Thioglycolate, enzyme

Mercuric chloride, other mercury-containing antibacterials and silver will inhibit enzymes in the membrane, and for that matter in the cytoplasm, which contain thiol, -SH, groups. A similar achon is shown by 2-bromo-2-nitropropan-l,3-diol (bronopol) and iso-thiazolones. Under appropriate condihons the toxic action on cell thiol groups may be reversed by addition of an extrinsic thiol compound, for example cysteine or thioglycollic aeid (see also Chapters 12 and 23). 

It appears that this enzyme (MW, 120,000) consists of six subunits, each with MW of 20,000. Size homogeneity of the subunits was further confirmed by the presence of a single band in polyacrylamide gel electrophoresis in 8M urea containing0.1 M thioglycolate (30). 

The keratin structure is destroyed through the use of proteolytic enzymes such as pronase and proteinase K. They are often used with chemical agents such as urea and thioglycolic to cleave the disulfide bonds and increase the dissolution rate of enzyme activity. The extracting procedures, using enzymatic digestion, last about 4-6 h and must be conducted at constant temperature and pH for providing maximum enzyme activity [155],... 

Free" heta-amylase is obtained on direct, aqueous extraction of the cereal, but treatment with salt, or thioglycolic acid, liberates further enzyme, namely, the bound heta-amylase. Evidence regarding the difference, if any, between these enzymes is limited. 

Many inhibitors may be present in biological samples or in buffers currently used for EIA. Pi is a competitive inhibitor of the enzyme and forms an intermediate with the enzyme which is indistinguishable from the intermediate formed during catalysis of the hydrolysis of phosphate esters (Caswell and Caplow, 1980). The K, (i.e. the for Pi) is lower than the for the substrate, typically KijK, = 0.3, i.e. a lower concentration of Pi than of the substrate is required to half-saturate the enzyme. Arsenate is a stronger competitive inhibitor than Pi, whereas phosphonates are weaker. Metal chelating products (EDTA, cysteine, thioglycolic acid) are also important inhibitors. Many amino acids show a mixed competitive or uncompetitive inhibition (Fernley, 1971). 

Numerous compounds which reduce Fe(III)—e.g., ascorbate, hy-droquinone, catechol, hydroxylamine, thioglycolate, cysteine, ferrocya-nide, and DOPA, whose oxidation was completely inhibited by iron chelators. Thus, they appear not to be directly oxidized by the enzyme, but function in an Fe-Cp coupled reaction and are iron-dependent substrates. 

McDermott et al. (163) have proposed that there are two groups of substrates for ceruloplasmin those substances for which non-chelated Fe is not required and therefore which interact directly with the enzyme and those substrates which require the presence of non-chelated Fe to be catal3dically oxidized. Some substrates belonging to the former group are epinephrine, PPD, norepinephrine, dopamine, and serotonin. Those falling in the second class are ascorbate, hydro-quinone, ferrocyanide, L-DOPA, hydroxylamine, thioglycolic, acid and cysteine. 

Specificity—Crook (41) reported that cysteine and thiolacetic acid could be substituted for GSH. The enzymatic reaction rate Avith cysteine Avas about half as great as the reaction rate AA ith GSH. With thiolacetic acid, the enzymatic reaction AA as only a little faster than the reaction in the absence of enzyme. Bukin (19), on the other hand, reported that sulfhydryl compounds such as thiolactic and thioglycollic acid could not substitute for GSH. Yamaguchi and Joslyn (51) were unable to measure an enzyme reaction AA ith cysteine and thioglycolate becau.se of the high blank reaction rate. They reported that dehydro-D-araboascorbic (dehydro-D-isoascorbic)... 

The thiol transacetylase that catalyzes this reaction has been partially purified from extracts of E. coli by Hager and Gunsalus (personal communication), and was shown to be relatively specific. We have recently discovered enzymes in extracts of C. kluyveri (47) and in pigeon-liver extracts (48) that catalyze similar acetyl transfers from acetyl-SCoA to 2-mercap-toethanol, thioglycollate, reduced lipoic acid, and hydrogen sulfide however, GSH and cysteine are relatively inert as acetyl acceptors. It remains to be demonstrated if one or more enzymes are involved in these acyl-transfer reactions. The enzyme-catalyzed reactions are to be differentiated from the nonenzymatic reactions in that they occur readily in more dilute solutions and also at neutral to slightly acid pH. 

The enzyme is specific for thiol esters of the type of S-acetyl and S-butyryl glutathione. Besides the three thiol esters already discussed, ethyl thiol-acetate, butyl thiolacetate, acetyl thioglycolic acid, acetyl mercapto propionic acid, S-acetyl-2 mercaptoethanol, acetyl thiomalic, acetyl coenzyme A, and butjryl coenzyme A were inactive with the purified enzyme. Crude extracts of liver attacked butyl thiolacetate and acetyl coenzyme A slowly. 

The dehydrogenase is indicated to be a sulfhydryl enzyme based on its inhibition by p-chloromercuribenzoate and the stimulation of purified yeast fractions of the enzyme with thioglycolate. 

Cystathionine /I-synthetase has a broad specificity with respect to the sulfur compound to be added to the serine moiety and also with respect to serine. Serine can be replaced by 0-acetylse-rine, cyanoalanine, chloroalanine and even by cysteine, while cysteamine, mercaptoethanol, thioglycolic acid, sulfide, cysteine and other thiol compounds could replace homocysteine (38, 39). This -replacement is so fast that it has been used to prepare a number of cysteine—S-derivatives by simply passing the appropriate reagents through a column of a bacterial synthetase bound to a rigid support (40). The table reported in the next page lists some of the substrates used by this enzyme. 

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