Physiological study of ergot: induction of alkaloid synthesis by tryptophan at the enzymatic level.

The enhancement of ergot alkaloid production by tryptophan and its analogues in both normal and high-phosphate cultures is more directly related to increased dimethylallyltryptophan (DMAT) synthetase activity rather than to a lack of regulation of the tryptophan biosynthetic enzymes. Thiotryptophan [beta-(1-benzo-thien-3-yl)-alanine] is rather ineffective in the end product regulation of tryptophan biosynthesis, whereas tryptophan and 5-methyltryptophan are potent effectors. The presence of increased levels of DMAT synthetase in ergot cultures supplemented with tryptophan or thiotryptophan, and to a lesser extent with 5-methyltryptophan, suggests that the induction effect involves de novo synthesis of the enzyme. Thiotryptophan and tryptophan but not 5-methyltryptophan can overcome the block of alkaloid synthesis by inorganic phosphate. The results with thiotryptophan indicate that the phosphate effect cannot be explained merely on the basis of a block of tryptophan synthesis.     

Physiological role of tryptophanase in control of tryptophan biosynthesis in Bacillus alvei

Hoch, J. A. (University of Illinois, Urbana), and R. D. DeMoss. Physiological role of tryptophanase in control of tryptophan biosynthesis in Bacillus alvei. J. Bacteriol. 91:667-672. 1966.-Indole excretion occurred early in the exponential growth phase, and derived mainly from biosynthetic intermediates of tryptophan. Tryptophan cleavage by tryptophanase contributed about 1.5% of the indole excreted. In the presence of exogenous tryptophan (5 to 10 mug/ml), excretion of early indole was not observed. Experiments with isotopically labeled indole and tryptophan showed that a low rate of endogenous tryptophan biosynthesis occurred constantly during growth. Both exogenously and endogenously supplied tryptophan were degraded by tryptophanase. As a consequence, the intracellular tryptophan concentration appeared to be maintained at a constant low level. It was suggested that the action of tryptophanase is an example of an enzymatic mechanism which controls the level of a specific metabolite pool.     

Physiological aspects of apoptosis

Study of apoptosis is a new stage in development of biomedical sciences. At the cellular level the process is realised on account of efferents supporting an equilibrium among proliferation, differentiation, and programmed cell death. Significance of each of the factors is determined by its role in physiological systems functioning. Cell death infringement may cause pathological conditions and diseases followed by both degenerative and proliferative changes.     

Physiological and pathophysiological aspects of ceramide

Activation of cells by receptor- and nonreceptor-mediated stimuli not only requires a change in the activity of signaling proteins but also requires a reorganization of the topology of the signalosom in the cell. The cell membrane contains distinct domains, rafts that serve the spatial organization of signaling molecules in the cell. Many receptors or stress stimuli transform rafts by the generation of ceramide. These stimuli activate the acid sphingomyelinase and induce a translocation of this enzyme onto the extracellular leaflet of the cell membrane. Surface acid sphingomyelinase generates ceramide that serves to fuse small rafts and to form large ceramide-enriched membrane platforms. These platforms cluster receptor molecules, recruit intracellular signaling molecules to aggregated receptors, and seem to exclude inhibitory signaling factors. Thus ceramide-enriched membrane platforms do not seem to be part of a specific signaling pathway but may facilitate and amplify the specific signaling elicited by the cognate stimulus. This general function may enable these membrane domains to be critically involved in the induction of apoptosis by death receptors and stress stimuli, bacterial and viral infections of mammalian cells, and the regulation of cardiovascular functions.     

