Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate beta-lyase

Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. The β-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine is an excellent aminotransferase and β-lyase substrate of rhGTK. Moderate aminotransferase and β-lyase activities occur with the chemopreventive agent Se-methyl-l-selenocysteine. l-3-(2-Naphthyl)alanine, l-3-(1-naphthyl)alanine, 5-S-l-cysteinyldopamine and 5-S-l-cysteinyl-l-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The α-keto acids generated by transamination/l-amino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTK-catalyzed β-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-l-cysteinyldopamine, but not with 5-S-l-cysteinyl-l-DOPA. The importance of transamination, oxidation and β-elimination reactions involving 5-S-l-cysteinyldopamine, 5-S-l-cysteinyl-l-DOPA and Se-methyl-l-selenocysteine in human tissues and their biological relevance are discussed.     

Cysteine S-conjugate β-lyases

Summary. Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess an electron-withdrawing group attached at the sulfur. The end products of the β-lyase reaction are pyruvate, ammonium and a sulfur-containing fragment. If the sulfur-containing fragment is reactive, the parent cysteine S-conjugate may be toxic, particularly to kidney mitochondria. Halogenated alkenes are examples of electrophiles that are bioactivated (toxified) by conversion to cysteine S-conjugates. These conjugates are converted by cysteine S-conjugate β-lyases to thioacylating fragments. Several cysteine S-conjugates found in allium foods (garlic and onion) are β-lyase substrates. This finding may account in part for the chemopreventive activity of allium products. This review (1) identifies enzymes that catalyze cysteine S-conjugate β-lyase reactions, (2) suggests that toxicant channeling may contribute to halogenated cysteine S-conjugate-induced toxicity to mitochondria, and (3) proposes mechanisms that may contribute to the antiproliferative effects of sulfur-containing fragments eliminated from allium-derived cysteine S-conjugates.     

Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH₃SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH₃SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH₃SeH and/or seleno-keto acid metabolites.     

Sulfate Assimilation in C(4) Plants: Intercellular and Intracellular Location of ATP Sulfurylase, Cysteine Synthase, and Cystathionine beta-Lyase in Maize Leaves

The activity of ATP sulfurylase, cysteine synthase, and cystathionine β-lyase was measured in crude leaf extracts, bundle sheath strands, and mesophyll and bundle sheath chloroplasts to determine the location of sulfate assimilation of C₄ plant leaves. Almost all the ATP sulfurylase activity was located in the bundle sheath chloroplasts while cysteine synthase and cystathionine β-lyase activity was located, in different proportions, in both chloroplast types.

A new spectrophotometric assay for measuring ATP sulfurylase activity is also described.

     

Induction of unscheduled DNA synthesis and micronucleus formation in Syrian hamster embryo fibroblasts treated with cysteine S-conjugates of chlorinated hydrocarbons

S-(chloroethyl)-cysteine (CEC) and S-(1,2-dichlorovinyl)cysteine (DCVO) have been proposed as intermediates in the metabolic transformation of the carcinogens 1,2-dichloroethane and 1,1,2-trichloroethylene. We have tested the ability of CEC and DCVC to induce DNA repair and genotoxic effects at the chromosomal level by comparative assessment of unscheduled DNA synthesis induction and micronucleus formation in Syrian hamster embryo fibroblasts. CEC induced a potent and dose-dependent response in both assays, whereas DCVC treatment resulted in a comparatively weak induction of DNA repair and failed to raise micronucleus formation above control rates. Inhibition of cysteine conjugate \gB-lyase diminished the effect of DCVC, but had no influence on the genotoxicity of CEC either in the unscheduled DNA synthesis or micronucleus assay.Abbreviations AOAA aminooxyacetic acid - CEC S-(chloroethyl)-cysteine; \gB-lyase, cysteine conjugate -lyase - DCE 1,2-dichloroethane - DCVC S(1,2-dichlorovinyl)-cysteine - GSH glutathione - HU hydroxyurea - IBR IBR-modified Dulbecco's Eagle's reinforced medium - MN2 micronuclei/2,000 cells - 4-NQO 4-nitroquinoline-1-oxide - SHE Syrian hamster embryo fibroblasts; ³H-Thd, ³H-thymidine - TCE 1,1,2-trichloroethylene - UDS unscheduled DNA synthesis     

Cysteine <Emphasis Type="Italic">S</Emphasis>-conjugate β-lyases: important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds,xenobiotics and anticancer agents

Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1–15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M _r cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Spectrophotometric measurement of the liberation of the alpha-amino group of cysteine residues in polypeptides

