Effect of Pleiotropic Carbohydrate Mutations (ctr) on Tryptophan Catabolism

The pleiotropic ctr mutation has been shown to affect tryptophan uptake and tryptophanase formation. Genetic reversions are of two types: (i) complete, restoring to wild type, located at 46 to 47 min; (ii) partial, restoring only tryptophanase synthesis, located at 73 min. In some strains the effect of ctr mutations could be reversed by cyclic adenosine 3',5'-monophosphate (cAMP) plus tryptophan. A mutant producing tryptophanase constitutively was suppressed by a ctr mutation. Production of tryptophanase in this suppressed strain was not restored by the addition of cAMP, but required cAMP plus tryptophan.     

Kinetics of the onset of catabolite repression in Escherichia coli as determined by lac messenger ribonucleic acid initiations and intracellular cyclic adenosine 3'',5''-monophosphate levels.

The rates of synthesis of beta-galactosidase (EC 3.2.1.23) and the intracellular levels of cyclic 3',5'-adenosine monophosphate (cAMP) soon after the addition of glucose or glycerol to exponentially growing cultures of Escherichia coli have been determined. Within 10 s of its addition, glucose, but not glycerol, lowered the apparent initiation frequency of lac messenger ribonucleic acid. The glucose-generated reduction in initiations is identified as catabolite repression by its reversibility with cAMP. The intracellular cAMP levels respond virtually identically to glucose and glycerol additions. Thus, no correlation was observed between the rate of messenger ribonucleic acid initiation and the level of cAMP.     

Catabolite repression of tryptophanase in Escherichia coli

Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression of beta-glactosidase. Induction of tryptophanase and beta-galactosidase in cultures grown with various carbon sources including succinate, glycerol, pyruvate, glucose, gluconate, and arabinose is affected differently by the various carbon sources. The extent of induction does not seem to be related to the growth rate of the culture permitted by the carbon source during the course of the experiment. In cultures grown with glycerol as carbon source, preinduced for beta-galactosidase or tryptophanase and made permeable by ethylenediaminetetraacetic acid (EDTA) treatment, catabolite repression of tryptophanase was not affected markedly by the addition of cAMP (3',5'-cyclic adenosine monophosphate). Catabolite repression by glucose was only partially relieved by the addition of cAMP. In contrast, under the same conditions, cAMP completely relieved catabolite repression of beta-galactosidase by either pyruvate or glucose. Under conditions of limited oxygen, induction of tryptophanase is sensitive to catabolite repression; under the same conditions, beta-galactosidase induction is not sensitive to catabolite repression. Induction of tryptophanase in cells grown with succinate as carbon source is sensitive to catabolite repression by glycerol and pyruvate as well as by glucose. Studies with a glycerol kinaseless mutant indicate that glycerol must be metabolized before it can cause catabolite repression. The EDTA treatment used to make the cells permeable to cAMP was found to affect subsequent growth and induction of either beta-galactosidase or tryptophanase much more adversely in E. coli strain BB than in E. coli strain K-12. Inducation of tryptophanase was reduced by the EDTA treatment significantly more than induction of beta-galactosidase in both strains. Addition of 2.5 x 10(-3)m cAMP appeared partially to reverse the inhibitory effect of the EDTA treatment on enzyme induction but did not restore normal growth.     

Effect of cyclic adenosine-3′,5′-monophosphate on the simultaneous synthesis of β-galactosidase and tryptophanase inEscherichia coli

When inducing simultaneously β-galactosidase and tryptophanase in a batch culture either the synthesis of tryptophanase or of both enzymes is decreased due to an insufficient cAMP concentration. The addition of this nucleotide can overcome this decrease. In a continuous culture both enzymes are synthesized at the maximum rate, as the amount of cAMP produced during carbon limitation of growth is probably sufficient for the simultaneous synthesis of both enzymes. In the β-galactosidase hyperproduction mutant cultivated continuously the level of β-galactosidase markedly decreases when tryptophanase is simultaneously induced. Also this decrease is caused by cAMP insufficiency and can be overcome by increasing its concentration. cAMP is thus an important regulatory factor of both enzymes and becomes a limiting factor in their simultaneous synthesis; a competition for this regulatory compound apparently occurs and probably also a different mutual affinity of the regulatory complex with the promoter site of the enzyme opérons is involved.     

