Role Of SH Groups Research Articles

The role of SH groups in the regulation of Gardos channels in glucose deficiency

Background. Disruption of the energy balance of erythrocytes under conditions of a decrease in the glycolysis level can cause a change in the ion permeability of their membrane. The aim. To study Ca2+-dependent potassium permeability of the erythrocytes membrane in the presence of SH group modifiers under conditions of glucose deficiency. Materials and methods. The study used precipitated erythrocytes obtained from the blood of 20 male Wistar rats. The change in the Ca2+-dependent potassium conductivity of the erythrocyte membrane was determined using the potentiometric method. The A23187-and redox-induced hyperpolarization responses of erythrocytes were evaluated. Results. Glucose deficiency in the medium, as well as the use of the glycolysis inhibitor 2-deoxyglucose, led to an increase in the amplitude of A23187-stimulated membrane hyperpolarization by the opening of the Gardos channels. At the same time, the redox-dependent hyperpolarization of the erythrocyte membrane turned out to be insensitive to a decrease in the glucose content in the medium and to the glycolysis inhibition. The effects of SH group modifiers in the normal incubation medium and under glucose deficiency turned out to be multidirectional and depended on the method of stimulation of Gardos channels. Conclusion. The results obtained indicate that metabolic disorders in erythrocytes under conditions of glucose deficiency lead to a change in the mechanisms of control of Gardos channels with the participation of SH groups of the proteins of these channels or their regulatory proteins.

Role of SH groups and chemisorbed hydrogen in the formation of sulfur vacancies on an edge of a molybdenum disulfide crystallite

The formation of sulfur vacancies on an edge of a molybdenum disulfide crystallite with the participation of SH groups has been considered using density functional theory with a plane-wave basis set and a PBE functional. The relative energy of a number of surface sulfur and hydrogen species has been determined. The profiles of the reactions of the SH group with molecular hydrogen have been investigated.

Use of Nitroxide Spin Probes and Electron Paramagnetic Resonance for Assessing Reducing Power of Beer. Role of SH Groups

Intensity of EPR spectra of stable organic free radicals, nitroxides, is decreasing with time if the radicals are dissolved in beer. The process is determined by a chemical reaction of nitroxide reduction by components naturally present in beer. Kinetics can be described as a simple irreversible first order with respect to both nitroxide and one reducing agent. Effective concentration of the reducing agent and the corresponding reaction rate constant has been determined. It is demonstrated that the nitroxide reduction is sensitive to the presence of solvent-accessible SH groups of proteins present in beer. It is proposed that quantitative analysis of reduction kinetics of small water-soluble nitroxide radicals such as TEMPO and TEMPOL can be used to assess the reducing power of beer. The effect of accelerated aging of beer achieved at elevated temperatures on nitroxide reduction kinetics is demonstrated.

Role of SH Groups in the Functioning of Ca 2+ -Transporting ATPases Regulating Ca 2+ Homeostasis and Exocytosis

