Interaction of lactoperoxidase with thiols and diiodotyrosine.

Glutathione and cysteine bind to the heme of lactoperoxidase, thereby causing a red shift of the Soret band which is reversed upon addition of iodide or guaiacol, two substrates for lactoperoxidase. The rate of formation of the enzyme-thiol complex is enhanced by diiodotyrosine. Binding of diiodotyrosine to lactoperoxidase does not cause a shift of the Soret band which indicates binding to the protein of the enzyme. At neutral pH and low ionic strength, lactoperoxidase is adsorbed on insolubilized diiodotyrosine (diiodotyrosine-agarose). It can be eluted at slightly increased ionic strength which shows that the binding is weak. In the presence of 5 X 10(-4) M glutathione, however, the binding of the enzyme to diiodotyrosine-agarose becomes much stronger so that a high salt concentration is required for elution. Lactoperoxidase is also adsorbed on insolubilized thiols (thiol-agarose). The presence of diiodotyrosine is not required for strong binding. A simple method for the preparation of lactoperoxidase from milk by affinity chromatography is based on the interactions of the enzyme with the two ligands, thiols and diiodotyrosine.     

Effect of thiols on macroconidia of Fusarium sulphureum.

Treatment of Fusarium sulphureum macroconidial cells with five thiols alters their morphology. Macroconidial cells incubated in dithiothreitol (DTT), dithioerythritol (DTE), or thiourea differentiate into thick-walled, chlamydospore-like cells (thiol-induced spores). These cells appear similar in size and shape to chlamydospores in the light microscope, but differ markedly in cell wall structure when viewed in the electron microscope (EM). Incubation of macroconidia with both DTT and DTE also leads to the formation of large swollen cells (giant cells) which have a parietal cytoplasm and electron-tranparent cell walls; most of these giant cells lyse within 3 to 7 days of incubation. Thiourea-induced spores are characterized by the deposition of a thick, electron-dense, extracellular layer and an accumulation of mitochondria. DTT and DTE, at the concentrations used, inhibit macroconidial germination while thiourea, mercaptoethanol, and cysteine do not. With the latter three thiols, the newly formed hyphal cells become elongated with either one or both ends swollen. Mercaptoethanol-treated cells contain an abundance of mitochondria. The DTT-induced spore differs from both macroconidia and chlamydospores with respect to cellular lipid and cell wall composition. While the thiols have different effects on the macroconidia, the fact that they all induce cell expansion suggests that they react at some common sites.     

Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis.

A simple and specific method for analyzing thiols and disulfides on the basis of the reversibility of N-ethylmaleimide (NEM) alkylation of thiols is described. When the adduct of NEM and glutathione (GSH) was electrolyzed at neutral pH, all of the GSH was recovered. When the adduct was exposed to pH 11.0 for 15 min at 30 degrees C before electrolysis, GSH was not detected. The same behavior was observed after protein thiols reacted with NEM. This pH-dependent production of thiol from the adduct was used to assay GSH and oxidized glutathione in yeast cells, to assay sulfhydryl groups and disulfide bonds in authentic proteins, and to protect thiols from oxidation during enzymatic digestion of protein. This method is useful for assay of thiols and disulfides of both small and large molecules and can be used to identify labile thiols in biological samples that are oxidized during extraction procedures.     

Mechanism for the interaction of thiols with methylcobalamin.

The reaction between methylcobalamin and ethane-thiol sulfonic acid (Co-enzyme M) has been studied under aerobic conditions. For this reaction evidence is presented for a catalytic cycle which promotes homolytic cleavage of the Cobalt-carbon sigma-bond to give Cob(II)alamin (B12-r) and methylcoenzyme M as the products. This reaction is especially pertinent to our understanding of the mechanism of methane-biosynthesis. In addition, we have used 220 MHZ 1H NMR and 13C NMR to show that thiols do not react with methylcorrinoids by displacing the base trans-axial to the cobalt-carbon bond. This NMR study is especially important since the co-ordination of thiols to cobalt has previously been reported to occur by a number of research groups including our own.     

Binding of thiols to microsomal cytochrome P-450.