Simultaneous induction of three catabolic enzymes inEscherichia coli

During a simultaneous induction of three enzymes which are subject to catabolite repression (β-galactosidase, tryptophanase and amylomaltase, or β-galactosidase, tryptophanase and D-serine deaminase) in a batch culture, the rates of synthesis of β-galactosidase and tryptophanase decreases, while the rates of synthesis of amylomaltase and D-serine deaminase remain unaffected. The addition of cAMP brings about a considerable increase of the rate of synthesis of D-serine deaminase and a partial synthesis rate increase of β-galactosidase while the synthesis rate of tryptophanase remains lowered and the synthesis rate of amylomaltase remains unaffected. In a continuous culture β-galactosidase, tryptophanase andD-serine deaminase are synthesized simultaneously at a maximum rate without mutual influence. The addition of cAMP increases the rate of synthesis of all three enzymes.     

Effect of cyclic AMP on fructose utilization, progressive motility and protein synthesis by ram spermatozoa

Ejaculated washed ram spermatozoa showed consistent increases in the intracellular concentration of cyclic 3', 5'-adenosine monophosphate (cAMP) after incubation for 15 minutes with the phosphodiesterase (PDE)-inhibitors, theophylline and caffeine. In vitro addition of cAMP or PDE-inhibitors to ram semen also stimulated and maintained sperm motility and enhanced the rate of fructose utilization. The same doses of cAMP or theophylline significantly stimulated the rate of protein synthesis by the washed spermatozoa, while the PDE-stimulator, imidazole, inhibited protein synthesis significantly. The stimulatory effect of cAMP on sperm protein synthesis was not affected by cycloheximide, but was abolished by the mitochondrial inhibitor, chloramphenicol. The present results indicate a positive correlation between the intracellular concentration of cAMP and the rates of progressive motility, fructose utilization, and protein synthesis by ram spermatozoa. The results suggest that the effect of cAMP is associated with the synthesis of mitochndrial proteins which may be involved with the observed enhancement of sperm motility and metabolism. The data also indicate that cAMP map act either as a first or a second messenger in mature spermatoza.     

The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.     

Effect of glucose on the fruiting body formation and adenosine 3',5'-cyclic monophosphate levels in Coprinus macrorhizus

The formation of fruiting bodies in the monokaryotic fis(c) strain and a dikaryon of Coprinus macrorhizus was inhibited by growth in high-glucose media. In high-glucose media the characteristic burst of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation during fruiting-body formation was absent. Enzymatic activity assays revealed that mycelia grown in high-glucose media contained relatively lower amounts of adenylate cyclase and cAMP-phosphodiesterase than mycelia grown in low-glucose media. The synthesis of inducible d-serine deaminase and tryptophanase was repressed in high-glucose media. A mutant (gluR) in which the glucose repression of fruiting-body formation is affected was isolated by selection in high-glucose media. The mutation caused the cAMP levels to be no longer affected by glucose and affected ability to synthesize the inducible d-serine deaminase and tryptophanase. The gluR mutant was partially dominant in dikaryons. It is suggested that cAMP may play important roles in inducing fruiting bodies and in controlling inducible enzyme synthesis in C. macrorhizus.     

Hormonal regulation of cytochrome P450 enzymes, cholesterol side-chain cleavage and 17 alpha-hydroxylase/C17-20 lyase in Leydig cells

Testosterone biosynthesis in Leydig cells is dependent on two cytochrome P450 enzymes, cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha]. The expression of these two enzymes is differentially regulated by LH acting via its second messenger, cyclic adenosine 3',5'-monophosphate (cAMP), and by specific steroid hormones. P450scc is constitutively expressed in normal mouse Leydig cells and in MA-10 tumor Leydig cells. Chronic cAMP stimulation increases the steady state levels of P450scc mRNA and de novo P450scc protein synthesis. In contrast, cAMP is obligatory for de novo synthesis of P450(17 alpha) in normal mouse Leydig cells; P450(17 alpha) synthesis ceases in the absence of luteinizing hormone or cAMP. MA-10 tumor Leydig cells do not express P450(17 alpha) even after treatment with cAMP. The amount of P450(17 alpha) in Leydig cells is negatively regulated by testosterone acting by two distinct mechanisms. At low concentrations, testosterone acts via the androgen receptor to repress cAMP-induced synthesis of P450(17 alpha), whereas at high concentrations this steroid increases the rate of degradation of the enzyme by an oxygen-mediated mechanism. Both constitutive and cAMP-induced synthesis of P450scc protein and steady state levels of mRNA are modulated by glucocorticoids. In normal mouse Leydig cells, glucocorticoids repress P450scc synthesis and steady state levels of P450scc mRNA, whereas glucocorticoids stimulate P450scc synthesis and levels of P450scc mRNA in the tumor Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Location of the sequences coding for capsid proteins VP1 and VP2 on polyoma virus DNA.

