Internal Metabolites Research Articles

A Method for Increasing the Concentration of a Specific Internal Metabolite in Steady‐State Systems

An approach is described by which it is possible to increase the concentration of any internal metabolite without affecting the concentrations of other metabolites and fluxes in the organism. This approach requires the manipulation of only a limited number of enzyme activities. The method shows which enzymes to manipulate and the extent of the manipulation required to achieve a given increase in a chosen metabolite. A case study involving tryptophan overproduction in Saccharomyces cerevisae is given as a practical example of how this method could be used.

Different signals control the activation of glycolysis in the yeast Saccharomyces cerevisiae.

The glycolytic pathway in Saccharomyces cerevisiae is activated by fermentable sugars at several steps. Mutants with deletions of genes coding for enzymes of the upper part of glycolysis were used to characterize the triggering mechanisms. Synthesis of fructose-2,6-bisphophate is catalysed by two 6-phosphofructo-2-kinase isoenzymes, one of which is activated by fermentable sugars while synthesis of the second enzyme is induced (Kretschmer and Fraenkel, 1991). Increase in the level of fructose-2,6-bisphosphate is demonstrated to depend on an internal metabolite upstream of the phosphoglucose isomerase reaction. The signalling process correlates with distinct temporal changes in the concentration of glucose-6-phosphate but not with its absolute level, indicating an adaptational mechanism. It is independent of the uptake and phosphorylation systems used by different sugars. Interestingly, this increase, although delayed, could also be observed in strains lacking the rapid cAMP increase after sugar addition which is thought to be responsible for the activating process. Synthesis of glucose-6-P and fructose-6-P is needed for the complete induction of pyruvate kinase and inactivation of fructose-1,6-bisphosphatase. On the other hand, induction of pyruvate decarboxylase depends mainly on a signal in the lower part of glycolysis.

Metabolic Pathway Characterization from Transient Response Data Obtained In Situ: Parameter Estimation in S-system Models

The actual values of internal metabolites and fluxes can be measured by a number of experimental techniques and they provide important information for evaluating the properties of a metabolic pathway in situ. In this paper we propose a strategy to properly exploit this information. The suggested approach permits estimation of a set of parameters on the whole system so that a useful model can be constructed and used to describe its components and systemic properties and to predict its behavior under new conditions. A simulated reference pathway is provided to validate this method and to show its utility in metabolic studies.

Osmotic adjustment of Zymomonas mobilis to concentrated glucose solutions

The physiological basis of the exceptionally high sugar tolerance of Zymomonas mobilis was investigated. Determinations of the internal metabolite concentrations of Z. mobilis showed that an increase in the extracellular glucose concentration was accompanied by a parallel rise in the intracellular glucose concentration, bringing about an almost complete osmotic balance between internal and external space. Studies of glucose transport confirmed that Z. mobilis has a facilitated diffusion system which enables a rapid equilibration between internal and external glucose concentrations. Studies using the non-metabolisable sugars maltose (impermeable) and xylose (permeable) revealed that these sugars were able to alter the osmotic pressure on the cytoplasmic membrane resulting in volume changes.

Leaf Carbon Metabolism and Metabolite Levels during a Period of Sinusoidal Light

Photosynthesis rate, internal CO(2) concentration, starch, sucrose, and metabolite levels were measured in leaves of sugar beet (Beta vulgaris L.) during a 14-h period of sinusoidal light, which simulated a natural light period. Photosynthesis rate closely followed increasing and decreasing light level. Chloroplast metabolite levels changed in a manner indicating differential activation of enzymes at different light levels. Starch levels declined during the first and last 2 hours of the photoperiod, but increased when photosynthesis rate was greater than 50% of maximal. Sucrose and sucrose phosphate synthase levels were constant during the photoperiod, which is consistent with a relatively steady rate of sucrose synthesis during the day as observed previously (BR Fondy et al. [1989] Plant Physiol 89: 396-402). When starch was being degraded, glucose 1-phosphate level was high and there was a large amount of glucose 6-phosphate above that in equilibrium with fructose 6-phosphate, while fructose 6-phosphate and triose-phosphate levels were very low. Likewise, the regulatory metabolite, fructose, 2,6-bisphosphate was high, indicating that little carbon could move to sucrose from starch by the triose-phosphate pathway. These data cast doubt upon the feasibility of significant carbon flow through the triose-phosphate pathway during starch degradation and support the need for an additional pathway for mobilizing starch carbon to sucrose.