Using a Ca2+-sensitive fluorescent indicator, Fura-2/AM, and a metallochromic dye, arsenazo, we measured the intracellular concentration of Ca2+ ([Ca2+] i ) and the content of total calcium in isolated acinar cells of the rat submandibular salivary gland. It was shown that the influence of a mercaptide-forming compound, sodium p-chloromercuribenzoate (pChMB), increased both the [Ca2+] i and content of total calcium but did not change the intensity of exocytosis. Such a situation is probably related to the fact that pChMB inhibits plasmalemmal Ca2+-ATPase (PMCA). The absence of changes in the exocytotic activity can be explained as follows: the influence of a pChMB-induced significant increase in the [Ca2+] i is neutralized due to the functioning of Ca2+-ATPases of the endoplasmic reticulum (SERCA), which pump Ca2+ into the store. Incubation of a microsomal fraction with pChMB resulted in suppression of the specific PMCA and SERCA activities with apparent constants of inhibition (I 50) 245 and 52 μM, respectively. Dithiothreitol (DTT, 0.1 mM) increased the PMCA and SERCA activities (probably facilitating the access of substrate to the active centers of ATPases at the expense of a decrease in the number of disulfide bonds, which is followed by changes in the conformation of intracellular hydrophilic loops of their molecules). Dithiothreitol also recovered the suppression of PMCA and SERCA activities induced by pre-incubation with pChMB (by 45 and 32%, respectively); these activities did not, however, reach the initial levels. A probable interpretation of this fact is that DTT shields from the action of pChMB only superficial but not sterically less accessible SH groups. Limited proteolysis of the microsomes by α-chymotrypsin decreased the specific PMCA and SERCA activities by 16 and 60%, respectively. Incubation of the microsomes in an α-chymotrypsin-containing medium (15 sec) with subsequent addition of 150 μM pChMB exerted almost no influence on the PMCA activity, whereas the SERCA activity dramatically increased (by 146%). This fact allows us to suggest that α-chymotrypsin is capable of eliminating the inhibitory effect of pChMB on the SERCA activity; the mechanism of this effect remains unknown. Therefore, functionally important SH groups are present in the catalytic and active centers of both PMCA and SERCA; superficial SH groups dominate in the PMCA molecules, whereas SERCA is controlled by more deeply localized SH groups.

Role of Sulfhydryl Groups in Functioning of Adenylyl Cyclase Signaling System

This review considers the literature data and author's own results on the role of SH-groups in functioning of the hormone-sensitive adenylyl cyclase system (ACS). It has been shown that the state of SH-groups affects crucially all main stages of the hormonal signal transudation: the ligand-binding properties of receptor and its coupling to G-proteins, interaction of G-proteins with adenylyl cyclase (AC) and its catalytic activity. It is noted that for the receptors, coupled to AC by a stimulating mode, the central aspect of the SH-dependent regulation of ACS is shifted to the receptor, while for the receptors coupled to AC by an inhibiting mode, it coincides with G-protein of the inhibiting type, which is sensitive to the SH-group state. Based on the performed comparative analysis of primary structures of signalling proteins—ACS components and of literature data, there are revealed the cysteine residues determining the functional activity of these proteins in the process of the hormonal signal transudation. The conclusion is made that the SH-group state (the ratio of free SH-groups and disulfide bonds) is the main factor determining the ACS reactivity to hormonal effects and selectivity of process of the signal transudation.

The effect of semen processing on human sperm DNA integrity is not associated with increased levels of sperm free thiols.

Objective: Some forms of sperm processing may adversely affect sperm DNA integrity but the mechanism remains unknown. We monitored the levels of sperm free thiols (SH groups) and DNA denaturation (DD) before and after density gradient centrifugation in order to explore a possible mechanism (the role of SH groups in the stabilization of sperm nuclear protamines) for the observed augmented sperm DD post-processing. Design: Prospective laboratory study. Materials/Methods: Semen samples (n = 43) were obtained from consecutive non-azoospermic men presenting for evaluation at our institution. Samples were processed by 2-layer density gradient centrifugation. Sperm motility, sperm chromatin structure (evaluated by flow cytometry analysis of acridine orange-treated spermatozoa) and sperm SH groups were compared before and after semen processing. Sperm chromatin integrity was expressed as the percentage of spermatozoa demonstrating DD. Results: Following density-gradient centrifugation, mean sperm motility (±SEM) improved significantly compared to whole semen (54 ± 3 vs. 43 ± 3%, p < 0.01). As previously observed, following density-gradient centrifugation, the percentage of sperm with denatured DNA increased compared to whole semen (29 ± 4 vs. 18 ± 2%, p < 0.001). However, the level of sperm free thiol groups was lower in processed compared to whole semen (268 ± 26 vs. 534 ± 43 nmol/106 spermatozoa, p < 0.01). Conclusions: Our data demonstrate that density gradient centrifugation adversely affects sperm DNA integrity despite improved sperm motility and reduced levels of sperm SH groups in the recovered sample. Increased levels of sperm DD post-processing do not appear to be due to a significant degree of sperm-specific disulfides to SH group conversion. Supported by: Kidney Foundation of Canada.