1. The reaction between β-bromopyruvic acid and SH groups of cysteine residues in reduced ribonuclease and in some other polypeptides was investigated. 2. One molecule of the acid was found to be necessary to block one SH group in reduced ribonuclease. The stoicheiometry of the interaction and the spectral characteristics of the compound formed suggested that the product is and S-oxalomethyl (R·S·CH₂·CO·CO₂H) derivative of reduced ribonuclease. 3. Digestion of reduced S-oxalomethylated ribonuclease by trypsin or chymotrypsin induced changes in the spectrum that could be attributed to the liberation of the α-amino group of S-oxalomethylated cysteine residues from peptide bonds. The spectral changes that accompanied the hydrolysis of specific peptide bonds in reduced S-oxalomethylated ribonuclease and S-oxalomethylated co-poly(l-Lys,l-CySH) allowed the kinetics of the digestion to be followed. 4. Possible applications of the spectrophotometric method in the study of protein structure are discussed.     

MenD as a versatile catalyst for asymmetric synthesis

The thiamine diphosphate (ThDP)-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) from Escherichia coli K12, formerly known as SHCHC-synthase, catalyses the decarboxylation of α-ketoglutarate and the subsequent addition of the resulting succinyl-THDP to isochorismate. Here, the enzyme is tested for unphysiologial C–C bond-forming reactions.Condensation of α-ketoglutarate after decarboxylation to a broad range of aldehydes gave α-hydroxyketones with isolated yields from 26 to 87% and 94 to 98% ee for addition to aromatic aldehydes. MenD accepts a wide range of aldehydes as acceptor substrates to produce chiral α-hydroxyketones with conserved regioselectivity where the activated succinylsemialdehyde serves selectively as the donor. Regioselectivity is inverted only for condensation of α-ketoglutarate with pyruvate (activated acetaldehyde) as donor. Besides α-ketoglutarate, pyruvate and oxalacetate are accepted as donors in combination with benzaldehyde and 2-fluorobenzaldehyde as acceptors, however with decreased activity of C–C bond formation.The physiological 1,4-addition of α-ketoglutarate to isochorismate was investigated for acceptor substrate variability. (2S,3S)-2,3-Dihydroxy-2,3-dihydrobenzoate (2,3-CHD), which lacks the pyruvyl found in isochorismate, is converted to (5S,6S)-2-succinyl-5,6-dihydroxycyclohex-2-enecarboxylate. In contrast to the addition to carbonyls, the active site of MenD does appear to impose specific constraints on the acceptor substrate for 1,4-addition with α,β-unsaturated carboxylic acids.     

Complete assimilation of cysteine by a newly isolated non-sulfur purple bacterium resembling Rhodovulum sulfidophilum (Rhodobacter sulfidophilus)

A rod-shaped, motile, phototrophic bacterium, strain SiCys, was enriched and isolated from a marine microbial mat, with cysteine as sole substrate. During phototrophic anaerobic growth with cysteine, sulfide was produced as an intermediate, which was subsequently oxidized to sulfate. The molar growth yield with cysteine was 103 g mol^–1, in accordance with complete assimilation of electrons from the carbon and the sulfur moiety into cell material. Growth yields with alanine and serine were proportionally lower. Thiosulfate, sulfide, hydrogen, and several organic compounds were used as electron donors in the light, whereas cystine, sulfite, or elemental sulfur did not support phototrophic anaerobic growth. Aerobic growth in the dark was possible with fructose as substrate. Cultures of strain SiCys were yellowish-brown in color and contained bacteriochlorophyll a, spheroidene, spheroidenone, and OH-spheroidene as major photosynthetic pigments. Taking the morphology, photosynthetic pigments, aerobic growth in the dark, and utilization of sulfide for phototrophic growth into account, strain SiCys was assigned to the genus Rhodovulum (formerly Rhodobacter) and tentatively classified as a strain of R. sulfidophilum. In cell-free extracts in the presence of pyridoxal phosphate, cysteine was converted to pyruvate and sulfide, which is characteristic for cysteine desulfhydrase activity (l-cystathionine γ-lyase, EC 4.4.1.1). Received: 15 December 1995 / Accepted: 1 April 1996     

Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 (ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies.     

