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1.
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.  相似文献   

2.
L-Cystathionine is oxidized by snake venom L-amino acid oxidase at a rate about half that with L-leucine at pH 8.5. The appearance of an absorbance at 296 nm and quantitation of the products of oxidation in the presence of catalase indicate formation in the solutions of a seven-membered ketimine ring produced by cyclization of the monoamino monoketo derivative of cystathionine. A limited double deamination has also been observed. In the absence of catalase, S-(carboxymethyl)homocysteine and S-(beta-carboxyethyl)cysteine have been identified together with ninhydrin-unreactive compounds yielding the above mentioned carboxy compounds upon hydrolysis with HCl. Authentic samples of the monoamino monoketo analogs of cystathionine have been prepared and compared with the enzymatic products. Cyclization of the synthetic products into the ketimine ring is pH-dependent as established by UV spectrum and other assays. Compounds derived from either the oxidation or the reduction of the ketimine have been prepared. It was found that many products of enzymatic and chemical changes of cystathionine and its ketimine described in the present paper are identical with those identified in the urine of cystathioninuric patients. This result indicates the occurrence in humans of secondary metabolic routes of cystathionine centered on the production of cystathionine ketimine, in equilibrium with the open form, which in cystathioninurics is revealed by the lack of cystathionase.  相似文献   

3.
Zygosaccharomyces rouxii is an important yeast in the formation of flavor in soy sauce. In this study, we investigated the separate effects of exogenous threonine, cystathionine, and the branched-chain amino acids on the metabolism of Z. rouxii. The addition of these amino acids had significant effects on both Z. rouxii growth and glycerol and higher alcohol production. It also seemed that Z. rouxii displayed the Crabtree effect, which was independent of the added amino acids. Furthermore, we investigated the regulation of the metabolism of alpha-ketobutyrate, which is a key-intermediate in Z. rouxii amino acid metabolism. Threonine and cystathionine were introduced separately to stimulate the formation rate of alpha-ketobutyrate and the branched-chain amino acids to inhibit its conversion rate. Enzyme activities showed that these amino acids had a significant effect on the formation and conversion rate of alpha-ketobutyrate but that the alpha-ketobutyrate pool size in Z. rouxii was in balance all the time. The latter was confirmed by the absence of alpha-ketobutyrate accumulation.  相似文献   

4.
2H-1,4-Thiazine-5,6-dihydro-3,5-dicarboxylic acid (trivial name: lanthionine ketimine) is a cyclic sulfur-containing imino acid detected in bovine brain extracts. This compound has been synthesized in a heavily labeled form starting from L-[35S]cysteine and purified by high performance liquid chromatography. We demonstrate the existence of a saturable and reversible binding of [35S]lanthionine ketimine to bovine brain membranes. A single population of binding sites with a concentration of 260 +/- 12 fmol/mg protein and a dissociation constant of 58 +/- 14 nM is present. Specific binding is competitively inhibited by other structurally similar imino acids, namely S-aminoethyl-L-cysteine ketimine and cystathionine ketimine. These results suggest a possible functional role for these ketimines in nervous system.  相似文献   

5.
Metabolism of beta-methylaspartate by a pseudomonad   总被引:1,自引:1,他引:0       下载免费PDF全文
A bacterium was isolated from soil which utilizes threo-beta-methyl-l-aspartate, certain other amino acids, and a variety of organic substances as single energy sources. It is, or closely resembles, Pseudomonas putida biotype B. The ability of this organism to rapidly decompose such amino acids is dependent on inducible enzyme systems. Dialyzed cell-free extracts of this bacterium metabolize beta-methylaspartate only when catalytic amounts of alpha-ketoglutarate, or pyruvate, and pyridoxal phosphate are also present. The main products formed from beta-methylaspartate under these conditions are alpha-aminobutyrate, carbon dioxide, and alpha-ketobutyrate. When l-aspartate is substituted for beta-methylaspartate in this system, it is converted mainly to alanine and carbon dioxide. beta-Methyloxalacetate is decarboxylated, and the resulting alpha-ketobutyrate is converted enzymatically in the presence of glutamate to alpha-aminobutyrate which accumulates. The added keto acids are converted, in part, to the corresponding amino acids probably by transamination. The data indicate that beta-methylaspartate is converted to alpha-aminobutyrate, and aspartate to alanine, by a circuitous transamination-beta-decarboxylation-transamination sequence rather than by a direct beta-decarboxylation.  相似文献   

