Bernardo Pensa

Hydrogen peroxide involvement in the rhodanese inactivation by dithiothreitol.

Abstract The conditions required to obtain rhodanese inactivation in the presence of dithiothreitol indicate the involvement of hydrogen peroxide produced by metal-ion catalyzed oxidation of dithiothreitol. Inhibition of dithiothreitol oxidation by a chelating agent, or by removal of hydrogen peroxide by catalase prevents the enzyme inactivation. The inactivated enzyme contains a disulfide bond resulting from the oxidation of the catalytic sulfhydryl group and another sulfhydryl group close to it. This disulfide might be formed via a sulfenic intermediate.

Transamination of L-cystathionine and related compounds by a bovine liver enzyme. Possible identification with glutamine transaminase

A transaminase which catalyses the monodeamination of L-cystathionine was purified 1100-fold with a yield of 15% from bovine liver. The monoketoderivative of cystathionine spontaneously produces the cyclic ketimine. Other sulfur-containing amino acids related to cystathionine such as cystine, lanthionine and aminoethylcysteine were also substrates for the enzyme. The relative molecular mass of the enzyme was determined to be 94 000 with a probable dimeric structure formed of identical subunits. The isoelectric point of the enzyme was at pH 5.0 and the maximal enzymatic activity was found at pH 9.0--9.2. Kinetic parameters for cystathionine and for the other sulfur amino acids as well as for some alpha-keto acids were also determined. Among the natural amino acids tested, glutamine, methionine and histidine were the best amino donors. The enzyme exhibited maximal activity toward phenylpyruvate and alpha-keto-gamma-methiolbutyrate as amino acceptors. The broad specificity of the enzyme leads us to infer that the cystathionine transaminase is very similar or identical to glutamine transaminase.

Transamination of l-cystathionine and related compounds by bovine brain glutamine transaminase

Glutamine transaminase (EC 2.6.1.15) has been purified 113 fold from bovine brain. The product is free of aspariate amino transferase (EC 2.6.1.1.) and other common transaminases. The enzyme shows a wide specificity similar to that reported from the same transaminase purified from bovine kidney and liver as regards both the amino donor and the amino acceptor. Of interest is the transamination and cyclization of l-cystathionine, l-lanthionine, l-cystine and S-aminoethylcysteine. The latter result indicates that the deamination and the cyclization of the sulfur containing diamino acids described for bovine liver and kidney enzyme is feasible also in the brain and suggests the possible endogenous origin of cyclothionine and thiomorpholine dicarboxylate recently detected in bovine brain.

Reaction of rhodanese with dithiothreitol

The reaction between bovine rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) and reduced dithiothreitol has been studied. This reagent, in the absence of thiosulfate, reduces the amount of sulfur carried by rhodanese with formation of sulfide and oxidized dithiothreitol: E-S-SH + reduced dithiothreitol replaced by E-SH + HS- + oxidized dithiothreitol, (E = enzyme). An inactivation was observed at high dithiothreitol/enzyme ratios or at very low enzyme concentrations. The inactivation was not observed in the presence of thiosulfate and can be reversed by cyanide or thiosulfate. A thiosulfate reduction activity of rhodanese was also found using dithiothreitol as reductant.

The titration of rhodanese with substrates

The transfer of sulfur catalyzed by rhodanese (EC 2.8.1 .l .) is thought to proceed through a double displacement mechanism where the enzyme is first charged with the donor sulfur and then discharged by the acceptor [ 1 ] . Earlier workers have reported that the enzyme accepts from 1 to 1.9 atoms of sulfur per mole of enzyme of mol. wt. of 37 000 (2-4). The large difference being mainly due to the procedure adopted for the purification of the enzyme and to the method used for the determination of transferable sulfur. As summarized in a recent review [S] the form of sulfur bound to rhodanese has been debated for a long time without receiving a satisfactory answer. In recent investigations carried out in our laboratory [6] it has been found that rhodanese, crystallized from beef kidney, exhibits a definite absorbancy in the form of a shoulder in the area of 335 nm which disappears on the addition of cyanide. This absorbancy, which has been ascribed to the presence of a persulfide group (R-SSH) in the active site of rhodanese, provides a new technical means for establishing the chemical form of transferable sulfur and for studying the catalytic mechanism of this enzyme. The present note describes the titration of rhodanese with cyanide and with thiosulfate, using the absorbancy at 335 nm as a guide, aimed at establishing the correlation between this absorbancy and the amount of sulfur loosely bound to the enzyme.

S- aminoethyl- l -cysteine transaminase from bovine brain: purification to homogeneity and assay of activity in different regions of the brain

A transminase acting on cystathionine, S-aminoethylcysteine and glutamine has been purified to homogeneity from bovine brain by ammonium sulfate precipitation. DE-52 chromatography, octyl-Sepharose chromatography, hydroxylapatite chromatography and gel filtration. The enzyme was purified 4700 times over the bovine brain homogenate and the overall recovery of the enzyme activity was about 18%. As demonstrated by polyacrylamide gel electrophoresis under native or denaturing conditions, the enzyme has a molecular mass of 100 kDa and is composed of two subunits with approximately identical weight. A single active peak was obtained at pH = 5.24 by chromatofocusing of a homogeneous enzyme preparation. K(m) values for S-aminoethylcysteine have been calculated using various ?-keto acids as amino acceptor and K(m) for glutamine has been determined with ?-keto-?-methiolbutyric acid as cosubstrate. The occurrence of the enzyme activity in some bovine brain regions was also studied.

Cyanylation of rhodanese by 2-nitro-5-thiocyanobenzoic acid

Cyanylation of rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) with a nearly stoichiometric amount of 2-nitro-5-thiocyanobenzoic acid produces a modification of the essential sulfhydryl group. Different S-cyano derivatives are obtained with the enzyme intermediate bearing transferable sulfur bound as a persulfide group (sulfur-rhodanese: E-S-SH) and with the sulfur-free rhodanese (E-SH). The interaction of a neighboring sulfhydryl group splits thiocyanate from the sulfur-rhodanese derivative and cyanide from the sulfur-free rhodanese derivative. In both cases an intramolecular disulfide bond is formed. Iodoacetate is effective on the modified enzyme only after cyanide addition which splits the disulfide so that the essential sulfhydryl group can be alkylated.

Preparation of 3-Mercaptolacttc Acid and S-Aminoethylmercaptolactic Acid

A simple and accurate method is described for synthesis of 3-mercaptolactic acid and its derivative, S-aminoethylmercaptolactic acid. Some spectrometric data of compounds are reported as well as their melting points, some colorimetric reactions and thin layer chromatographic behaviour. S-Aminoethylmercaptolactic acid is also determined by amino acid analyzer.

Determination of rhodanese activity using a pH-STAT apparatus

Abstract A new method for the assay of the rhodanese activity is described. The method uses a pH-STAT apparatus to monitor the rates of enzyme catalyzed reaction under controlled conditions. A standard kinetic analysis of the bovine kidney enzyme, including pH and temperature dependence, gave results which are in agreement with those obtained by the classic spectrophotometric method.

Determination of 3-mercaptolactic acid by amino acid analyzer after aminoethylation☆

A quantitative determination of 3-mercaptolactic acid was performed after its conversion into S-aminoethylmercaptolactic acid by reacting with excess of 2-bromoethylamine. S-aminoethylmercaptolactic acid was quantitated by an amino acid analyzer. Other thiols were shown not to interfere with the determination of 3-mercaptolactic acid. The sensitivity of the method was at the nanomoles level. The application of the method to the determination of 3-mercaptolactic acid in human urine is also reported.