Nicholas M. Kredich

Role of S-adenosylhomocysteine in adenosine-mediated toxicity in cultured mouse T lymphoma cells

Abstract S-adenosylhomocysteine (SAH) is known to be a potent inhibitor of S-adenosylmethionine (SAM)-mediated reactions, of which SAH itself is a product. The immediate metabolic fate of SAH involves its hydrolysis to adenosine and L-homocysteine by the enzyme SAH hydrolase, but the reversibility of this reaction and its extremely low K eq in the hydrolytic direction suggest that under certain conditions of adenosine excess, SAH might accumulate with significant cytotoxic effects. We have used a model system consisting of cultured S49 mouse lymphoma cells together with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), to determine whether SAH is a mediator of adenosine cytotoxicity. Cells rendered resistant to adenosine-induced pyrimidine starvation by the addition of exogenous uridine or by the mutational loss of adenosine kinase are still sensitive to adenosine at concentrations >15 μM. We find that this effect is appreciably enhanced by the addition of L-homocysteine thiolactone to the culture medium. Cytotoxic concentrations of adenosine also cause significant elevations in intracellular levels of SAH, which are increased an additional several fold by 100μM exogenous L-homocysteine thiolactone. A fair correlation exists between a single time point determination of intracellular SAH and the degree of growth inhibition after 72 hr, but complicated time-dependent variations in SAH make it difficult to compare results obtained in the absence and presence of exogenous L-homocysteine thiolactone. In vivo DNA methylation in S49 cells is markedly inhibited by exposure of cells to concentrations of adenosine known to cause uridine-resistant cytotoxicity. This inhibition of methylation has been measured with short-term pulses of radiolabel, and correlates well with intracellular concentrations of SAH at all tested combinations of adenosine and L-homocysteine thiolactone. The results suggest that the uridine-resistant cytotoxic effects of adenosine on ADA-inhibited S49 cells are secondary to the inhibition of SAM-mediated methylation reactions by the adenosine metabolite SAH.

Biosynthesis of Cysteine.

The synthesis of L-cysteine from inorganic sulfur is the predominant mechanism by which reduced sulfur is incorporated into organic compounds. L-cysteineis used for protein and glutathione synthesis and serves as the primary source of reduced sulfur in L-methionine, lipoic acid, thiamin, coenzyme A (CoA), molybdopterin, and other organic molecules. Sulfate and thiosulfate uptake in E. coli and serovar Typhimurium are achieved through a single periplasmic transport system that utilizes two different but similar periplasmic binding proteins. Kinetic studies indicate that selenate and selenite share a single transporter with sulfate, but molybdate also has a separate transport system. During aerobic growth, the reduction of sulfite to sulfide is catalyzed by NADPH-sulfite reductase (SiR), and serovar Typhimurium mutants lacking this enzyme accumulate sulfite from sulfate, implying that sulfite is a normal intermediate in assimilatory sulfate reduction. L-Cysteine biosynthesis in serovar Typhimurium and E. coli ceases almost entirely when cells are grown on L-cysteine or L-cystine, owing to a combination of end product inhibition of serine transacetylase by L-cysteine and a gene regulatory system known as the cysteine regulon, wherein genes for sulfate assimilation and alkanesulfonate utilization are expressed only when sulfur is limiting. In vitro studies with the cysJIH, cysK, and cysP promoters have confirmed that they are inefficient at forming transcription initiation complexes without CysB and N-acetyl-L-serine. Activation of the tauA and ssuE promoters requires Cbl. It has been proposed that the three serovar Typhimurium anaerobic reductases for sulfite, thiosulfate, and tetrathionate may function primarily in anaerobic respiration.

The molecular basis for positive regulation of cys promoters in Salmonella typhimurium and Escherichia coli

Most genes required for cysteine biosynthesis in Salmonella typhimurium and Escherichia coli are positively regulated by cysB, which encodes a transcriptional activator belonging to the LysR family of regulatory proteins. CysB protein binds just upstream of the ‐35 region of positively regulated promoters, where in the presence of inducer it facilitates formation of a transcription initiation complex. CysB protein also autoregulates its own synthesis by binding to the cysB promoter as a repressor. Cysteine down‐regulates the pathway by inhibiting synthesis of O‐acetylserine, a direct cysteine precursor and possibly an inducer of gene expression. O‐Acetylserine spontaneously isomerizes to N‐acetyl‐serine, which is clearly an inducer. Sulphide and thio‐sulphate provide additional regulation by acting as anti‐inducers. Inducer stimulates CysB protein binding to sites involved in positive regulation, and inhibits binding to the negatively autoregulated cysB promoter. For three sites with unknown function, binding is stimulated at one and inhibited at the other two.