6.
The oxidative decarboxylation and subsequent production of glucose from alpha-ketobutyrate were studied using perfused livers from fasted rats. The production of 14CO2 from alpha-keto-[1-14C]butyrate increased monotonically while the production of glucose from alpha-ketobutyrate was biphasic as the perfusate concentration of alpha-ketobutyrate was increased. The biphasic gluconeogenic response using alpha-ketobutyrate as the gluconeogenic precursor was similar to that observed with propionate. The decarboxylation of alpha-ketobutyrate was found to be exquisitely sensitive to the effects of the monocarboxylate transport inhibitor, alpha-cyanocinnamate. Infusion of beta-hydroxybutyrate caused a substantial inhibition of alpha-ketobutyrate decarboxylation while dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, did not stimulate the metabolism of alpha-ketobutyrate but was inhibitory. The effects of alpha-ketobutyrate infusion on pyruvate decarboxylation were tested and it was found that at low perfusate pyruvate concentrations (ca. 0.25 mM) increasing alpha-ketobutyrate led to increasing inhibition of pyruvate decarboxylation, while at high perfusate pyruvate concentrations (ca. 2.5 mM) an initial inhibition was apparent which did not increase substantially with increasing alpha-ketobutyrate concentrations. The results obtained indicate that the regulation of alpha-ketobutyrate metabolism by oxidative decarboxylation differs significantly from that of pyruvate. In addition, while the rate of gluconeogenesis using alpha-ketobutyrate as a precursor was remarkably similar to that using propionate as a gluconeogenic precursor, the effects of alpha-ketobutyrate on the oxidative decarboxylation of pyruvate were qualitatively different from the effects of propionate on pyruvate metabolism.  相似文献   

7.
Aminoethylcysteine, lanthionine, cystathionine and cystine are mono-deaminated either by L-amino-acid oxidase or by a transaminase exhibiting the properties described for glutamine transaminase. The deaminated products cyclize producing the respective ketimines. Authentic samples of each ketimine were prepared by reacting the appropriate aminothiol compound with bromopyruvate, except cystine ketimine which required the interaction of thiopyruvate with cystine sulfoxide. Reduction of the first three mentioned ketimines with NaBH4 yields the respective derivatives with the saturated rings of thiomorpholine and hexahydrothiazepine. The same reduction is carried out enzymically by a reductase extracted from mammalian tissues. Properties of the members of this family of compounds are described. Gas chromatography followed by mass spectrometry permits the identification of most of these products. HPLC is very useful for the determination of the ketimines by taking advantage of specific absorbance at 380 nm obtained by prior derivatization with phenylisothiocyanate. Adaptation of these and other analytical procedures to biological samples disclosed the presence of most of these compounds in bovine brain and in human urine. By using [35S]lanthionine ketimine as a representative member of the ketimine group, the specific, high-affinity, saturable and reversible binding to bovine brain membranes has been demonstrated. The binding is removed by aminoethylcysteine ketimine and by cystathionine ketimine indicating the occurrence in bovine brain of a common binding site for ketimines. The reduced ketimines are totally ineffective in competing with [35S]lanthionine ketimine. Alltogether these findings are highly indicative for the existence in mammals of a novel class of endogenous sulfur-containing cyclic products provided with a possible neurochemical function to be investigated further.  相似文献   

8.
Bovine brain ketimine reductase   总被引:2,自引:0,他引:2  
We report the purification from bovine brain of an NAD(P)H-dependent reductase which actively reduces a new class of cyclic unsaturated compounds, named ketimines. Ketimines arise from the transamination of some sulphur-containing amino acids, such as L-cystathionine, S-aminoethyl-L-cysteine and L-lanthionine. The enzyme also reduces delta 1-piperidine 2-carboxylate, the carbon analog of aminoethylcysteine ketimine. Some kinetic and molecular properties of this enzyme have been determined. Subcellular localization and regional brain distribution have also been studied. The ketimine reductase activity was found to be associated with the soluble fraction, and was located prevalently in the cerebellum and cerebral cortices. Cyclothionine and 1,4-thiomorpholine-3,5-dicarboxylic acid, the enzymatic reduction products of cystathionine ketimine and lanthionine ketimine, respectively, have been detected in bovine brain, thus suggesting a role of this enzyme in their biosynthesis.  相似文献   