Lipophilic thiol compounds interact spectrally with liver microsomes from phenobarbital-pretreated rats by formation of unusual optical difference spectra with peaks at 378, 471, 522 and 593 nm in the oxidized state. The binding kinetics were biphasic. The EPR spectrum of cytochrome P-450 was slightly modified but the magnitude of the low-spin signal was unchanged. n-Octanethiol competitively displaced metyrapone and n-octane from the active site of cytochrome P-450. Other thiols behaved similarly with variations in the magnitude and the affinity of the binding process. Tertiary thiols caused the formation of the high-spin cytochrome P-450 substrate complex, and model studies with myoglobin revealed that steric hindrance prevented the liganding of the tertiary thiol group to the ferric cytochrome P-450. Addition of thiols to dithionite reduced microsomes resulted in relatively small spectral changes with maxima at 449 nm typical for ligand complexes of the ferrous cytochrome. It was concluded that lipophilic thiols can be bound as ligands by at least two species of oxidized cytochrome P-450 which represent, however, not more than about one fifth of the total cytochrome P-450 content in liver microsomes from phenobarbital-pretreated rats.     

The reaction of carbonyl cyanide phenylhydrazones with thiols.

Carbonyl cyanide phenylhydrazone and its ring-substituted analogs react with thiols (thioglycolic acid, 2-mercaptoethanol, dithiothreitol) and aminothiols (cysteine, glutathione) to give corresponding N-(substituted phenyl)-N'-(alkylthiodicyano)-methylhydrazine derivatives. These addition products decompose to the original components in alkaline solution. On the other hand, in the presence of an excess of thiols in aqueous buffered systems the addition reactions are practically quantitative with respect to phenylhydrazones, follow pseudo-first-order kinetics and can be investigated spectrophotometrically. These reactions are of the bimolecular AdN type where the non-dissociated form of carbonyl cyanide phenylhydrazones function as an electrophilic component, while the RS- ion plays the role of nucleophilic component in the case of thiols (the attack of the azomethine group). The reactivitiy of carbonyl cyanide phenylhydrazones with respect to thiols increases in the order carbonyl cyanide phenylhydrazone less than carbonyl cyanide m-chlorophenylhyrazone less than carbonyl cyanide p-trifluoromethoxyphenylhydrazone which corresponds to the order of decreasing values of the pKa constants. On the other hand, the reactivity of thiols increases with their basicity. The reactivity of carbonyl cyanide phenylhydrazone with thiols is comparable with the reactivity of phenyl isothiocyanate and N-ethylmaleimide. It was demonstrated that carbonyl cyanide phenylhydrazone is an efficient inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). The results obtained are discussed in relation to the biological activity of carbonyl cyanide phenylhydrazones.     

On the modification of adenosine kinase by thiols.

The catalytic activity of adenosine kinase (EC 2.7.1.20) from yeast is very labile. Even incubation with thiols provokes a loss of two thirds of its enzymatic activity. Concomitantly, two SH-groups appear on the enzyme in addition to the single SH-group already present in the untreated enzyme, the latter being absolutely essential for activity. Treatment of adenosine kinase with thiols does not substantially affect the binding of the substrates adenosine and ATP-Mg2. The reactivity of the two newly formed SH-groups is diminished in the presence of ATP-Mg2, whereas adenosine has no influence. The opposite holds for the reactivity of the single SH-group essential for enzymatic activity. Complete reactivation of the enzymatic activity after incubation of adenosine kinase with thiols can be achieved by reoxidation of the enzyme in presence of high concentrations of adenosine. These observations suggest the notion that adenosine kinase contains an essential SH-group close to the adenosine-binding site and a disulfide bridge near to the binding site of ATP-Mg2, the latter being easily accesible to the reduction by thiols.     

Myeloperoxidase oxidation states involved in myeloperoxidase-oxidase oxidation of thiols.

The human red-blood-cell glyoxalase system was modified by incubation with high concentrations of glucose in vitro. Red-blood-cell suspensions (50%, v/v) were incubated with 5 mM- and 25 mM-glucose to model normal and hyperglycaemic glucose metabolism. There was an increase in the flux of methylglyoxal metabolized to D-lactic acid via the glyoxalase pathway with high glucose concentration. The increase was approximately proportional to initial glucose concentration over the range studied (5-100 mM). The activities of glyoxalase I and glyoxalase II were not significantly changed, but the concentrations of the glyoxalase substrates, methylglyoxal and S-D-lactoylglutathione, and the percentage of glucotriose metabolized via the glyoxalase pathway, were significantly increased. The increase in the flux of intermediates metabolized via the glyoxalase pathway during periodic hyperglycaemia may be a biochemical factor involved in the development of chronic clinical complications associated with diabetes mellitus.     

Role of thiols in degradation of proteins by cathepsins.