The 19S and 16S polyoma virus late mRNAs have been separated on sucrose-formamide density gradients and translated in vitro. The 16S RNA codes only for polyoma capsid protein VP1, while the 19S RNA codes in addition for capsid protein VP2. Since the 19S and 16S species have been previously mapped on the viral genome, these results allow us to deduce the location of the sequences coding for VP1 and VP2. Comparison of the chain lengths of the capsid proteins with the size of the viral mRNAs coding for them suggests that VP1 and VP2 are entirely virus-coded. Purified polyoma 19S RNA directs the synthesis of very little VP1 in vitro, although it contains all the sequences required to code for the protein. The initiation site for VP1 synthesis which is located at an internal position on the messenger is probably inactive either because it is inaccessible or because it lacks an adjacent "capped" 5' terminus. Similar inactive internal initiation sites have been reported for other eucarotic viral mRNAs (for example, Semliki forest virus, Brome mosaic virus, and tobacco mosaic virus), suggesting that while eucaryotic mRNAs may have more than one initiation site for protein synthesis, only those sites nearer the 5' terminus of the mRNA are active.     

Requirement of chain initiation factor 3 and ribosomal protein S1 in translation of synthetic and natural messenger RNA.

Amino acid incorporation directed by poly(A), poly(U) or R17 RNA has been examined in S1-depleted protein synthesizing systems. We observe that the translation of either synthetic or natural messenger RNA is strictly dependent on the presence of chain initiation factor 3 and ribosomal protein S1. With poly(A) or poly(U) both IF-3 and S1 stimulate amino acid incorporation at least 25-fold, and with R17 RNA the stimulation is approximately 15-fold. More than one copy of S1 per ribosome decreases amino acid incorporation directed by poly(U) or R17 RNA. Initiation complex formation with R17 RNA is also stimulated optimally by the addition of one copy of S1 per ribosome. The function of IF-3 and S1 in protein synthesis is considered.     

Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli

1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).     

Effects of 8-substituted analogs of cyclic adenosine 3'',5''-monophosphate on in vivo and in vitro syntheses of beta-galactosidase in Escherichia coli.

Several 8-substituted alkylthio and alkylamino cyclic adenosine 3',5'-monophosphate (cAMP) derivatives were tested for their ability to stimulate beta-galactosidase synthesis in Estherichia coli in vivo and in vitro and to inhibit the cAMP phosphodiesterase activity of E. coli. Stimulation of beta-galactosidease synthesis in vivo by cAMP derivatives decreased with increasing length of the unbranched carbon chain of the substituent. On the other hand, the stimulation in vitro was increased as the carbon chain elongated. The 8-decylthio- and 8-dodecylthio-cAMP compounds stimulated beta-galactosidase synthesis almost eight-fold compared with cAMP, whereas 8-undecyl-, 8-dodectyl-, and 8-tridecylamino-cAMP stimulated beta-galactosidase synthesis about threefold. However, in in vitro experiments with a phosphodiesterase-deficient strain of E. coli, the Crooks strain, the stimulatory effects of the derivatives disappeared, except for 8-dodecylthio cAMP which stimulated beta-galactosidase about 1.4- to 1.6-fold. All derivatives were quite resistant to hydrolysis by phosphodiesterase. Most derivatives competitively inhibited the hydrolysis of cAMP by phosphodiesterase.     