Determination of underivatised efaroxan and idazoxan in blood plasma by capillary gas chromatography with mass-selective detection.

Sensitive and specific methods for the determination of efaroxan and idazoxan in blood plasma have been developed based on solvent extraction, chromatographic separation and quantification by selected-ion monitoring using a quadruple mass-selective detector. The use of a short non-polar bonded-phase capillary gas chromatography (GC) column facilitated rapid separation of the compounds of interest from internal standards, metabolites and endogenous material. Of equal significance was the ability to chromatograph these basic compounds without prior derivatisation. The application of bonded-phase capillary GC coupled to selected-ion monitoring resulted in robust analytical procedures with sub-ng ml(-1) sensitivity and high selectivity.

Interdependence between cooperativity and control coefficients

Influence of the concentration of internal metabolites on the control coefficient (defined as fractional change in flux per fractional change in enzyme activity) and regulatory properties of a given enzyme have been studied theoretically using a cyclic model of three enzymes. This model is useful to investigate the properties of the flux control coefficient for an enzyme following different rate equations. Enzymes can have high or low values of control coefficient irrespective of the type of kinetic equation, but the results obtained show that the sensitivity of these values to substrate variations is strongly dependent on its rate equation. These results help identify which kinetic equation allows the best control of a given metabolic pathway. These results have been applied to the purine nucleotide cycle. It is demonstrated that the best control of the cycle is reached when the irreversible reaction catalyzed by AMP deaminase follows a rate law that corresponds to a rational function of 2 : 2 degree with respect to AMP concentration.

The role of glutamate in modulating the synthesis and degradation of NADP-glutamate dehydrogenase from Saccharomyces cerevisiae

NADP-glutamate dehydrogenase (NADP-GDH) from Saccharomyces cerevisiae has a lower activity in yeast grown on glutamate as nitrogen source than when grown on ammonium. With the use of the immunotitration method, it was found that the difference in activity was parallel to the difference in immunoprecipitable material. By isotope incorporation studies, it was established that the decrease in NADP-glutamate dehydrogenase levels in glutamate-grown cells was brought about by an increase in the degradation rate and a decrease in the synthesis constant of the enzyme. The degradation rate of NADP-glutamate dehydrogenase is further increased in carbon-starved cells. The possible role of internal metabolites in modulating NADP-glutamate dehydrogenase degradation is discussed.

The role of glutamate in modulating the synthesis and degradation of NADP-glutamate dehydrogenase from Saccharomyces cerevisiae

NADP-glutamate dehydrogenase (NADP-GDH) from Saccharomyces cerevisiae has a lower activity in yeast grown on glutamate as nitrogen source than when grown on ammonium. With the use of the immunotitration method, it was found that the difference in activity was parallel to the difference in immunoprecipitable material. By isotope incorporation studies, it was established that the decrease in NADP-glutamate dehydrogenase levels in glutamate-grown cells was brought about by an increase in the degradation rate and a decrease in the synthesis constant of the enzyme. The degradation rate of NADP-glutamate dehydrogenase is further increased in carbon-starved cells. The possible role of internal metabolites in modulating NADP-glutamate dehydrogenase degradation is discussed.

Does osmotic regulation of amylase activity in bean cotyledons exist?

The hypothesis that the promotive effect of the embryo axis of the germinating bean seed on amylase activity in the cotyledons is mediated by an osmoregulative mechanism was examined. After 2 days of germination the action of the axis on amylolytic activity was already clearly revealed, whereas at the same time it did not have any influence on osmotic pressure in the cotyledons. When the axis was attached to one cotyledon during 4 days of incubation, osmotic pressure in the cotyledon was lower than its value in the cotyledons of the intact seedling, whereas amylolytic activity was similar in both treatments. It was concluded that the tested hypothesis is not valid in the case of the bean seedling. External osmotic agents brought about a decrease in the level of amylase in the cotyledons, but this does not prove that osmotic changes which are brought about by production of internal metabolites are involved in the regulation of amylase synthesis.