Modulation influence of p-chloromercuribenzoate on plasma membrane Na+-Ca2+ exchanger of the secretory cells of chironomus larvae salivary gland.

Investigations the role of SH-groups of Ca2+-transport systems molecules in secretory cells functioning have special significance because of: (i) Ca2+ cations have extraordinary importance in secretory process; (ii) SH-groups have importance to maintain of lipid bilaer structure [5,11]; (iii) one of the most sensitive links, obviously, for all membrane Ca2+transporting systems are SH-groups of aminoacid’s residues since its: * have high reactive ability [9]; ** determine fourth structure of protein molecule [9], besides its are form part of regulatory domains of these transport systems and especially Na+-Ca2+-exchanger molecule dogs cardiomyocites [1]. It has also known that SH-groups are form part of large intracellular loop f of Na+-Ca2+exchanger molecule [10]. Availability of SH-groups in the Nat Ca2+exchanger molecule of secretory cell membrane in Chironomus larvae salivary gland we have supposed on the base of changes in Na+-Ca2+exchange current at alkalisation of external solution [6,8] and influence of Cd2+,Ni2+ and La3+[3]. Therefore the target of this study was to investigate the influence of SH-groups specific blocator — p-chloromercuribenzoate (PCMB) on the Na+-Ca2+exchange in the secretory cell membrane of investigated glands.

Alanine transport across the human placental brush border membrane and the role of SH groups in carrier function.

We have determined the kinetic characteristics of alanine transport into brush border membrane vesicles (BBMV) of human full term placenta and identified functional groups of the carrier proteins that are important for transport function. Alanine influx into BBMV was found to be mediated by two transport systems with different kinetic features and distinct substrate specificities. An uphill operating electrogenic Na(+)-dependent cotransport system could be kinetically separated from a Na(+)-independent facilitated diffusion system. The Na(+)-dependent transporter mediates Na(+)-alanine cotransport with a 1:1 flux coupling ratio (Hill coefficient 1.13 +/- 0.12) and a Km for alanine of 0.45 +/- 0.06 mmol/l. Half-maximal stimulation of Na(+)-dependent alanine influx was observed at a Na+ concentration (NaCl) of 51.4 +/- 1.3 mmol/l. A variety of group specific reagents were used to identify functional groups in the transport proteins. Only compounds reacting with SH-residues (NEM, DTNB, PCMBS) or NH2-groups (PITC) were found to affect Na+ dependent and Na+ independent alanine transport. The EC50 value for inhibition of alanine influx by PCMBS was 450 +/- 48 mumol/l. Chemical modifications of SH-groups by PCMBS caused a significant reduction (p < 0.005) in the Vmax for Na(+)-dependent alanine influx from 0.57 +/- 0.06 to 0.16 +/- 0.05 nmol.mg protein-1.10s-1 without affecting significantly the Km value. Inhibition by PCMBS was reversed by treatment of BBMV with DTT. When the substrate binding site of the transporter was protected by alanine or leucine, PCMBS still, blocked transport function, indicating that the crucial SH groups are not located within the substrate binding site of the transport proteins.