Purification and substrate characterization of α-ketobutyrate decarboxylase from Pseudomonas putida

α-Ketobutyrate decarboxylase encoded in the -methionine catabolism operon of Pseudomonas putida is homologous with the E1 component of pyruvate dehydrogenase complex from gram-negative bacteria. The enzyme was purified to homogeneity from the cell extract of an Escherichia coli transformant. The purified enzyme was homodimeric with a subunit of M_r 93,000 on SDS-PAGE. The enzyme activity was activated by the addition of both thiamine pyrophosphate (TPP) and a divalent cation, such as Mg²⁺, Mn²⁺, and Co²⁺. The enzyme showed high activity for α-ketobutyrate and α-keto-n-valerate rather than pyruvate, but the α-keto acids with increasing length of the side chain as well as branching, such as α-keto-n-caproate and α-keto-3-methylvalerate, were not used by the enzyme. The K_m values for α-ketobutyrate and pyruvate were 0.016 and 0.147 mM, respectively, and the k_cat/K_m value (10.69 s⁻¹ mM⁻¹) for α-ketobutyrate was 29-fold greater than that for pyruvate. Thus, α-ketobutyrate decarboxylase is distinguished from the pyruvate dehydrogenase E1 component with respect to the substrate specificity, although their structural and enzymological properties were similar. These results suggest that the unique substrate specificity of α-ketobutyrate decarboxylase is due to a slight difference in the highly conserved active sites of both enzymes.     

Simultaneous synthesis of enantiomerically pure (S)-amino acids and (R)-amines using α/ω-aminotransferase coupling reactions with two-liquid phase reaction system

An efficient simultaneous synthesis of enantiopure (S)-amino acids and chiral (R)-amines was achieved using α/ω-aminotransferase (α/ω-AT) coupling reaction with two-liquid phase system. As, among the enzyme components in the α/ω-AT coupling reaction systems, only ω-AT is severely hampered by product inhibition by ketone product, the coupled reaction cannot be carried out above 60 mM substrates. To overcome this problem, a two-liquid phase reaction was chosen, where dioctylphthalate was selected as the solvent based upon biocompatibility, partition coefficient and effect on enzyme activity. Using 100 mM of substrates, the AroAT/ω-AT and the AlaAT/ω-AT coupling reactions asymmetrically synthesized (S)-phenylalanine and (S)-2-aminobutyrate with 93% (>99% ee^S) and 95% (>99% ee^S) of conversion yield, and resolved the racemic α-methylbenzylamine with 56% (95% ee^R) and 54% (96% ee^R) of conversion yield, respectively. Moreover, using 300 mM of 2-oxobutyrate and 300 mM of racemic α-methylbenzylamine as substrates, the coupling reactions yielded 276 mM of (S)-2-aminobutyrate (>99% ee) and 144 mM of (R)-α-methylbenzylamine (>96% ee) in 9 h. Here, most of the reactions take place in the aqueous phase, and acetophenone mainly moved to the organic phase according to its partition coefficient.     

17α-Hydroxylase/17,20-lyase defects

Congential adrenal hyperplasia due to 17α-hydroxylase/17/20-lyase deficiency is caused by genetic defects in the gene encoding P450c17 (CYP17). To date, 18 different mutations in 27 individuals have been identified and all of them are located in the coding region of CYP17. Several mutations have been reconstructed in human P450c17 cDNA and expressed in COS cells to characterize the kinetic properties of 17α-hydroxylase and 17,20-lyase activities. The molecular bases of cases clinically reported as 17α-hydroxylase deficiency have turned out to result from complete or partial combined deficiencies of 17α-hydroxylase/17,20-lyase. The elucidation of the molecular bases generally explains the patient's clinical profiles including the sexual phenotype of the external genitalia. In one case initially reported as isolated 17,20-lyase deficiency, the molecular basis was found to be partial combined deficiency of both activities, somewhat discordant with the patient's clinical profile. However, the patient was subsequently found to have 17α-hydroxylase deficiency, suggesting involvements of age-dependent unknown factors affecting P450c17 activity.     

Immobilization of Saccharomyces cerevisiae Cystathionine γ-Lyase and Application of the Product to Cystathionine Synthesis

Cystathionine γ-lyase of Saccharomyces cerevisiae was immobilized to aminohexyl-Sepharose through the cofactor pyridoxal 5′-phosphate and was characterized with respect to its cystathionine γ-synthase activity. The immobilized product was so stable that it repeatedly catalyzed as many as five cycles of the reaction without losing activity.     

Unusual intramolecular N→O acyl group migration occurring during conjugation of (−)-DHMEQ with cysteine

Previously we found that (−)-DHMEQ, a specific NF-κB inhibitor, covalently bound to a specific cysteine of NF-κB component proteins. In the course of formation of the (−)-DHMEQ and protected cysteine conjugate, we observed an unusual intramolecular N→O acyl group migration.     