Regulation of Glucose Metabolism in Growing Yeast Cells

Publisher Summary This chapter discusses two distinct groups of yeasts—namely, glucose insensitive and glucose-sensitive yeasts. It is important to compare both yeast types and to determine whether there exist principal regulatory differences or whether there are distinct steps in the enzyme pattern, which lead to the differences in the response toward glucose. The reason for glucose repression is still obscure. A model is outlined in this review that represents a reasonable working hypothesis, but needs to be verified in the yeast system. The phenomenological similarities of the cells when other limitations are applied need to be investigated with respect to similarities or dissimilarities in the underlying molecular events. The differences discussed in the transport systems indicate that there are possibly differences in internal metabolite concentrations, which in turn may be expressed in the different regulatory responses. These observations require an extension of regulatory studies to the investigation of transport systems with respect to glucose-insensitivity and glucose-sensitivity. The current status of the research on glucose metabolism in yeasts has reached a point where new approaches are needed for elucidation of the different regulatory phenomena.

Effect of Photosynthetic Intermediates on the Magnesium Inhibition of Oxygen Evolution by Barley Chloroplasts

Millimolar concentrations of Mg(2+) inhibited CO(2)-dependent O(2) evolution by barley (Hordeum vulgare L.) chloroplasts and also prevented the activation of NADP-glyceraldehyde-3-phosphate dehydrogenase, ribulose-5-phosphate kinase, and fructose-1,6-diphosphatase by light in intact chloroplasts. When added in the dark, 3-phosphoglycerate prevented the inhibition of O(2) evolution by Mg(2+) and reduced the Mg(2+) inhibition of enzyme activation by light. Fructose 1,6-diphosphate and ribulose 5-phosphate also prevented the inhibition of O(2) evolution by Mg(2+) whereas glucose 1-phosphate, glucose 6-phosphate, ribulose 1,5-diphosphate, and citrate had no effect. Phosphoenolpyruvate gave an intermediate response. Metabolites that prevented the Mg(2+) inhibition of O(2) evolution shortened the lag phase of CO(2)-dependent O(2) evolution in the absence of M(2+). Loading chloroplasts in the dark with 3-phosphoglycerate reduced both the lag phase of O(2) evolution and the inhibition of O(2) evolution by Mg(2+). The results suggested that Mg(2+) inhibition was lessened either by external metabolites that compete with inorganic phosphate for transport into the chloroplast or by a high concentration of internal metabolites.

A thin-layer chromatographic method for the determination of acebutolol and its major metabolite in serum

A sensitive and specific thin-layer chromatographic method for the simultaneous determination of acebutolol [DL-1-(2-acetyl-4-n-butyramidophenoxy)-2-hydroxy-3-isopropylaminopropane] and its major metabolite [DL-1-(2-acetyl-4-acetamidophenoxy)-2-hydroxy-3-isopropylaminopropane] is described. A 2-ml volume of serum with 350 ng of quinidine as internal standard was extracted at pH 10, the solvent was evaporated off and the residue was dissolved in 50 mul of methanol. A 10-mul volume of the solution was spotted on a thin-layer plate and after elution (ethyl acetate-methanol-ammonia, 75:20:5) the plate was dried at 90 for 15 min and, after cooling, dipped in a 10% paraffin wax solution. The fluorescence was measured using a spectrofluorimeter with a thin-layer scanning attachment. The peak-height ratios of acebutolol to internal standard and metabolite to internal standard were used to quantitate acebutolol and the metabolite, respectively.

Pleiotropic effects of mutations involved in the regulation of Escherichia coli K-12 alkaline phosphatase.

Induction of alkaline phosphatase in wild-type Escherichia coli K-12 leads to the appearance of three new proteins in addition to alkaline phosphatase in the periplasmic space of the bacteria. These proteins are detected in autoradiograms of sodium dodecyl sulfate-acrylamide gel electropherograms of extracts from cells labeled with [(35)S]methionine. Studies with constitutive mutants defective in the three genes phoS, phoT, and phoR that have been shown to regulate alkaline phosphatase synthesis indicate that the three periplasmic proteins are coregulated with alkaline phosphatase. A mutant that has a deletion in the alkaline phosphatase structural gene phoA produces the three proteins, but a newly discovered mutant phoB that has a defect in the expression of alkaline phosphatase fails to produce the three proteins. phoB mutants are shown here to be unable to make detectable amounts of alkaline phosphatase polypeptides, as measured by immunoprecipitins or acrylamide gel electropherograms. On the basis of these results we suggest a new model for the regulation of alkaline phosphatase biosynthesis. In this model, a ternary complex composed of phoB(+) and phoR(+) gene products and an internal metabolite functions as a positive control element to regulate the transcription of several cistrons coding for periplasmic proteins.