Vagal nerve and the gastric mucosal defense

An essential role for an intact vagal nerve has been proven in the development of gastric mucosal cyto- and general protection. On the other hand, chemically-induced (ethanol, HCl, indomethacin) gastric mucosal damage is enhanced after acute surgical vagotomy. The aims of this paper were to study the possible mechanisms of the vagal nerve in the development of gastric mucosal defense. The following questions were addressed: 1) effect of surgical vagotomy on the development of ethanol- (ETOH), HCl-, and indomethacin (IND)-induced gastric mucosal damage; 2) changes in the gastric mucosal defense by scavengers, prostacyclin and other compounds (small doses of atropine and cimetidine: 3) changes in the gastric mucosal vascular permeability due to chemicals; 4) effect of indomethacin in the ETOH and HCl models with and without surgical vagotomy; 5) changes in the gastric mucosal content of prostacyclin and PGE 2 in the ETOH and HCl models after surgical vagotomy; and 6) changes in the role of SH-groups in gastric mucosal defense after surgical vagotomy. It was found that: 1) the gastric mucosal damage produced by chemicals (ETOH, HCl, and indomethacin) was enhanced after surgical vagotomy; 2) the cyto- and general gastric protective effects of β-carotene, prostacyclin, and small doses of atropine and cimetidine disappeared after surgical vagotomy; 3) the vascular permeability due to chemicals (ETOH, HCl, indomethacin) significantly increased after surgical vagotomy in association with an increase in both number and severity of gastric mucosal lesions; 4) IND alone (in animals with an intact vagus) did not produce gastric mucosal lesions (in 1-h experiments), but it aggravated ETOH-induced gastric mucosal damage (both its number and severity); 5) the gastric mucosal levels of prostacyclin and PGE 2 decreased after surgical vagotomy; 6) IND application (after surgical vagotomy) decreased further the tissue levels of prostacyclin and PGE 2 in association with an increase of gastric mucosal damage; and 7) the gastric mucosal protective effects of SH-groups were abolished by surgical vagotomy.

The effects of hexavalent chromium on the activity of alkaline phosphatase in the intestine of rainbow trout ( Oncorhynchus mykiss)

This work is a contribution to the study of the effects of hexavalent chromium on intestinal alkaline phosphatase activity in fish. The enzyme activity was studied in intact tissues of rainbow trout ( Oncorhynchus mykiss). incubated at 25°C with paranitrophenyl phosphate. Substrate hydrolysis has an optimum at pH 10 and is linear with time up to, at least, l h. Preincubation at a basic pH in the absence of substrate for 30 min or more, leads to a decrease of the enzyme activity. This inhibition is lower when phosphate is added to the preincubation medium. The mucosal layer has a special influence on V max which is higher when the intestinal tissue is blotted on filter paper. Chromium dichromate leads to a decrease of the enzyme activity when the tissue is first preincubated in the absence of substrate. This inhibition is dependent upon the metal concentration up to 1 mM. At higher concentrations an attenuation of this effect has been shown. Preincubation at alkaline pH stimulates the effect of chromium. The inhibition is time dependent. However, for longer preincubations (60 min) the chromium effect seems to be progressively reversed. Mucus does not protect the enzyme from the metal. Dichromate ions have a special influence on V max and a minor one on the K m, of the enzyme. The substrate (paranitrophenyl phosphate) effectively protects the enzyme even at low concentrations (0.18 mM). Phosphate has the same effect but especially al a high concentration (100 mM). After a reduction by ascorbate, chromium does not inhibit phosphatase activity. The inhibition has been thought to be due to the oxidation of specific molecules. The role of SH groups has been postulated since DTT protects the enzyme, and partially restores its activity after treatment with chromium.

Contribution to the knowledge of the role of SH-groups in the molecule of pea alcohol dehydrogenase

p-Chloromercuribenzoate irreversibly inactivates alcohol dehydrogenase (ADH) isolated from germinating pea seeds. The reaction follows the first order kinetics. The inactivation of pea ADH is pH-dependent and is maximal at pH 9.0. NAD protects the enzyme from inactivation by p-chloromercuribenzoate; the higher the concentration of the coenzyme and the longer the period of incubation of NAD with the enzyme, the lower the degree of inactivation. Ethanol does not prevent the enzyme from inactivation. o-Phenanthroline in a concentration of 1 . 10-3 mol l-1 decreases the degree of inactivation of the enzyme by p-chloromercuribenzoate by 20%; imidazole is without effect on the reaction. Zn2+-ions in concentration of 1 . 10-5 mol l-1 also partly protect the enzyme from inactivation by p-chloromercuribenzoate. The results obtained show that the SH-groups sensitive to labeling with p-chloromercuribenzoate are localized in the active center of the enzyme, probably in the coenzyme-binding site. The protective action of Zn2+-ions and of o-phenanthroline against this inactivation confirms the assumption that the SH-group acts as a zinc ligand in the active center of the enzyme.

