Aminoethyl Cysteine Research Articles

Aminoethylcysteine can replace the function of the essential active site lysine of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The biodegradative 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Pseudomonas mevalonii catalyzes the NAD(+)-dependent conversion of (S)-HMG-CoA to (R)-mevalonate. Crystallographic analysis of abortive ternary complexes of this enzyme revealed lysine 267 located at a position in the active site, suggesting that it might serve as the general acid/base for catalysis. Site-directed mutagenesis and subsequent chemical derivatization were therefore employed to investigate this active site lysine. Replacement of lysine 267 by alanine, histidine, or arginine resulted in mutant enzymes that lacked detectable activity. Lysine 267 was next replaced by the lysine analogues aminoethylcysteine and carboxyamidomethylcysteine. Using instead of the wild-type enzyme the fully active, cysteine-free mutant enzyme C156A/C296A, lysine 267 was first replaced by cysteine. Cysteine 267 of mutant enzyme C156A/C296A/K267C was then treated with bromoethylamine or iodoacetamide to insert aminoethylcysteine (AEC) or carboxyamidomethylcysteine at position 267. The carboxyamidomethylcysteine derivative was inactive, whereas mutant enzyme C156A/C296A/K267AEC exhibited high catalytic activity. That aminoethylcysteine, but not other basic amino acids, can replace the function of lysine 267 documents both the importance of this residue and the requirement for a precisely positioned positive charge at the active site of the enzyme.

A Potential Screening System for Identifying Sorghum Ecotypes with Increased Lysine in the Seeds

AbstractClassical “high‐lysine” sorghum lines are characterized by smaller seeds than average, due to a decrease in prolamin synthesis and a subsequent decrease in yield. To exploit the natural variation in lysine content and to identify ecotypes with a seed lysine content higher than average, characterized by plump seeds, a method was developed based on root‐growth inhibition of seeds growing on a medium containing aminoethylcysteine (AEC), a lysine analogue. By using a collection of sorghum mutants and ecotypes a correlation coefficient of 0.926 between root length and lysine content was established. This method, which uses the root length of plants growing on aec to indicate which lines have a potential elevated lysine content, can be applied for the screening of sorghum germplasm. Since this is a non‐destructive method it can also be used at the individual seed level, for example for screening progenies of regenerated plants from in vitro culture to exploit the somaclonal variation.

Aspartokinase genes lysCα and lysCβ overlap and are adjacent to the aspartate β-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum

A 2.1 kb DNA fragment of the recombinant plasmid pCS2, isolated from an aminoethyl cysteine (AEC)-resistant and lysine-producing Corynebacterium glutamicum mutant strain, and which confers AEC resistance and lysine production on the wild-type G. glutamicum ATCC 13032 was analysed. DNA sequence analysis of this fragment revealed three large open reading frames (ORFs). The incomplete ORF1 does not contain the 5' end of the coding region. ORF2, which uses the same reading frame as ORF1, is identical to the 3' end of ORF1 and encodes a putative protein of 172 amino acids (aa) and of Mr 18,584. ORF3 encodes a putative protein of 344 aa and of Mr 36,275. The amino acid sequences deduced from ORF1 and ORF2 display strong homologies to those of the alpha- and beta-subunits of the Bacillus subtilis aspartokinase II. It is therefore proposed that the incomplete ORF1, termed lysC alpha, encodes part of the alpha-subunit of the C. glutamicum aspartokinase whereas the complete ORF2, termed lysC beta, encodes the beta-subunit of the same enzyme. ORF2 is responsible for AEC resistance and lysine production due to a feedback-resistant aspartokinase. The amino acid sequence deduced from ORF3, termed asd, is highly homologous to that of the Streptococcus mutans aspartate beta-semialdehyde dehydrogenase (ASD). Plasmids carrying the C. glutamicum asd gene complemented Escherichia coli asd mutants. Increase in ASD activity by a factor of 30-60 was measured for C. glutamicum cells harbouring high copy-number plasmids with the C. glutamicum asd gene.

Cloning of a DNA fragment from Corynebacterium glutamicum conferring aminoethyl cysteine resistance and feedback resistance to aspartokinase.

The Corynebacterium glutamicum/Escherichia coli shuttle vector plasmid pZ1 was used to clone the S-(2-aminoethyl)-D,L-cysteine (AEC)-resistance gene from a lysine-excreting, AEC-resistant strain of C. glutamicum, the aspartokinase activity of which was released from feedback inhibition by mixtures of lysine and threonine or AEC and threonine respectively. A recombinant plasmid designated pCS2 carrying a 9.9-kb chromosomal insert that conferred AEC resistance and the ability to excrete lysine to its host was isolated. The aspartokinase activity of the pCS2-carrying strain was resistant towards inhibition by mixtures of lysine and threonine or AEC and threonine respectively. By deletion analysis the DNA region conferring AEC resistance to the host and feedback resistance to its aspartokinase activity could be confined to a 1.2-kb DNA fragment.

Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins.

Cultured rat embryo cells were stimulated to rapidly release a small group of proteins that included several heat-shock proteins (hsp110, hsp71, hscp73) and nonmuscle actin. The extracellular proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. Heat-shocked cells released the same set of proteins as control cells with the addition of the stress-inducible hsp110 and hsp71. Release of these proteins was not blocked by either monensin or colchicine, inhibitors of the common secretory pathway. A small amount of the glucose-regulated protein grp78 was externalized by this pathway. The extracellular accumulation of these proteins was inhibited after they were synthesized in the presence of the lysine analogue aminoethyl cysteine. It is likely that the analogue-substituted proteins were misfolded and could not be released from cells, supporting our conclusion that a selective release mechanism is involved. Remarkably, actin and the squid heat-shock proteins homologous to rat hsp71 and hsp110 are also among a select group of proteins transferred from glial cells to the squid giant axon, where they have been implicated in neuronal stress responses (Tytell et al.: Brain Res., 363:161-164, 1986). Based in part on the similarities between these two sets of proteins, we hypothesized that these proteins were released from labile cortical regions of animal cells in response to perturbations of homeostasis in cells as evolutionarily distinct as cultured rat embryo cells and squid glial cells.

