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A previous study showed that significantly less taurine is excreted in the urine by epileptics than by control subjects. The difference is ascribed to genetic variation in taurine transport governed by a pair of codominant polymorphic alleles. The present study of plasma taurine concentrations and urinary taurine output confirms previous findings among epileptics and provides evidence that some anticonvulsant medications may affect taurine transport. The posited codominant alleles represent the first single-locus component in the polygenic complexes creating susceptibility to seizures and epitomizes the small additive effects classically attributed to such genes.

The embryonic development of skeletal muscle proceeds by the adherence and fusion of myoblast cells to form multinucleated myotubes. In the present study, enzymes in the dolichol pathway for asparagine-linked glycoprotein synthesis and oligosaccharide chain composition were characterized in myoblasts and myotubes derived from the C2 (mouse) muscle cell line. The N-acetylglucosaminyltransferase responsible for chain initiation and the mannosyl- and glucosyltransferases for Dol-P-Man and Dol-P-Glc synthesis were characterized with respect to substrate, cation, and detergent dependence. Time course studies in the absence and presence of exogenous Dol-P revealed that myoblasts had a two- to threefold higher capacity than myotubes for Dol-sugar synthesis. Pulse-chase experiments following the elongation of the Dol-oligosaccharide by intact cells showed myoblasts to label oligosaccharide intermediates approximately fourfold greater than myotubes; myotubes, however, were more efficient than myoblasts for converting the intermediates to the glucosylated Dol-tetradecasaccharide. Oligosaccharide chains isolated from sarcolemma glycopeptides were analyzed by Con A, WGA, and QAE chromatography. There were no differences between myoblast and myotube oligosaccharides with respect to the proportion of tri-tetraantennary complex, biantennary complex, and high mannose chains. Hybrid chains were not detected. The major high mannose chain contained nine mannose residues. Sialyltransferase activity was identical. The results suggest that higher levels of Dol-P and protein acceptor contribute to the greater degree of protein glycosylation in myoblast vs myotube muscle cells.

Supplementing the salts-glucose medium of Escherichia coli with adenine initiates induction of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), growth inhibition, and an increased potential for the net deamination of adenine. The extent and duration of these events are proportional to the initial adenine concentration and are dependent upon adenylate pyrophosphorylase and repression of histidine biosynthesis for maximal expression. The conversion of adenine to hypoxanthine, though limited in rate, occurs concurrently with induction and accounts for the progressively decreasing rate of deaminase induction, since hypoxanthine is a relatively ineffective inducer. The subsequent decrease in deaminase activity is due to dilution by continued cell division and by enzyme inactivation which occurs during the late-log and early-stationary phases. The partially purified deaminase is labile to a number of environmental conditions, particularly to phosphate buffers of pH 6.8 or less. A disproportionately slow rate of adenine deamination by cells utilizing lactate permits a more prolonged period of induction and, consequently, a greater quantity of enzyme to be synthesized; cell division, but not enzyme inactivation, reduces enzyme concentration. The adenosine deaminases of Aerobacter aerogenes and Salmonella typhimurium are not inducible.

This study is concerned with the isolation and characterization of the enzyme, S-adenosylmethionine:ribosomal ribonucleic acid-adenine (N(6-)) methyl-transferase [rRNA-adenine (N(6)-) methylase] of Escherichia coli strain B, which is responsible for the formation of N(6)-methyladenine moieties in ribosomal ribonucleic acids (rRNA). A 1,500-fold purified preparation of the species-specific methyltransferase methylates a limited number of adenine moieties in heterologous rRNA (Micrococcus lysodeikticus and Bacillus subtilis) and methyl-deficient homologous rRNA. The site recognition mechanism does not require intact 16 or 23S rRNA. The enzyme does not utilize transfer ribonucleic acid as a methyl acceptor nor does it synthesize 2-methyladenine or N(6)-dimethyladenine moieties. Mg(2+), spermine, K(+), and Na(+) increase the reaction rate but not the extent of methylation; elevated concentrations of the cations inhibit markedly. The purified preparations utilize 9-beta-ribosyl-2,6-diaminopurine (DAPR) as a methyl acceptor with the synthesis of 9-beta-ribosyl-6-amino-2-methylaminopurine. A comparison of the two activities demonstrated that one methyltransferase is responsible for the methylation of both DAPR and rRNA. This property provides a sensitive assay procedure unaffected by ribonucleases and independent of any specificity exhibited by rRNA methyl acceptors.