Physiological aspects of the regulation of ketogenesis

The importance of ketone bodies (acetoacetate and 3-hydroxybutyrate) as substrates for peripheral tissues, especially nervous tissue, of man is now firmly established. This has renewed interest in the factors that control the production of ketone bodies by the liver in various physiological situations, such as alterations of dietary status, stage of development or alteration in demand for circulating substrates (e.g. in exercise or lactation). In the discussion of the regulation of ketogenesis in the present paper, distinction is made between extrahepatic and intrahepatic control. The former is mainly concerned with the factors (e.g. hormonal status of animals) that alter the flux of non-esterified fatty acids to the liver, whereas intrahepatic regulation involves the fate (esterification versus beta-oxidation) of fatty acids within the liver. Emphasis is placed on the fact that alterations in blood glucose concentrations are indirectly responsible, via effects on insulin secretion, for the extrahepatic control of ketogenesis. By analogy, it is postulated that the carbohydrate status of the liver may play a role in the intrahepatic regulation of ketogenesis. Some support for this postulate is provided by comparison of measurements of blood ketone-body concentrations in various inborn errors of hepatic carbohydrate metabolism (e.g. deficiencies of glucose 6-phosphatase, fructose 1,6-bisphosphatase and glycogen synthase) in man and by experiments with isolated rat hepatocytes. Present information on the short- and long-term factors that may be responsible for the altered rates of ketogenesis during the foetal-neonatal and suckling-weanling transitions, in lactation, on feeding a high-fat diet and post-exercise is discussed. It is concluded that the major factors involved in the regulation of ketogenesis in these situations are (a) flux of non-esterified fatty acids to the liver and (b) the partitioning of long-chain acyl-CoA between the esterification and beta-oxidation pathways.     

Stereoelectronic control of bond formation in Escherichia coli tryptophan synthase: substrate specificity and enzymatic synthesis of the novel amino acid dihydroisotryptophan

The reactions of the indole analogues indoline and aniline with the Escherichia coli tryptophan synthase alpha-aminoacrylate Schiff base intermediate have been characterized by UV-visible and 1H NMR absorption spectroscopy and compared with the interactions of indole and the potent inhibitor benzimidazole. Indole, via the enamine functionality of the pyrrole ring, reacts with the alpha-aminoacrylate intermediate, forming a transient quinonoid species with lambda max 476 nm as the new C-C bond is synthesized. Conversion of this quinonoid to L-tryptophan is the rate-limiting step in catalysis [Lane, A., & Kirschner, K. (1981) Eur. J. Biochem. 120, 379-398]. Both aniline and indoline undergo rapid N-C bond formation with the alpha-aminoacrylate to form quinonoid intermediates; benzimidazole binds rapidly and tightly to the alpha-aminoacrylate but does not undergo covalent bond formation. The indoline and aniline quinonoids (lambda max 464 and 466 nm, respectively) are formed via nucleophilic attack on the electrophilic C-beta of the alpha-aminoacrylate. The indoline quinonoid decays slowly, yielding a novel, new amino acid, dihydroisotryptophan. The aniline quinonoid is quasi-stable, and no new amino acid product was detected. We conclude that nucleophilic attack requires the precise alignment of bonding orbitals between nucleophile and the alpha-aminoacrylate intermediate. The constraints imposed by the geometry of the indole subsite force the aromatic rings of indoline, aniline, and benzimidazole to bind in the same plane as indole; thus nucleophilic attack occurs with the N-1 atoms of indoline and aniline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic diversity of the tryptophan pathway in chlamydiae: reductive evolution and a novel operon for tryptophan recapture

Background  

Complete genomic sequences of closely related organisms, such as the chlamydiae, afford the opportunity to assess significant strain differences against a background of many shared characteristics. The chlamydiae are ubiquitous intracellular parasites that are important pathogens of humans and other organisms. Tryptophan limitation caused by production of interferon-γ by the host and subsequent induction of indoleamine dioxygenase is a key aspect of the host-parasite interaction. It appears that the chlamydiae have learned to recognize tryptophan depletion as a signal for developmental remodeling. The consequent non-cultivable state of persistence can be increasingly equated to chronic disease conditions.     

The molecular pathway for the allosteric regulation of tryptophan synthase

The pyridoxal 5'-phosphate (PLP)-dependent tryptophan synthase is a alpha(2)beta(2) complex. The alpha-beta subunit interaction plays a critical role both in the reciprocal activation of the individual subunits and in the allosteric regulation. We have investigated whether mutations of alpha loop6 Gly(181) and beta helix6 Ser(178) affect intersubunit communication. The loss of the hydrogen bond between these residues, achieved by proline substitution, does not significantly influence the intersubunit catalytic activation, but completely abolishes ligand-induced intersubunit signaling. The comparison of the crystal structure of the wild type and beta Ser(178)Pro mutant, in the absence and presence of alpha-subunit ligands, indicates that the removal of the interaction between beta Ser(178) and alpha Gly(181) strongly affects the equilibrium between active (closed) and inactive (open) conformations of the alpha-active site, the latter being stabilized in both mutants.