Regulation of cell division and of tyrosinase in B16 melanoma cells by imidazole: a possible role for the concept of metabolite gene regulation in mammalian cells

Results of hemacytometer cell counts and of tyrosinase measurements made by the Pomerantz method demonstrate that imidazole added to the medium of cultured B16 mouse melanoma cells can stimulate tyrosinase specific activity and inhibit cell division. These effects are greater than with adenosine 3',5' cyclic monophosphate (cAMP) or the cAMP-phosphodiesterase inhibitor theophylline. The effects of imidazole on cell division and tyrosinase are enhanced by theophylline and antagonized by cAMP. Cyclic AMP-phosphodiesterase activity in cell-free extracts can be inhibited by theophylline and stimulated by imidazole. However, imidazole does not affect cAMP-phosphodiesterase specific activity in vivo, nor does it affect intracellular cAMP concentrations as determined by competitive protein-binding assays. In contrast, the specific activity of cAMP-phosphodiesterase in vivo is stimulated by cAMP and theophylline, supporting the hypothesis that cAMP and agents which increase intracellular cAMP concentrations induce the synthesis of cAMP-phosphodiesterase. Studies with actinomycin-D and cycloheximide support the hypothesis that cAMP can also mediate posttranslational activation of tyrosinase. Similar experiments suggest that imidazole, or a derivative thereof, can induce the synthesis of tyrosinase at the pretranslational level of control. We hypothesize that this type of regulation (pretranslational) by imidazole may define a role for the concept of "Metabolite Gene Regulation" (MGR), in mammalian cells.     

Differential and synergistic actions of nerve growth factor and cyclic AMP in PC12 cells

When a clonal line of rat pheochromocytoma (PC12) was exposed to beta-nerve growth factor (beta NGF), N6, O2-dibutyryl adenosine 3':5' cyclic monophosphate (Bt2cAMP), or a combination of the two, 10, 26, or 70% of the cell clumps, respectively, displayed neurites after 1.d. Increases in the cellular RNA concentration were also found to be additive or greater when both agents were present. Neurites induced by Bt2cAMP alone were not maintained after replacement with beta NGF. The degree of potentiated neurite outgrowth was a function of the time of simultaneous exposure to both agents. The initiation of neurite outgrowth in the presence of Bt2cAMP was independent of RNA synthesis, in contrast to that induced by beta NGF alone. We conclude that beta NGF-induced initiation of morphological differentiation of these cells is not mediated by a cAMP-dependent mechanism. Consideration of Bt2cAMP effects upon other cell lines suggest that Bt2cAMP causes a rapid, but unstable, reorganization of the PC12 cytoskeleton, resulting in the initiation of neurite outgrowth from these cells. In contrast, beta NGF alone achieves a more stable cytoskeleton reorganization by an RNA synthesis-dependent mechanism.     

Biochemical signals transmitted by Fc gamma receptors: triggering mechanisms of the increased synthesis of adenosine-3',5'-cyclic monophosphate mediated by Fc gamma 2a- and Fc gamma 2b- -receptors of a murine macrophage-like cell line (P388D1)

The specific binding of IgG2a or IgG2b subclass monoclonal anti-sheep erythrocyte antibodies to P388D1 cell surface Fc gamma 2aR3 or Fc gamma 2bR, respectively, triggered the synthesis of adenosine-3'5'-monophosphate (cAMP) to an approximately same extent by the mechanisms that are apparently unique for each type of Fc gamma Rs. Fc gamma 2aR appeared to trigger directly, upon binding of IgG2a antibodies, the adenylate cyclase system without requiring the participation of guanine nucleotide-binding (G/F) regulatory protein, because the Fc gamma 2aR-triggered cAMP synthesis, which reached maximum within 30 min, was not significantly affected by an uncoupler, Mn++ or by addition of guanosine triphosphate (GTP) analog, 5'-guanylylimidodiphosphate (Gpp(NH)p). In contrast, Fc gamma 2bR appeared to stimulate indirectly the G/F regulatory requiring-adenylate cyclase system by generating prostaglandins, since the cAMP synthesis, which required 90 min to reach plateau after binding of IgG2b to Fc gamma 2bR, was totally suppressed by phospholipase A2 inhibitor (p-bromophenacylbromide) or cyclo-oxygenase inhibitor (indomethacin), partially suppressed by Mn++, and slightly increased by Gpp(NH)p. Furthermore, the inhibition of phagocytic process by cytochalasin D increased cAMP synthesis mediated by Fc gamma 2aR (about 70% at 2 micrograms/ml), but did not affect Fc gamma 2bR-mediated cAMP synthesis. In addition, our data suggested that both Fc gamma 2aR- and Fc gamma 2bR-mediated cAMP synthesis are independent from beta-adrenergic receptor-mediated stimulation of the adenylate cyclase system, since either beta-agonist (isoproterenol) or beta-antagonist (propranolol) did not affect significantly the levels of cAMP produced in response to EA-stimulation.