and beta subunits, and is distinctly different from the number reported for animal tubulin.     

OBSERVATIONS ON THE BASEMENT MEMBRANES IN RAT KIDNEY

Basement membranes in the kidney are made up of a homogeneous matrix. In argyria, silver passes from the blood in the ionic form and diffuses into the kidney basement membranes in which it is precipitated. X-ray diffraction studies of "silver-stained" rat kidneys show that most of the silver in the kidneys is combined with some form of sulfur. Histochemical staining for sulfhydryls and disulfides demonstrates the presence of these groups in basement membranes. It appears that silver ions combine with either or both the sulfhydryl or disulfide groups in the basement membranes and also in mitochondria (when the silver diffuses into a cell).     

A modulating role of mercurials-sensitive sulfhydryl groups in synaptic GABA receptor binding

The specific binding of GABA (γ-aminobutyric acid) agonist ³H-muscimol, to synaptic membranes from the rat brain showed a significant increase, when the membranous preparations were treated with a low concentration (10^?4–10^?5M) of mercurial sulfhydryl reagents such as p-chloromercuribenzoate and mercuric chloride. This activation in GABA receptor binding was bicuculline-sensitive, and was partially restored by subsequent treatments with 10 mM cysteine, penicillamine, or mercaptoethanol. Scatchard analysis of the binding revealed that this activation was due to the increase in the affinity of both high and low affinity bindings sites but not in the B_max values. On the other hand, the treatment of synaptic membranes with hydrophilic sulfhydryl reagents such as N-ethylmaleimide and iodoacetate had no effect on the binding. These hydrophilic sulfhydryl reagents, however, induced an increase of the binding following the pretreatment of synaptic membranes with 0.01% Triton X-100 or 0.5 U/mg prot. of phospholipase A₂ (EC 3.1.1.4.). These results suggest that mercurials-sensitive sulfhydryl groups, which are normally masked by membrane lipids, may play a modulating role in GABA receptor binding at central synapses.     

Mercuric and cadmium ions stimulate phosphorylation of band 4.2 protein on human erythrocyte membranes

In this study, we found that Hg2+ and Cd2+ enhanced the phosphorylation of human erythrocyte membranous proteins, especially band 4.2 protein, which was hardly phosphorylated in the absence of the metal ions. p-Chloromercuribenzenesulfonate and p-chloromercuribenzoate had effects similar to those of Hg2+ and Cd2+ on band 4.2 protein phosphorylation, while other metal ions and sulfhydryl agents, such as N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), or iodoacetate, did not. The Hg2+-stimulated phosphorylation of band 4.2 protein required a millimolar concentration of Mg2+, and it was inhibited by Ca2+ dose-dependently. Phosphoserine was identified from a hydrolysate of the phosphorylated band 4.2 protein by high-voltage electrophoresis. A specific protein inhibitor against cAMP-dependent protein kinase decreased the Hg2+-stimulated phosphorylation of band 4.2 protein. This protein had more binding sites for 203Hg2+ than any other membrane proteins. A spectrin complex from the Hg2+-treated membranes contained the band 4.2 protein, which was not detected in the complex from untreated membranes. Furthermore, protein kinase, which could phosphorylate the band 4.2 protein, was also contained in the cytoskeletal fraction from the Hg2+-treated membranes. These results suggest that Hg2+ may bind certain sulfhydryl groups of band 4.2 and other proteins to make band 4.2 protein susceptible to the endogenous cAMP-dependent protein kinase.     

The bspA locus of Lactobacillus fermentum BR11 encodes an L-cystine uptake system

BspA is a basic surface-exposed protein from Lactobacillus fermentum BR11. Sequence comparisons have shown that it is a member of family III of the solute binding proteins. It is 89% identical to the collagen binding protein, Cnb, from Lactobacillus reuteri. Compared with the database of Escherichia coli proteins, BspA is most similar to the L-cystine binding protein FliY. To investigate the function of BspA, mutants depleted for BspA were generated by homologous recombination with a temperature-sensitive plasmid. These mutants were significantly impaired in their abilities to take up L-cystine. Uptake rates of L-glutamine, L-histidine, and L-lysine, which are substrates for other binding proteins with similarity to BspA, were unaffected. Evidence was obtained that BspA is necessary for maximal resistance to oxidative stress. Specifically, inactivation of BspA causes defective growth in the presence of oxygen and sensitivity to paraquat. Measurements of sulfhydryl levels showed that incubation of L. fermentum BR11 with L-cystine resulted in increased levels of sulfhydryl groups both inside and outside the cell; however, this was not the case with a BspA mutant. The role of BspA as an extracellular matrix protein adhesin was also addressed. L. fermentum BR11 does not bind to immobilized type I collagen or laminin above background levels but does bind immobilized fibronectin. Inactivation of BspA did not significantly affect fibronectin binding; therefore, we have not found evidence to support the notion that BspA is an extracellular matrix protein binding adhesin. As BspA is most probably not a lipoprotein, this report provides evidence that gram-positive bacterial solute binding proteins do not necessarily have to be anchored to the cytoplasmic membrane to function in solute uptake.     

Succinate and phenylsuccinate as modifiers of sulfhydryl groups of inner mitochondrial membrane protein. Study by EPR

Paramagnetic labels specific for sulfhydryl (SH) groups have been used to study the conformational changes of the inner mitochondrial membrane. The EPR spectra of the SH-groups spin-labeled with maleimide or iodoacetamide show the existence of two populations of sulfhydryl groups, differing in their mobility (one weakly, the other strongly immobilized). The incubation with succinate or phenylsuccinate decreased the binding of these labels of the weakly immobilized sites while the number of total SH groups was the same before and after the incubation. These results suggest that succinate or phenylsuccinate induce a reversible change in protein conformation or in protein arrangement within the inner mitochondrial membrane. This change is concomitant to the protein movement between inner membrane and perimembranal space induced by either of these two molecules.