9.
McCormick, N. G. (University of Washington, Seattle), E. J. Ordal, and H. R. Whiteley. Degradation of pyruvate by Micrococcus lactilyticus. I. General properties of the formate-exchange reaction (J. Bacteriol. 83:887-898. 1962.-At an alkaline pH, extracts of Micrococcus lactilyticus(2) catalyze the phosphoroclastic degradation of pyruvate to formate and acetyl phosphate and the rapid exchange of formate into the carboxyl group of pyruvate. At an acid pH, hydrogen, carbon dioxide, and acetyl phosphate are produced, and carbon dioxide is exchanged into the carboxyl group of pyruvate. A concentration of approximately 1 m phosphate is required for the phosphoroclastic reaction and formate exchange; the production of carbon dioxide and hydrogen is greatly inhibited by high concentrations of phosphate. Formate exchange requires a divalent metal ion and is stimulated by reducing agents and an atmosphere of hydrogen. Inhibition by p-chloromercuribenzoate, Zn(++), Cd(++), and arsenite indicates that sulfhydryl groups on the enzyme are involved in the reaction; the inhibition by arsenite and Cd(++) may be relieved by 2,3-dimercaptopropanol, suggesting that vicinal dithiols may be required. Inhibition by hypophosphite may reflect a competition with formate for a site on the enzyme.At an alkaline pH, alpha-ketobutyrate is degraded to propionate and formate, whereas alpha-ketoglutarate is fermented to succinate, propionate, carbon dioxide, hydrogen, and formate. Formate is exchanged into the carboxyl groups of alpha-ketobutyrate and alpha-ketoglutarate under these conditions. Only traces of alpha-ketovalerate and alpha-ketoisovalerate are fermented at an alkaline pH and the exchange of formate into these compounds is very low.The addition of viologen dyes under the conditions used for formate exchange causes a reduction of pyruvate, alpha-ketobutyrate, alpha-ketovalerate, and alpha-ketoisovalerate to the corresponding alpha-hydroxy acids.  相似文献   

10.
The sulfur containing imino acids cystathionine ketimine (CK) and lanthionine ketimine (LK) have been detected in the human brain by an HPLC procedure. The HPLC procedure takes advantage of the selective absorbance at 380 nm of the phenylisothiocyanate-ketimine adduct. Quantitation of cystathionine ketimine and lanthionine ketimine indicates a mean concentration (mean ± SD, n = 4) of 2.3 ± 0.8 nmol/g for CK and of 1.1 ± 0.3 nmol/g for LK in four human cerebral cortex samples of neurosurgical source. The identification of these cyclic ketimine derivatives of L-cystathionine and L-lanthionine as normal human metabolites in human nervous tissue may have interesting metabolic and physiological implications.  相似文献   

11.
Human peripheral blood polymorphonuclear leukocytes were preincubated with cystathionine and cystathionine metabolites found in the urine of patients with cystathioninuria. Among the cystathionine metabolites, cystathionine ketimine and N-acetyl-S-(3-oxo-3-carboxy-n-propyl) cysteine (NAc-OCPC) significantly enhanced the N-formylmethionylleucylphenylalanine (fMLP)-induced superoxide generation, but cystathionine, NAc-cystathionine, and cyclothionine did not enhance the superoxide generation. Cystathionine ketimine and NAc-OCPC also enhanced superoxide generation induced by opsonized zymosan (OZ) but not that induced by arachidonic acid (AA) and phorbol 12-myristate 13-acetate (PMA). Superoxide generation induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein, an inhibitor of tyrosine kinase, and was enhanced by 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C. Cystathionine ketimine and NAc-OCPC markedly also increased phosphorylation of 45-kDa protein in human neutrophils and the phosphorylation depended on the concentrations of cystathionine ketimine and NAc-OCPC. The phosphorylation of 45-kDa protein induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein and herbimycin A, inhibitors of tyrosine kinase, but was not inhibited by H-7 and staurosporine, inhibitors of protein kinase C. Cystathionine metabolites and l-cystathionine sulfoxides were separated into two diastereoisomers, CS-I and CS-II. CS-I enhanced the superoxide generation induced by AA and PMA but not that induced by fMLP and OZ. In contrast, CS-II enhanced the superoxide generation induced by fMLP and OZ, but not that induced by AA and PMA.  相似文献   