In vitro characterization of constitutive CysB proteins from Salmonella typhimurium

Expression of the cysteine regulon in Salmonella typhimurium and Escherichia coli is controlled by the LysR‐type transcriptional activator CysB and by the inducer N‐acetyl‐l‐serine. Sulphide and thiosulphate are anti‐inducers. Two highly purified constitutive CysB proteins, CysB(T149M) and CysB(T149P), were found to bind to the cysJIHcysK and cysP promoters, to activate transcription from the cysJIH and cysK promoters in the absence of N‐acetyl‐l‐serine, and to be insensitive to the effects of anti‐inducers. At 10mM MgCl2, the in vitro transcription activity of CysB(T149M) was maximal without N‐acetyl‐l‐serine, but that of CysB(T149P) was increased by inducer. At 2mM MgCl2, both proteins were fully active without inducer. A third mutant protein, CysB(W166R), was totally inactive at 10mM MgCl2, but gave constitutive expression of the cysK and cysJIH promoters at 2 mM MgCl2. Surprisingly, wild‐type CysB was also constitutive for the cysK promoter at 2mM MgCl2 but not at 10mM MgCl2; it required inducer for cysJIH promoter activation at both concentrations. Mutagenic studies indicated that this difference between promoters is due to the distance between activation site half‐sites, which are separated by 1 bp in the cysJIH promoter and by 2 bp in the cysK promoter. We speculate that inducer acts to decrease the distance between the binding domains of two CysB subunits that interact with an activation site. In vitro activities of wild‐type and mutant CysB proteins correlated much better with in vivo behaviour at 2mM than at 10mM MgCl2, suggesting that the former is the more physiological concentration.

Oral dipyridamole increases plasma adenosine levels in human beings.

Plasma adenosine levels in five healthy volunteers for 5 consecutive days showed far less intrasubject than intersubject variation (p < 0.0001), indicating that plasma adenosine levels are relatively constant during this period. Plasma adenosine levels were then measured in a different group of five healthy subjects for a 5‐day control period and during a 5‐day course of oral dipyridamole at a dose of 100 mg every 6 hours. Intrasubject comparisons showed that plasma adenosine levels were significantly higher during the 5 days of dipyridamole administration than during the control period (p = 0.017) and that this increase was most significant after 48 hours of drug (p < 0.001) administration. The average increase was 0.133 µmol/L (60%) with a range of 0.063 to 0.197 µmol/L (37% to 212%) during the last 3 days. A significant positive correlation was noted between plasma adenosine and dipyridamole levels (p = 0.001). We conclude that adenosine levels are relatively stable for an individual and are maximally increased after 2 days of oral dipyridamole.

CHROMOSOMAL LOCALIZATION AND CATALYTIC PROPERTIES OF THE RECOMBINANT ALPHA SUBUNIT OF HUMAN LYMPHOCYTE METHIONINE ADENOSYLTRANSFERASE

Human lymphocyte methionine adenosyltransferase (HuLy MAT) consists of heterologous subunits α and β. The cDNA sequence of the α subunit of HuLy MAT from Jurkat leukemic T cells was identical to that of the human kidney α subunit and highly homologous to the sequence of the extrahepatic MAT from other sources. The 3′-untranslated sequence was found to be highly conserved, suggesting that it may be important in regulating the expression of MAT. The extrahepatic α subunit of MAT was found to be expressed also in human liver, and no differences were found in the sequence of the α subunit from normal and malignant T cells. The sequence of two unspliced introns found in the cDNA clones from the Jurkat library enabled us to isolate genomic clones harboring the human extrahepatic α subunit gene and to localize it to the centromere on chromosome arm 2p, an area that corresponds to band 2p11.2. Expression of the α subunit cDNA in Escherichia coli yielded two peptides with the immunoreactivity and mobilities of authentic α/α‘ subunits from HuLy. The K of the recombinant α subunit was 80 μM, which is 20-fold higher than found for the (αα′) β holoenzyme purified from leukemic lymphocytes and 4-10-fold higher than found for the normal lymphocyte enzyme. The data suggest that the α/α‘ subunits mediate the enzyme catalytic activity and that the β subunit may be a regulatory subunit of extrahepatic MAT.