Inhibitory effects of aspartate-derived amino acids and aminoethylcysteine, a lysine analog, on the growth of sorghum seedlings; relation with three enzymes of the aspartate-pathway

The effects of the aspartate-derived amino acids, lysine and threonine on the root growth of Sorghum bicolor (L.) Moench. seedlings have been investigated. Lysine and threonine, when added separately, are not severe inhibitors. However, when present simultaneously, lysine and threonine (each 2 mM) can inhibit the root growth up to 90%. This growth inhibition can be relieved by methionine (or homoserine). The lysine analog, S-2- aminoethylcysteine (AEC) appears to be a strong inhibitor: the root growth decreases to 14% when 0.2 mM AEC is used. This inhibition can be relieved by adding lysine to the culture medium. These effects can be related to the feedback regulation exerted by lysine (or AEC) and threonine on key enzymes of the aspartate-pathway. The activity of aspartate kinase is inhibited for 78% by 0.5 mM lysine, for 25% by 0.5 mM threonine and by a combination of both these amino acids (each 0.5 mM) up to 90%. Diohydrodipicolinate synthase (DHDPS), the first enzyme unique to the lysine synthesis, is strongly inhibited by lysine (80% at 6 μM). Threonine is found to inhibit homoserine dehydrogenase activity up to 74%, using NADH as cofactor. Threonine has no effect on homoserine dehydrogenase when NADPH is used as cofactor.

Amino acid analogs inhibit murine xenotropic retrovirus expression and cell cycle progression.

Certain functional analogs of amino acids were examined for their capacity to inhibit chemically induced expression of endogenous xenotropic retrovirus from Kirsten sarcoma virus transformed BALB/c (K-BALB) mouse cells. Partially synchronized cells cultured with aminoethylcysteine (AEC), parafluorophenylalanine (PFA), or valinol, and subsequently induced with either 5-iododeoxyuridine (IUdR), cycloheximide, or histidinol, showed inhibition of virus activation. Inhibition was concentration- and time-dependent (1 to 4 h) and not a consequence of cytotoxicity. Inhibition was competed out by the analogous amino acid and was specific to the induction process. After a 4 h analog treatment, heteronuclear RNA synthesis was reduced 24, 38, and 35% by PFA, AEC, and valinol, respectively, whereas cycloheximide or actinomycin D reduced synthesis by 60 and 90%, respectively; therefore, the analogs did not seem to inhibit induction through a general transcriptional block. Culture of cells in the presence of the analogs resulted in an abrupt reduction (70 to 90%) in DNA synthesis. Using synchronized cells, it was found that 0.1 mM AEC added in G1 phase and followed by IUdR induction almost totally inhibited virus expression. No inhibition was observed when AEC was added during S phase concomitantly with the inducer. AEC added to synchronous cells in G1 phase inhibited the progression of cells into S phase and the onset of DNA synthesis. The results show that K-BALB cells have an AEC-sensitive restriction point in G1 phase that might relate to the effects amino acid analogs have on cell replication and S phase dependent gene expression, as well as subsequent differentiation.

Effect of Aspartate‐derived Amino Acids and Aminoethyl Cysteine on Growth of Excised Mature Embryos of Maize1

Twenty‐one amino acids were tested individually for their effects on growth of roots from excised mature embryos of maize (Zea mays L.). Among these, tryptophan, tyrosine, threonine, and homoserine were partially inhibitory to root growth at 1 mM concentrations. Length and dry weight data showed that maize roots both from embryos and from kernels were synergistically inhibited by lysine + threonine but not other paired combinations of aspartate‐derived amino acids. Roots from embryos, however, were more sensitive to inhibition. The synergistic inhibition of embryo root length was near maximal at 1 mM lysine + 1 mM threonine. This synergism was not observed with other paired combinations of aspartatederived amino acids. Methionme and homoserine specifically relieved lysine + threonine inhibition, whereas 17 other amino acids were ineffective. These results are compatible with a model and published enzymology studies which suggest that exogenously added lysine and threonine may act as feedback inhibitors of aspartokinase and homoserine dehydrogenase, respectively, early enzymes in the aspartate biosynthetic pathway.The lysine analog, S‐2‐aminoethyl‐L‐cysteine (AEC) was a potent inhibitor of root growth in maize seedlings. Among 21 amino acids tested, lysine was most effective in relieving AEC inhibition while arginine and methionine were partially effective.

The complete amino-acid sequence of guinea-pig -lactalbumin.

Reduced, S-aminoethylated guinea-pig α-lactalbumin was cleaved with cyanogen bromide at its single methionyl residue. The products were separated gel by filtration and characterized by amino acid analysis and N-terminal group determination, as a 90-residue peptide from the N-terminus of the protein (N-terminal lysine) and a 33-residue peptide from the C-terminus of the protein (N-terminal aminoethyl cysteine). The amino acid sequences of both these peptides were determined by examination of the peptides obtained by digestion of the purified fragments with trypsin, chymotrypsin or thermolysin. The complete amino acid sequence of guinea-pig α-lactalbumin thus obtained is discussed in relation to the known sequences of other members of the lysozyme-α-lactalbumin group.