The effect of feeding upon the phototactic behaviour of cirripede nauplii

In laboratory experiments, nauplii of Elminius modestus and Balanus balanoides were made more negative to light by prior feeding with algal cultures. This difference was observed whether or not the surrounding water contained algal metabolites. In the plankton such behaviour would result in well fed individuals leaving the surface water rich in phytoplankton for darker conditions at greater depths, where they would be less exposed to visually-directed predation. Hardy's (1936) hypothesis of animal exclusion thus receives some support but the effect is due to internal metabolites rather than external ones. Both fed and starved larvae showed similar trends, in which peak numbers became negative to light in the middle of the day, followed by a photopositive trend in the afternoon. This persisted under constant environmental conditions. The internal physiological state therefore seems to be important in controlling the behaviour of these larvae. The photonegative swimming during the day time is similar to that seen in vertical migrations of many other planktonic animals, whilst photopositive movements may be related to the need for food.

Observations on the regulation of the synthesis of the tricarboxylic acid cycle enzymes in [formula omitted], Marburg

This chapter discusses two distinct groups of yeasts—namely, glucose insensitive and glucose-sensitive yeasts. It is important to compare both yeast types and to determine whether there exist principal regulatory differences or whether there are distinct steps in the enzyme pattern, which lead to the differences in the response toward glucose. The reason for glucose repression is still obscure. A model is outlined in this review that represents a reasonable working hypothesis, but needs to be verified in the yeast system. The phenomenological similarities of the cells when other limitations are applied need to be investigated with respect to similarities or dissimilarities in the underlying molecular events. The differences discussed in the transport systems indicate that there are possibly differences in internal metabolite concentrations, which in turn may be expressed in the different regulatory responses. These observations require an extension of regulatory studies to the investigation of transport systems with respect to glucose-insensitivity and glucose-sensitivity. The current status of the research on glucose metabolism in yeasts has reached a point where new approaches are needed for elucidation of the different regulatory phenomena.

INFLUENCE OF BIOLOGICAL METHYLATION ON THE BIOSYNTHESIS OF MITOMYCIN A.

Kirsch, E. J. (Lederle Laboratories, Pearl River, N.Y.), and J. D. Korshalla. Influence of biological methylation on the biosynthesis of mitomycin A. J. Bacteriol. 87:247-255. 1964.-Methionine-methyl-C(14) was shown to contribute radioactive carbon to the mitomycin antibiotic complex synthesized by Streptomyces verticillatus in a simple synthetic medium containing glucose and inorganic salts. The position of radioactivity in mitomycin A was determined by selective hydrolysis of the 7 and 9a methoxyl functions. Essentially all of the radioactivity incorporated was distributed evenly between these two substituent groups. Mitomycin A, synthesized by washed resting cells of S. verticillatus at the expense of internal metabolites, also incorporated methyl label. When the methionine antagonist, d,l-ethionine, was added to resting cells at a concentration causing 65% inhibition of antibiotic synthesis, incorporation of radioactive methyl groups was reduced to the same extent. Synthetic medium supplemented with d,l-ethionine supported about 90% maximal growth of the culture, but antibiotic biosynthesis was markedly inhibited. The addition of the inhibitor during the period of rapid antibiotic synthesis resulted in cessation of further increases in antibiotic titer. l-Methionine was shown to be capable of reversing ethionine inhibition; the extent of reversal was dependent on the concentration, as well as on the time of addition of amino acid. The data suggest the critical nature of a methyl transfer system in the biogenesis of biologically active mitomycins.

Note on Rashevsky's equation for cellular growth

The equation for growth by intussusception involves the product of the internal concentrations ofn metabolites. This products is shown, in general, to satisfy a certain algebraic equation of then-th degree. Approximate solutions are exhibited for a somewhat wider class of cases than are considered by Rashevsky.