12.
Summary S-(2-oxo-2-carboxyethyl)homocysteine (OCEHC), produced by the enzymatic monodeamination of cystathionine, is known to cyclize producing the seven membered ring of cystathionine ketimine (CK) which has been recognized as a cystathionine metabolite in mammals. Studies have been undertaken in order to find the best conditions of cyclization of synthetic OCEHC to CK and for the preparation of solid CK salt product. It has been found that ring closure takes place at alkaline pH and is highly accelerated in 0.5 M phosphate buffer. The sodium salt of CK has been prepared by controlled additions of NaOH to water-ethanol solution of OCEHC under N2 atmosphere. A solid product is obtained which, dissolved in water, shows the spectral features of CK. Solutions of the sodium salt of CK show the presence of a pH depending reversible equilibrium with the open OCEHC form. Plot of the absorbance at 296 nm in function of pH indicates that at pH 9 the compound is completely cyclized while at pH 6 is totally in the open OCEHC form. At intermediate pHs variable ratios between the two forms occur. According to the results obtained by the spectral analysis, HPLC assays of the sodium salt of CK show different patterns depending on the pH of the elution buffer.Abbreviations CK cystathionine ketimine - OCEHC S-(2-oxo-2-carboxyethyl) homocysteine - HPLC high performance liquid chromatography  相似文献   

13.
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.  相似文献   

14.
A study of the sulphur amino acids of rat tissues   总被引:2,自引:2,他引:0       下载免费PDF全文
1. In a study of the metabolism of l-[(35)S]methionine in vivo, the labelled sulphur compounds of rat liver and brain were separated first by ion-exchange chromatography into two fractions containing (i) free sulphur amino acids such as methionine, cystathionine, cyst(e)ine and homocyst(e)ine and (ii) glutathione. 2. Two-dimensional paper chromatography with butan-1-ol-acetic acid or propionic acid-water in the first direction and 80% acetone or acetone-ethyl methyl ketone-water in the second direction was found superior to other solvent systems for separating the sulphur amino acids. 3. At 10min. after injection of [(35)S]methionine only a small part of the (35)S was found combined in free methionine or other free sulphur amino acids. 4. Evidence was obtained of the presence of adenosyl[(35)S]methionine and adenosyl[(35)S]homocysteine in perchloric acid extracts of rat liver and brain. 5. The trans-sulphuration pathway was active in brain as well as in liver.  相似文献   

15.
Detection of cystathionine and lanthionine ketimines in human urine   总被引:1,自引:0,他引:1  
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.  相似文献   

16.
2H-1,4-Thiazine-5,6-dihydro-3-carboxylic acid (trivial name: aminoethylcysteine ketimine) is a cyclic sulfur-containing imino acid detected in bovine brain extracts by means of three different procedures. Gas liquid chromatography of protein-free extracts of five bovine brains revealed the presence of this compound at concentrations ranging from 2 to 3 nmol/g wet weight of tissue. The enzymatic method based on the inhibition of D-amino acid oxidase activity by aminoethylcysteine ketimine together with an high-performance liquid chromatography procedure confirm the identification and quantitations obtained with gas liquid chromatography. The discovery of this compound structurally similar to pipecolic acid opens the question of its physiological role in the central nervous system.  相似文献   

17.
Decarboxylation rates for a series of C-3 to C-6 alpha-keto acids were determined in the presence of resting cells and cell-free extracts of Streptococcus lactis var. maltigenes. The C-5 and C-6 acids branched at the penultimate carbon atom were converted most rapidly to the respective aldehydes in the manner described for alpha-carboxylases. Pyruvate and alpha-ketobutyrate did not behave as alpha-carboxylase substrates, in that O(2) was absorbed when they were reacted with resting cells. The same effect with pyruvate was noted in a nonmalty S. lactis, accounting for CO(2) produced by some "homofermentative" streptococci. Mixed substrate reactions indicated that the same enzyme was responsible for decarboxylation of alpha-ketoisocaproate and alpha-ketoisovalerate, but it appeared unlikely that this enzyme was responsible for the decarboxylation of pyruvate. Ultrasonic disruption of cells of the malty culture resulted in an extract inactive for decarboxylation of pyruvate in the absence of ferricyanide. Dialyzed cell-free extracts were inactive against all keto acids and could not be reactivated.  相似文献   