Modification of α-crystallin subunit chains and formation of α-neoprotein molecules: Role of SH groups

Immunoadsorbents with bound antibodies restricted to determinants dependent on α-crystallin's quaternary structure permitted the fractionation of the population of 125I-labeled α-crystallin molecules, treated by iodoacetic acid, into molecules in which the native structure was still preserved and molecules with a completely different quaternary structure than the native protein. Parallel experiments with [ 14C]iodoacetic acid yielded information on the percentage of blocked SH groups in each of the above two fractions. The presence of molecules formed by A with B-chain association was established by sequential binding first to an immunoadsorbent with antibodies restricted to determinants located on α-crystallin's A-subunit chains as ligand and second, after desorption, to an immunoadsorbent with antibodies to B chains as ligand. With the aid of these techniques, it was established that (i) The modified α-crystallin molecules with quaternary determinants of the native protein contained a maximum of 23% blocked SH groups, indicating that the carboxymethylation involved only the fastreacting surface SH groups. (ii) The modified α-crystallin molecules without the native protein's quaternary structure were built by a different association between A and B subunits than in α-crystallin, indicating formation of α-neoprotein molecules. (iii) Monomeric A chains with all SH groups carboxymethylated, and monomeric B chains in a ratio of 1A:5B, 2A:1B, and 5A:1B in urea solution, associate on dialysis, forming α-neoprotein molecules.

Distribution of sulfhydryl and disulfide groups in ocular tissues.

The localization and concentration of protein-bound sulfhydryl groups and disulfide linkages in mouse ocular tissues were examined with a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM). Corneal epithelium and endothelium, conjunctival epithelium, lens cortex, retinal plexiform layers and inner segment and pigment epithelium were rich in SH groups. Descemet's membrane and lens capsule contained many S-S linkages. Corneal stroma, sclera and retinal outer segment contained a moderate number of S-S linkages. There was a unique distribution of SH groups and S-S linkages in the eye; that is, tissues abundant in SH groups contained few S-S linkages, and tissues abundant in S-S linkages contained few SH groups. The role of SH groups and S-S linkages in the eye was discussed.

Grafting onto Wool. XXI. Graft Copolymerization of Methyl Methacrylate onto Reduced Wool by Use of Manganese Acetylacetonate as Initiator

Abstract In an attempt to ascertain the role of SH groups of wool fiber in graft copolymerization, methyl methacrylate (MMA) was graft co-polymerized onto reduced wool by using manganese acetylacetonate (Mn(acac)3) as the initiator in an aqueous medium. The reduction of wool was carried out with thioglycollic acid (TGA) in an aqueous solution. The percentage of grafting was determined as a function of the concentration of Mn(acac)3, MMA, and nitric acid; the extent of reduction of wool by TGA; and the time and temperature. Under optimum conditions methyl methacrylate was grafted to the reduced wool to the extent of 57.0%. Unreduced wool under optimum conditions was reported earlier to afford a maximum grafting of MMA to the extent of 82.5%. The reduction of wool does not promote grafting of MMA onto wool in the presence of Mn(acac)3.

O-128 - Potent analgesics and opioid receptor 29 : The role of SH groups in opioid-receptor interactions

O-128 - Potent analgesics and opioid receptor 29 : The role of SH groups in opioid-receptor interactions

33] Sulfhydryl chemistry of rhodopsin

This chapter reviews the sulfhydryl chemistry of rhodopsin, followed by methodological directions for various relevant procedures. The data on the sulfhydryl chemistry of rhodopsin are almost exclusively restricted to the bovine species, because of the easy availability in large amounts. The six sulfhydryl groups of bovine rhodopsin exhibit clearly different reactivity/accessibility on treatment with SH reagents. These differences can be analyzed in great detail, as they are more or less dependent on certain conditions: (1) the type of reagent used; (2) the microenvironment of rhodopsin; and (3) whether or not the preparation is illuminated. This experimental versatility provides information on how the sulfhydryl chemistry of rhodopsin reflects its structural and functional integrity and allows important, though still tentative conclusions on the role of SH groups in structure and function of rhodopsin. It is evident that sulfhydryl modification studies on rhodopsin present a number of intriguing observations, which are, however, difficult to interpret unambiguously.