The effects of thiols on the breakdown of 125I-labelled insulin, albumin and formaldehyde-treated albumin by highly purified rat liver cathepsins B, D, H and L at pH 4.0 and 5.5 were studied. At both pH values degradation was strongly activated by the thiols cysteamine, cysteine, dithiothreitol, glutathione and 2-mercaptoethanol, and its rate increased with increasing thiol concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect the rate of degradation by cathepsin D or L, and determination of free thiol groups after incubation of the proteins in the presence of glutathione but without cathepsin showed that their disulphide bonds were stable under the incubation conditions. Sephadex G-75 chromatography of the acid-soluble products of insulin digestion by cathepsin D or L suggested that thiols can reduce disulphide bonds in proteins after limited proteolysis. The resultant opening-up of the protein structure would lead to further proteolysis, so that the two processes (proteolysis and reduction) may act synergistically. By using the osmotic protection method it was shown that, at a physiological pH, cysteamine, and its oxidized form cystamine, can cross the lysosome membrane and thus may well be the physiological hydrogen donor for the reduction of disulphides in lysosomes. The results are discussed in relation to the lysosomal storage disease cystinosis.     

The reaction between thiols and 8-azidoadenosine derivatives.

Thiols react at room temperature in dilute solution with 8-azidoadenosine and its nucleotides to give the corresponding 8-aminoadenosine derivatives. The reaction which takes place in the dark is base-catalysed and is particularly rapid when dithiols, e.g. dithiothreitol are used.     

Protein thiols in spermatozoa and epididymal fluid of rats.

Thiol (SH) oxidation to disulphides (SS) is thought to be involved in sperm chromatin condensation and tail structure stabilization, which occur during maturation of spermatozoa. Previously developed procedures, using the fluorescent labelling agent monobromobimane (mBBr), enabled us to study the thiol-disulphide status of spermatozoa. Electrophoretic separation of labelled sperm proteins from the caput and cauda regions showed that during maturation thiol oxidation occurs in many protein fractions from the tail and that the magnitude of oxidation differs between proteins. Among the protein bands, one major band (MPB), probably a dense fibre constituent, is quantitatively prominent. N-Ethylmaleimide (NEM) or mBBr alkylation (of intact spermatozoa) changes the mobility of the caput MPB, but not that of the cauda MPB. The results indicated that the altered mobility of MPB is mainly due to a change in its shape, possibly resulting from the alkylation of a few critical SH groups. Epididymal fluid proteins contain both SH and SS. The thiol and disulphide content of the various epididymal proteins appears similar, although some diminution in fluorescence is seen in epididymal fluid proteins from the cauda region as compared with those from the caput region. The prominent changes in thiol status occur in the spermatozoa.     

Free thiols of platelet thrombospondin. Evidence for disulfide isomerization

The free thiols of platelet thrombospondin (TSP) were derivatized with labeled N-ethylmaleimide (NEM) or iodoacetamide (IAM). When Ca2+ was chelated with EDTA, 2.9 mol of NEM or 2.6 mol of IAM reacted/mol of native TSP. No additional thiols were found after denaturation with urea. Since TSP has three apparently identical polypeptide chains, this suggests one free thiol/polypeptide chain. Ca2+ protected all of the thiols from reaction with IAM. In Ca2+ about half the thiols reacted normally with NEM and the others were unreactive, indicating that the thiols of TSP are not identical. The number of reactive thiols as a function of [Ca2+] revealed a sigmoidal curve with a transition midpoint of 207 microM. The ability of analogs of NEM to compete for derivatization of the thiols with labeled NEM was greater with larger, more hydrophobic agents. Gel electrophoretic separation of labeled TSP that had been partially digested with thrombin and trypsin indicated that some of the label was in the C-terminal tryptic fragment but that most was in the adjacent trypsin-sensitive region. After cyanogen bromide cleavage of the labeled and reduced protein, four labeled fractions were obtained from a gel filtration column. With subsequent combinations of tryptic digestion and reversed-phase high performance liquid chromatography, labeled peptides were purified from these four fractions, and the amino acid sequences were determined. Twelve labeled cysteines were identified, each with a specific radioactivity less than that of the thiol labeling reagent, indicating that only a fraction of that cysteine in a population of TSP molecules was a free thiol at the time of derivatization. While 2 labeled cysteines are in the non-repeating C-terminal portion of the molecule, the other 10 labeled cysteines are in the adjacent trypsin-sensitive type 3 repeats proposed (Lawler, J., and Hynes, R. O. (1986) J. Cell. Biol. 103, 1635-1648) as the calcium-binding region of the molecule. The disulfide bonds most sensitive to reduction by dithioerythritol were also stabilized by Ca2+, implying location in the Ca2(+)-sensitive part of the molecule. It is proposed that one equivalent of free thiol/polypeptide chain is distributed among 12 different cysteine residues through an intramolecular thioldisulfide isomerization.