18.
Initial velocity studies in the absence and presence of product and dead-end inhibitors suggest a steady-state random mechanism for malic enzyme in the direction of reductive carboxylation of pyruvate. For this quadreactant enzymatic reaction (Mn2+ is a pseudoreactant), initial velocity patterns were obtained under conditions in which two substrates were maintained at saturating concentrations while one reactant was varied at several fixed concentrations of the other. Data from the resulting reciprocal plots, analyzed in terms of a bireactant mechanism, are consistent with a sequential mechanism with an obligatory order of addition of metal prior to pyruvate. NAD is competitive against NADH whether pyruvate and CO2 are maintained at low or high concentrations, whereas it is noncompetitive against pyruvate and CO2. Thio-NADH, alpha-ketobutyrate, and nitrite were used as dead-end analogs of NADH, pyruvate, and CO2, respectively. Thio-NADH is competitive against NADH, whereas it is noncompetitive against pyruvate and CO2, in accordance with a random mechanism. alpha-Ketobutyrate and nitrite gave noncompetitive inhibition against all substrates. The noncompetitive patterns observed for alpha-ketobutyrate versus pyruvate and nitrite versus CO2 suggest binding of the inhibitor to both the E.Mn.NADH and E.Mn.NAD complexes. Primary deuterium isotope effects are equal on all kinetic parameters, in agreement with the random mechanism, and suggest equal off-rates for NAD from E.Mn.NAD as well as pyruvate and NADH from E.Mn.NADH.pyruvate. Data are consistent with an overall symmetry in the malic enzyme reaction in the two reaction directions with a requirement for metal bound prior to pyruvate and malate.  相似文献   

19.
Cystathionine gamma-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-L-homoserine (OSHS) and L-cysteine via a gamma-replacement reaction. In the absence of L-cysteine, OSHS undergoes an enzyme-catalyzed, gamma-elimination reaction to form succinate, alpha-ketobutyrate, and ammonia. Since elimination of the gamma-substituent is necessary for both reactions, it is reasonable to assume that the replacement and elimination reaction pathways diverge from a common intermediate. Previously, this partitioning intermediate has been assigned to a highly conjugated alpha-iminovinylglycine quininoid (Johnston et al., 1979a). The experiments reported herein support an alternative assignment for the partitioning intermediate. We have examined the gamma-replacement and gamma-elimination reactions of cystathionine gamma-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy. The gamma-elimination reaction is characterized by a rapid decrease in the amplitude of the enzyme internal aldimine spectral band at 422 nm with a concomitant appearance of a new species which absorbs in the 300-nm region. A 485-nm species subsequently accumulates in a much slower relaxation. The gamma-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (approximately 3 ms). This relaxation is followed by a decrease in absorbance at 425 nm that is tightly coupled to the appearance of a species which absorbs in the 300-nm region. Reaction of the substrate analogues L-alanine and L-allylglycine with cystathionine gamma-synthase results in bleaching of the 422-nm absorbance and the appearance of a 300-nm species. In the absence of L-cysteine, L-allylglycine undergoes facile proton exchange; in the presence of L-cysteine, L-allylglycine undergoes a gamma-replacement reaction to form a new amino acid, gamma-methylcystathionine. No long-wavelength-absorbing species accumulate during either of these reactions. These results establish that the partitioning intermediate is an alpha-imino beta,gamma-unsaturated pyridoxamine derivative with lambda max congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.  相似文献   

20.
D-Amino acid aminotransferase, purified to homogeneity and crystallized from Bacillus sphaericus, has a molecular weight of about 60,000 and consists of two subunits identical in molecular weight (30,000). The enzyme exhibits absorption maxima at 280, 330, and 415 nm, which are independent of the pH (5.5 to 10.0), and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. One of the pyridoxal-5'-P, absorbing at 415 nm, is bound in an aldimine linkage to the epsilon-amino group of a lysine residue of the protein, and is released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive form, which is reactivated by addition of pyridoxal 5'phosphate, still has a 330 nm peak and contains 1 mol of pyridoxal 5'-phosphate. Therefore, this form is regarded as a semiapoenzyme. The holoenzyme shows negative circular dichroic bands at 330 and 415 nm. D-Amino acid aminotransferase catalyzes alpha transamination of various D-amino acids and alpha-keto acids. D-Alanine, D-alpha-aminobutyrate and D-glutamate, and alpha-ketoglutarate, pyruvate, and alpha-ketobutyrate are the preferred amino donors and acceptors, respectively. The enzyme activity is significantly affected by both the carbonyl and sulfhydryl reagents. The Michaelis constants are as follows: D-alanine (1.3 and 4.2 mM with alpha-ketobutyrate and alpha-ketoglutarate, respictively), alpha-ketobutyrate (14 mM withD-alanine), alpha-ketoglutarate (3.4 mM with D-alanine), pyridoxal 5'-phosphate (2.3 muM) and pyridoxamine 5'-phosphate (25 muM).  相似文献   

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