The role of SH-groups in the concentrative transport of D-glucose into brush border membrane vesicles

The role of SH-groups in the concentrative transport of D-glucose into brush border membrane vesicles

Platelet Aggregation by Thimerosal

Thimerosal, a sulphydryl inhibitor, induces aggregation of normal platelet rich plasma over a wide range of concentrations. Low doses induce a monophasic response preceded by a lag phase, high doses produce an immediate biphasic response. Thimerosal induces platelet aggregation through its binding by sulphydryl groups. Thimerosal induced aggregation is not mediated by ADP, it is not influenced by fibrinogen, von Willebrand factor, calcium, and magnesium ions of the medium. Thimerosal induced platelet aggregation is normal in patients affected by thrombocytopathia (defect of ADP release) but not in patients affected by Glanzmann's thrombasthenia. Mercaptopropionglycine, a substance which tends to preserve SH groups, inhibits platelet aggregation induced by thimerosal, thrombin, collagen, and ADP. A mechanism is proposed for thimerosal induced aggregation and the role of SH groups also in ADP, thrombin and collagen induced aggregation is indicated.

Kinetics of opiate receptor inactivation by sulfhydryl reagents: evidence for conformational change in presence of sodium ions.

The role of SH groups in opiate-receptor interactions has been further examined. In activation by N-ethylmaleimide of sterospecific opiate binding by rat brain membrane fractions follows pseudo-first order kinetics and exhibits strong temperature dependence. The kinetics indicate that alkylation of a single SH group suffices to block opiate binding. Considerable protection from SH group inactivation is observed when treatment with N-ethylmaleimide is carried out in the presence of an opiate or an antagonist, suggesting close proximity of the SH group to the opiate binding site. The rate of inactivation of receptor binding by N-ethylmaleimide is markedly slower in buffers containing 100 mM NaCl (t1/2 equals 30 plus or minus 1.4 min) than in sodium-free buffers (t1/2 equals 10 plus or minus 1.0 min). Since the rate of alkylation of model SH compounds is unaffected by sodium ions, this protection seems best explained by a conformational change in the receptors that renders the SH groups less accessible to alkylation. The rate of inactivation is not affected by K+, Rb+, or Cs+ and only slightly by Li+. This cation specificity as well as the concentration-response to Na+ are remarkably similar to those previously shown to lead to increased antagonist and decreased agonist binding. We suggest that the same conformational change is involved in the two phenomena.

Iodoacetic-acid-resistant alcohol dehydrogenase of an Escherichia coli mutant.

Nitrosoguanidine‐induced mutants of Escherichia coli were tested in vivo (tetrazolium staining assay) for iodoacetic acid resistance of their alcohol dehydrogenase. One mutant was isolated (mutant 2773) which has a fully iodoacetic‐acid‐resistant alcohol dehydrogenase. Mutant 2773 contains two species of alcohol dehydrogenase; one (the enzyme of the mutant strain, wild‐type fraction i.e. wild‐type‐mutant enzyme) has the same characteristics as the enzyme of the parental strain (wild‐type enzyme), including the inhibition by iodoacetic acid; the other species (enzyme of the mutant strain, mutant fraction i.e. the mutant enzyme) however, is iodoacetic acid resistant, has a high molecular weight (&gt; 600000) and is not related to the wild‐type enzyme as detected by immunological methods.The role of SH‐groups taking part in the reaction at the active center of the alcohol dehydrogenase and the occurrence of several enzyme species are discussed.