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D Cavallini

Publications and source records attributed to D Cavallini.

At least 55 records · Page 3Linked to original sources
PubMed1988

Purification and characterization of a ketimine-reducing enzyme.

An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500-fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping-pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto-open forms. As previously described, ketimines arise from the deamination of a number of sulfur-containing amino acids, i.e. L-cystathionine, L-lanthionine and S-aminoethyl-L-cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4-thiomorpholine 3,5-dicarboxylic acid and 1,4-thiomorpholine 3-carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible role of this enzyme in their biosynthesis.

Amino Acids, Sulfur↗
PubMed1988

Detection of cystathionine and lanthionine ketimines in human urine.

A recently developed HPLC procedure for the determination of cystathionine ketimine (CK) and lanthionine ketimine (LK) has been applied to the detection of these compounds in human urine. The assay has taken advantage of the selective production of an absorbance at 380 nm, not seen with other amino acids, when the two ketimines are reacted with phenylisothiocyanate. Coelution with authentic phenylthiohydantoin derivatives of CK and LK and the identical absorption spectra establish the identity of the compounds found in the urine with the synthetic products. Quantitation of the two ketimines by HPLC indicates that the excretion of CK and LK is respectively 606 micrograms and 84 micrograms per g of creatinine as mean values of 10 healthy subjects of both sexes, 20-40 years old, in the early morning voided urine.

Adult↗
PubMed1987

Hexahydro-1,4-thiazepine-3,5-dicarboxylic acid and thiomorpholine-3,5-dicarboxylic acid are present in normal human urine.

Hexahydro-1,4-thiazepine-3,5-dicarboxylic acid and thiomorpholine-3,5-dicarboxylic acid, simply referred to as cyclothionine and TMDA, respectively, are two cyclic sulfur-containing imino acids detected in bovine brain. Human urine has been investigated to establish the occurrence of these imino acids as common constituents under normal conditions. The morning urine of healthy subjects has been analyzed for enrichment of these compounds by using an ion-exchange procedure. Gas/liquid chromatography of the final extracts revealed the presence of peaks coeluting with authentic cyclothionine and TMDA. The latter compound eluted very close to an unknown sulfur-containing compound. A resolved peak of TMDA has been obtained by high-performance liquid chromatography of the final extracts derivatized with phenylisothiocyanate. Selected ion monitoring with multiple-ion detection applied to the compounds separated by gas chromatography revealed the presence of the respective molecular ions and of the decarboxylated fragments, thus confirming the identification of cyclothionine and TMDA in human urine.

Adult↗
PubMed1987

Cysteamine oxygenase: possible involvement of superoxide ion in the catalytic mechanism.

The reaction catalyzed by cysteamine oxygenase on cysteamine in the presence of phenazine methosulphate as cofactor like compound is inhibited by nitroblue tetrazolium, a scavenger of superoxide ions. The reaction is not inhibited by superoxide dismutase and allyl alcohol and it is not activated by superoxide ions produced in solution. Nitroblue tetrazolium is reduced by cysteamine or mercaptoethanol and phenazine methosulphate. This reaction is completely inhibited by superoxide dismutase. In the presence of cysteamine oxygenase the reduction with mercaptoethanol is greatly enhanced and it is only partially inhibited by superoxide dismutase. According to these data a reaction mechanism is proposed in which superoxide ions and thiyl radicals are produced at the active site during catalysis.

1-Propanol↗
PubMed1986

The transamination of L-cystathionine, L-cystine and related compounds by a bovine kidney transaminase.

An enzyme which actively transaminates L-cystathionine, L-cystine, L-lanthionine and S-aminoethyl-L-cysteine has been purified from bovine kidney. The transaminase appears to be pure up to 90% and probably consists of two subunits of similar molecular mass of about 47 kDa. The enzymatic products arising from the transamination of L-cystathionine and related compounds spontaneously cyclize into ketiminic structures, which are the immediate precursors of unusual imino acids recovered in biological materials. The specificity towards other amino acid and oxo acid acceptors is similar to the specificity exhibited by rat kidney glutamine transaminase. This suggests that the sulfur amino acid transaminations that have been described could be performed by the bovine kidney glutamine transaminase.

Alanine↗
PubMed1986

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.

Amino Acids↗
PubMed1986

Interaction of pantetheinase with sulfhydryl reagents and disulfides.

The effect of many thiol reagents and disulfides on pantetheinase (E.C. 3.5.1.-; pantetheine hydrolase) was studied in the presence or absence of S-pantetheine-3-pyruvate as substrate. Iodoacetamide, iodoacetate, bromopyruvate and N-ethylmaleimide irreversibly inactivate the enzyme at very different rates. Inactivation constants, corrected for the different reactivity of halogeno derivatives with non-protein thiols, suggest the presence of an essential sulfhydryl group in the enzyme and a negatively charged environment near this group. p-Chloromercuribenzoate is the most effective inhibitor; 2-nitro-5-thiocyanobenzoate, o-iodosobenzoate and hydrogen peroxide give a biphasic inhibition pattern, indicating the existence of two sulfhydryl groups whose modification affects activity. Organic arsenicals decrease activity to about 50%. Neutral and positively charged disulfides are effective inhibitors. Substrate protects the enzyme from inactivation, except in the case of negatively charged disulfides, where the presence of substrate enhances the inhibitory effect. Titration with Ellman's reagent or 4,4'-dithiodipyridine under various experimental conditions demonstrated the existence of two sulfhydryls and three disulfides in the fully active enzyme. Pantetheinase may become inactive during purification with concomitant loss of one titrable sulfhydryl group.

Alkylating Agents↗
PubMed1986

The oxidation of cyclothionine by D-aspartate oxidase.