Role of transmethylation reactions in cellular motility and phagocytosis

The chemotactic responses of such eukaryotic cells as human polymorphonuclear leukocytes, monocytes and guinea pig macrophages require transmethylation reactions mediated by S-adenosyl methionine. Inhibition of methylation with inhibitors of adenosine deaminase plus adenosine and homocysteine produces as much as a 92% depression of chemotactic responsiveness. While the exact transmethylation reactions required for leukocyte chemotaxis are unknown, alterations of phospholipid methylation appear to be involved. Chemotactic agonists, but not antagonists, depress the methylation of phosphatidylethanolamine in chemotactically responsive cells. Immune phagocytosis, on the other hand, does not depress phospholipid methylation. It is speculated that occupancy of chemotactic factor receptors by chemotactic agonists alters the phospholipid composition, and thus biophysical properties, of newly synthesized membrane at local sites surrounding the receptors. Such asymmetrical alteration of leukocyte membranes may be necessary for sustained polarized locomotion.

Residue threonine‐149 of the Salmonella typhimurium CysB transcription activator: mutations causing constitutive expression of positively regulated genes of the cysteine regulon

In both Salmonella typhimurium and Escherichia coli, CysB is a LysR family transcriptional activator, which regulates genes of the cysteine regulon. Transcription activation of cys genes also requires an inducer, W‐acetyl‐L‐serine, and cysB mutants that do not require Inducer are termed constitutive, i.e. cysBc. After finding that two independently isolated cysBc mutants are substituted at amino acid residue threonine‐149 (T149), we isolated the other 17 single‐amino‐acid substitutions by site‐directed mutagenesis. Of the 19 mutant alleles, 11 supported normal growth on sulphate, and nine of these were cysBc. Four other mutants were‘leaky’cysB+, and four were cysB−. Insertions of up to 14 amino acids were also tolerated at T149, and two of three such mutants were cysBc. An allele containing a TAG translation terminator at codon 149 had no detectable function in a δcysB strain, but gave a constitutive phenotype when introduced into either wild‐type S. typhimurium or the E. coll strain NK1, which contains a cysB− mutation in a predicted helix‐turn‐helix region that interferes with specific binding of CysB to DNA and with autoregulation ot cysB. The peptide encoded by the T149ter allele is proposed to interact with the wild‐type CysB peptide or with the NK1 mutant peptide to form hetero‐oligomers that do not require N‐acetyl‐L‐serine for cys gene activation.

Regulation of carbon utilization by sulfur availability in Escherichia coli and Salmonella typhimurium

Different pleiotropic transcriptional regulators are known to function in the coordination of regulons concerned with carbon, nitrogen, sulfur, phosphorus and iron metabolism, but how expression profiles of these different regulons are coordinated with each other is not known. The basis for the effects of cysB mutations on carbon utilization in Escherichia coli and Salmonella typhimurium was examined. cysB mutations affected the utilization of some carbon sources more than others and these effects could be partially, but not completely, reversed by the inclusion of cysteine or djenkolate in the growth medium. Assays of transport systems and enzymes concerned with glucitol and alanine utilization showed that these activities were depressed in cysB mutants relative to isogenic wild-type strains, and cysteine or djenkolate present in the growth media partially restored these activities. Using transcriptional fusions to the fdo (formate dehydrogenase) and gut (glucitol) operons, it was shown that decreased expression resulted from defects at the transcriptional level. Furthermore, the effects of loss of CysB were much less pronounced under conditions of catabolite repression than in the absence of a catabolite-repressing carbon source, and cAMP largely reversed the effect of the loss of CysB. Comparable effects were seen for E. coli lacZ gene expression under the control of its own native promoter, and sulfur limitation in a cysB mutant depressed net cAMP production in a cAMP phosphodiesterase mutant. Adenylate cyclase thus appears to be responsive to sulfur deprivation. These observations may have physiological significance allowing carbon and sulfur regulon coordination during the growth of enteric bacteria in response to nutrient availability.

Purification and properties of rat lens methionine adenosyltransferase

Methionine adenosyltransferase (MAT) has been partially purified from rat lenses using a combination of ammonium sulfate fractionation and hydrophobic chromatography on phenyl Sepharose columns. The partially purified enzyme resembles purified Type II MAT from non-hepatic tissues. The Km for methionine is 3.0 microM, and the Km for ATP is 80 microM. The enzyme is activated by potassium ions (25-50 mM), and inhibited by higher concentrations of potassium. A divalent cation (magnesium or manganese) is essential for activity. The Vmax with magnesium is about five times higher than with manganese, but the optimal manganese concentration is around 2.0 mM, compared with 10-20 mM for magnesium. The enzyme is active over a broad pH range, with optimal activity between pH 7.0 and 8.0. The enzyme is inhibited by all three of its products, phosphate, pyrophosphate, and S-adenosylmethionine. Individually phosphate and pyrophosphate are weak inhibitors, but in combination they show a marked synergistic inhibitory effect. Tripolyphosphate is also an effective inhibitor. The inhibition of the enzyme by the cataractogenic agent, dimethylsulfoxide, further confirmed the similarity to Type II MAT.