The inhibition of Escherichia coli strain B and strain W-11 by 6-methylpurine depended on the formation of 6-methylpurine ribonucleotide by the action of adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.7). 6-Methylpurine ribonucleotide inhibited the de novo synthesis of purines, presumably via pseudofeedback inhibition of phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14). The same mechanism accounted for its inhibition of adenylosuccinate synthetase [IMP: l-aspartate ligase (GDP), EC 6.3.4.4]. Adenine and 6-methylaminopurine prevented inhibition by competing for the action of adenine phosphoribosyltransferase. In addition, adenine reversed this inhibition by replenishing the AMP to bypass both sites of inhibition. Nonproliferating suspensions of strain B-94, which lacked adenylosuccinate lyase (EC 4.3.2.2), converted exogenous hypoxanthine and aspartate to succinoadenine derivatives which accumulated in the medium. Compounds which inhibited adenylosuccinate synthetase inhibited accumulation of the succinoadenine derivatives. A method was described for the isolation of mutants which potentially possessed an altered adenylosuccinate synthetase.

These investigations are concerned with the ionic and substrate-site requirements of ribosomal ribonucleic acid (rRNA)-adenine (N(6)-) methylase of Escherichia coli B. The methylase was essentially inactive in solutions of low ionic strength. The addition of MgCl(2) (optimal at 5 mM) or; to a lesser degree, KCl (optimal at 45 mM) stimulated the rate of methylation; the combination of MgCl(2) and KCl stimulated methylation to an extent equivalent to the sum of the stimulation of each acting alone. The extent of nonspecific binding of the methylase to rRNA decreased as the ionic strength of the solution increased. In the absence of ions, dimethylsulfoxide (DMSO), a nucleic acid denaturing agent, had little influence on the rate of methylation; however, DMSO plus KCl synergistically increased both the rate and the extent of methylation to a greater degree than the combination of Mg(2+) plus K(+). NH(4) (+) was less effective than K(+), and the divalent Mg(2+) offered little stimulation. Monovalent anions (acetate, nitrate, and chloride) were equally effective, whereas divalent SO(4) (2-) was decidedly inhibitory. The appropriate ionic milieu of mono- and divalent cations was required to provide the appropriate conformation of the rRNA and to facilitate specific interactions of the methylase and its recognition sites in the rRNA, while decreasing nonspecific ionic binding of the methylase to rRNA. DMSO may facilitate methylation by increasing the number of substrate sites exposed in single-stranded regions of the rRNA. Nonmethylatable rRNA species served as competitive inhibitors, whereas the polyanions deoxyribonucleic acid, transfer RNA, and polyadenylic acid were inactive. Micrococcus lysodeikticus and Bacillus subtilis rRNA, methylated by the methylase, each contained two distinct heptanucleotides containing newly synthesized 6-methyladenine moieties. The data are consistent with the view that E. coli strain B possesses two species of rRNA-adenine (N(6)-) methylases, each of which recognizes a specific adenine moiety in a unique pentapurine nucleotide sequence in a single-stranded region of rRNA.

Love, Samuel H. (Bowman Gray School of Medicine, Wake Forest College, Winston-Salem, N.C.), and Charles N. Remy. Metabolism of methylated purines in Escherichia coli: derepression of purine biosynthesis. J. Bacteriol. 91:1037-1049. 1966.-Various methylated purines were examined for their effects on growth of purine-requiring mutants of Escherichia coli, strains W-11 and B-96, and for their effects on purine biosynthesis. 6-Methylaminopurine and 6-methoxypurine stimulated the accumulation of purine precursor derivatives (ribosyl-5-aminoimidazole and ribosyl-5-amino-4-imidazole carboxamide) beyond their ability to support growth. Information obtained from in vivo and in vitro systems demonstrated that the metabolism of 6-methylaminopurine and 6-methoxypurine utilized identical pathways. The riboside derivatives are formed either by direct ribosidation via nucleoside phosphorylase or, indirectly, by dephosphorylation of the 5'-phosphoribosyl derivatives which are synthesized via adenylate pyrophosphorylase. Information obtained with the aid of strain W-11/DAP (lacking adenylate pyrophosphorylase) demonstrated that both pathways were important to the growing cells. Regardless of the metabolic pathway by which they are synthesized, the ribosyl derivatives are demethylaminated (demethylated) by adenosine deaminase to yield inosine. The final conversion of inosine to inosinic acid via the intermediate formation of hypoxanthine accounts for the net conversion of the methylated bases to inosinic acid. The utilization of the bases is sufficiently rate-limiting to cause derepression of the early enzymes required for the de novo synthesis of purine, thus accounting for the elevated accumulation of purine precursors originally observed.