Cyclothionine was found to be a substrate for bovine kidney D-Aspartate oxidase. The substrate, prepared chemically as a mixture of the possible stereoisomers, exhibits an inhibition at elevated concentrations. Compounds structurally related to cyclothionine, like TMDA and alpha-alpha'-iminodipropionic acid, have also been assayed with the enzyme.

Amino Acid Oxidoreductases↗
PubMed1985

Gas-chromatographic mass-spectrometric detection of 1,4-hexahydrothiazepine-3,5-dicarboxylic acid (cyclothionine) in bovine brain.

Cystathionine has been reported to undergo enzymatic changes leading to the formation of seven membered cyclic products (Ricci, G., Santoro, L., Achilli, M., Matarese, R. M., Nardini, M., and Cavallini, D. (1983) J. Biol. Chem. 258, 10511-10517; Cavallini, D., Costa, M., Pensa, B., and Coccia, R. (1985) Biochem. Int. 10, 641-646). Gas-chromatographic and mass-spectrometric evidence reported in this paper indicates that the cyclic derivative of cystathionine, 1,4-hexahydrothiazepine-3,5-dicarboxylic acid, here simply named cyclothionine, is a normal component of bovine brain. This finding together with the detection of the same compound in the urine of cystathioninuric patients (Kodama, H., Sasaki, K., Mikasa, H., Cavallini, D., and Ricci, G. (1984) J. Chromatogr. 311, 183-188) supports the conclusion that cystathionine, apart from its role in trans-sulfuration, is converted also into cyclic compounds whose biochemical significance is as yet unknown.

Animals↗
PubMed1985

1,4-Thiomorpholine-3,5-dicarboxylic acid, a novel cyclic imino acid detected in bovine brain.

Gas-liquid chromatography of enriched bovine brain extract revealed the occurrence of several sulfur-containing compounds. By co-chromatography with authentic product and by mass-spectrometric analysis, one of these compounds has been identified as 1,4-thiomorpholine-3,5-dicarboxylic acid (TMDA). The possible derivation of TMDA from lanthionine is discussed. This represents the second S-containing cyclic amino acid so far discovered in a mammalian brain whose physiological significance has not yet been explored.

Animals↗
PubMed1985

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.

Amino Acids, Sulfur↗
PubMed1985

The conversion of L-cystathionine into the cyclic ketimine form by heated rat liver extracts containing cystathionase and transaminase activities.

Rat liver homogenates heated for 10 min at 60 degrees C incubated with L-cystathionine yield cystathionine ketimine which was identified by its typical UV spectrum and by cochromatography with authentic samples on the amino acid analyzer. Alanine and alpha-amino butyric acid have been also detected among the final products. The reaction is due to heat stable gamma-cystathionase and transaminases present in the extracts. Cystathionase produces alpha-keto butyric acid and pyruvic acid which are then used for the transamination of the remaining cystathionine to yield the ketimine. This is the first report indicating the occurrence in a mammalian tissue of an enzymatic system using cystathionine for reactions differing from the traditional transulfuration to cysteine.

Animals↗
PubMed1985

New enzymatic changes of L-cystathionine catalyzed by bovine tissue extracts.

L-Cystathionine was used as substrate for enzyme systems prepared by heating bovine tissue extracts in the presence of pyruvate at 60 degrees C for 10 min. Analysis of the products indicated that the systems converted L-cystathionine into the cyclic ketimine form which was detected by its spectral properties and by chromatography on the amino acid analyzer. Alanine, alpha-aminobutyrate and cystine were also produced. Pyruvate and alpha-ketobutyrate enhance the production of the ketimine by liver, kidney and heart extracts, and are necessary for the brain extracts: alpha-Ketoglutarate is much less effective and its presence favors the production of homocystine by all the extracts. Homocystine was found in the brain incubates when any of the ketoacids assayed were added. The overall reaction is explained by the action of heat stable cystathionine gamma-lyase and beta-synthase which produce alpha-ketobutyrate and pyruvate used for the transamination of the remaining cystathionine to the monoketoacid. This last compound cyclizes spontaneously into the ketimine form thus avoiding the removal of the second amino group. This represents a new nontransulfurative path leading to the production of a seven membered etherocyclic product whose biochemical implications are yet unexplored.

Amino Acids↗
PubMed1985

Enzymatic production of S-(2-hydroxy-2-carboxyethyl)homocysteine.

The monodeamination product of L-cystathionine, S-(2-oxo-2-carboxyethyl)homocysteine is enzymatically reduced by human and hog L-lactate dehydrogenase to S-(2 hydroxy-2-carboxyhethyl)homocysteine which has been found in the urine of cystathioninuric patients. Km values are 6.6 mM and 13 mM for human and hog enzymes respectively. The reaction is not complete at alkaline pH values because of cyclization of the ketoacid into a 1,4 thiazepine derivative ring. By a coupled enzymatic system of L-amino acid oxidase and L-lactate dehydrogenase S-(2-hydroxy-2-carboxyethyl)homocysteine is also formed from L-cystathionine.

Animals↗
PubMed1984

Continuous spectrophotometric assay of pantetheinase activity.

A continuous spectrophotometric assay for pantetheinase determination using S-pantetheine-3-pyruvate as substrate is described. The enzymatic hydrolysis of this new substrate leads to the formation of S-cysteamine-3-pyruvate, which cyclizes in a non-rate-limiting step to give 2H-1,4-thiazin-5,6-dihydro-3-carboxylic acid (aminoethylcysteine ketimine), a compound exhibiting a strong absorption at 296 nm. The assay is optimized with respect to pH, buffer, and substrate concentration. Prereduction of the enzyme and some properties of the reaction are also studied. The assay is simple, rapid, very sensitive, and specific.

Amidohydrolases↗