The functional role of cysteine residues for c-Abl kinase activity

S-glutathionylation, the formation of mixed disulfides of glutathione with cysteine residues of proteins, is a broadly observed physiological modification that occurs in response to oxidative stress. Since cysteine residues are particularly susceptible to oxidative modification by reactive oxygen species, S-glutathionylation can protect proteins from irreversible oxidation. In this study, we show that the kinase activity of the non-receptor tyrosine kinase c-Abl is inhibited by in vitro thiol modification; specifically, the cysteine residues of c-Abl are modified by S-glutathionylation and by thiol alkylating agents such as 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid and N-ethylmaleimide. Modification of cysteine residues of c-Abl tyrosine kinase using glutathione disulfide and thiol alkylating agents corresponds to a concomitant loss of kinase activity. We also demonstrate that S-glutathionylation of c-Abl can be reversed using a physiological system involving glutaredoxin and this reversal restores c-Abl kinase activity. To our knowledge, these are the first data to show S-glutathionylation of c-Abl, and this modification may represent a mechanism of regulation of c-Abl kinase activity in cells under oxidative stress.     

Conjugations with glutathione. The enzymic conjugation of some chlorocyclohexenes

1. α-3,4,5,6-Tetrachlorocyclohex-1-ene and γ-2,3,4,5,6-pentachlorocyclohex-1-ene are conjugated with glutathione in vitro by a rat-liver enzyme that is probably glutathione S-aryltransferase. 2. Chlorocyclohexane and the α-, β-, γ- and δ-isomers of hexachlorocyclohexane were not substrates for rat-liver glutathione S-aryltransferase. 3. Glutathione-S-aryltransferase activity was present in tissue preparations of houseflies of insecticide-resistant and -susceptible strains. More activity was found in a dieldrin-resistant strain of houseflies fed on dieldrin than in either a dieldrin-resistant strain not fed on dieldrin or a control strain of dieldrin-susceptible houseflies. 4. Housefly soluble supernatant preparations converted S-(2-chloro-4-nitrophenyl)glutathione into the corresponding cysteine and mercapturic acid derivatives.     

Boron deprivation decreases liver S-adenosylmethionine and spermidine and increases plasma homocysteine and cysteine in rats

Two experiments were conducted with weanling Sprague–Dawley rats to determine whether changes in S-adenosylmethionine utilization or metabolism contribute to the diverse responses to boron deprivation. In both experiments, four treatment groups of 15 male rats were fed ground corn-casein based diets that contained an average of 0.05 mg (experiment 1) or 0.15 mg (experiment 2) boron/kg. In experiment 2, some ground corn was replaced by sucrose and fructose to increase oxidative stress. The dietary variables were supplemental 0 (boron-deprived) or 3 (boron-adequate) mg boron/kg and different fat sources (can affect the response to boron) of 75 g corn oil/kg or 65 g fish (menhaden) oil/kg plus 10 linoleic acid/kg. When euthanized at age 20 (experiment 1) and 18 (experiment 2) weeks, rats fed the low-boron diet were considered boron-deprived because they had decreased boron concentrations in femur and kidney. Boron deprivation regardless of dietary oil increased plasma cysteine and homocysteine and decreased liver S-adenosylmethionine, S-adenosylhomocysteine, and spermidine. Plasma concentration of 8-iso-prostaglandin F_2α (indicator of oxidative stress) was not affected by boron, but was decreased by feeding fish oil instead of corn oil. Fish oil instead of corn oil decreased S-adenosylmethionine, increased spermidine, and did not affect S-adenosylhomocysteine concentrations in liver. Additionally, fish oil versus corn oil did not affect plasma homocysteine in experiment 1, and slightly increased it in experiment 2. The findings suggest that boron is bioactive through affecting the formation or utilization of S-adenosylmethionine. Dietary fatty acid composition also affects S-adenosylmethionine formation or utilization, but apparently through a mechanism different from that of boron.     

A large-scale synthesis of somatostatin for clinical use by a novel alternating solution/solid-phase procedure

A large-scale synthesis of somatostatin was developed. A stepwise C→N approach in solution was used, employing N(α)-t-butoxycarbonyl amino acid active esters. The scheme of semipermanent protection utilized 2-(methylsulfonyl)-ethoxycarbonyl for the -amino group of lysine; acetamidomethyl for the β-thiol groups of cysteine; the orange-colored 2-[4-(phenylazo)-phenylsulfonyl]-ethoxy group for the C-terminal carboxy group of cysteine. All condensations and N(α)-deprotections were carried out in homogeneous solution, while isolation and purification of peptides carrying the colored group was achieved by precipitation and washing of the solid products. Thus, the “alternating solution/solid-phase peptide synthesis” combines advantages of both the classical solution synthesis and the Merrifield solid-phase technique. The overall yield was 5%, or 16 g of somatostatin from 100 g of the novel amino acid derivative, N(α)-t-butoxycarbonyl-S-acetamidomethyl- -cysteine 2-[4-(phenylazo)-phenylsulfonyl]-ethyl ester. An improved method for the preparation of S-acetamidomethyl- -cysteine, free of thiazolidine carboxylic acid, is described.