Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Starch Binding Domain-containing Protein 1/Genethonin 1 Is a Novel Participant in Glycogen Metabolism

Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.     

Affinity Purification of Synenkephalin-Containing Peptides Including a Novel 23.3-Kilodalton Species

Abstract: Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1 -70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7–3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6- kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182–203) and [Met⁵]-enkephalin-Arg⁶-Gly⁷-Leu⁸. The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3- kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1–206), containing [Leu⁵]- enkephalin at the C-terminus.     

Expression of the HNK-1/NC-1 epitope in early vertebrate neurogenesis

Summary A family of glycoconjugates has recently been shown to share a common carbohydrate epitope recognized by the mouse monoclonal antibody HNK-1. The specificity of HNK-1 was found to be similar to that of another monoclonal antibody, NC-1. These two IgM monoclonal antibodies were raised after immunization of mice with a human T-cell line and avian neural crest-derived ganglia, respectively. The antigens recognized by these antibodies include the myelin-associated glycoprotein, MAG, a glycolipid of defined structure, and a set of molecules involved in cell adhesion. The timing and pattern of appearance of these antigens are distinct. Moreover, the epitope may be absent on an antigen at a given stage or in a given tissue. Therefore, although the molecules able to carry the NC-1/ HNK-1 epitope are numerous and expressed in various tissues, the use of the monoclonal antibodies on tissue sections has proven adequate for following the migration of avian neural crest cells, the major cell lineage recognized by NC-1 and HNK-1 during early embryogenesis. Analogies in several other species have been found on the basis of HNK-1 reactivity. In this study we show that NC-1 and HNK-1 can be used successfully to label migrating neural crest cells in dog, pig and human. On the other hand, the NC-l/HNK-1 epitope was not present on migrating crest cells in amphibians or mice and was found only transiently on the neural crest of rats.     

Mannitol metabolism in cultured plant cells

Non-structural storage carbohydrates were measured in 9-day-old barley ( Hordeum vulgare L. cv. Brant) primary leaves. Accumulation rates of starch, sucrose and total non-structural carbohydrates (TNC) were approximately linear when measured between 2- and 12-h of light. Progressively higher TNC accumulation rates were observed at higher irradiance levels (i.e., comparing 250, 550 and 1050 ·mol m⁻² s⁻¹). Synthesis of a low-molecular-weight fructan also was enhanced by high irradiances. Low irradiance treatments decreased leaf sucrose levels and there was a corresponding increase in the lag period preceding starch synthesis in the light. Increased starch accumulation rates were usually observed when sucrose concentrations were high. These and other results suggested that cytosolic sucrose concentrations affected starch metabolism in the chloroplast. However, sucrose accumulation rates increased and starch storage decreased when barley seedlings were transferred from 20 to 10°C during the light period. Lowering the night temperature from 20 to 10°C for a single dark period 8-days after planting increased the TNC content of barley primary leaves at the beginning of day nine. In this experiment, TNC accumulation rates of treated and untreated leaves were similar. Changes in the accumulation rate of TNC were usually observed within 2- to 4-h after barley seedlings were exposed to altered environmental conditions. Monitoring rapid changes in leaf carbohydrate levels is a sensitive method for assessing the effects of environmental treatments on photosynthetic metabolism.     

Effects of photoperiodicity and light irradiance on phosphate-limited Oscillatoria agardhii in chemostat cultures

The cyanobacterium Oscillatoria agardhii was grown in continuous culture under various light conditions in order to study the interactions of light on phosphorus-limited growth. Under severe P-limiting (light-saturating) conditions, a low chlorophyll a and C-phycocyanin content was found. In addition, the light-harvesting capacity, reflected in the values of P _max (maximum light-saturated oxygen production rate) and (photosynthetic affinity), were low compared to light-limited cultures.Reduction of the light climate, either by reduction of the length of the photoperiod or light-intensity, resulted in an increase in light-harvesting capacity (higher pigment content, P _m and ) during growth under P-limiting conditions. Light-induced changes in P _max and could be related to the relative growth rate, being the actual growth rate as a fraction of the growth rate which would be observed under light-limiting conditions.Under P-limiting conditions, reduction of the light-climate caused a reduction in dry weight of the culture. This decrease was mainly due to a decrease in carbohydrate content of the cells. Under all conditions tested, carbohydrates were found to accumulate during the light-period and to be consumed during the dark-period.Evaluation of carbohydrate consumption in the dark yielded a specific maintenance rate constant of 0.001 h^-1. This observation leads to the conclusion that the specific maintenance rate constant is independent on the character of the growth rate limiting nutrient for O. agardhii.     

An unusual pattern of carbohydrate utilization in Corallococcus (Myxococcus) coralloides (Myxobacterales)

The myxobacterium, Corallococcus (Myxococcus) coralloides strain Cc c127, could not utilize mono- and disaccharides, but maltotriose and the polysaccharides starch, amylose, amylopectin, and pullulan stimulated growth. Radioactive CO₂ was set free from ¹⁴C-labeled starch. When starch was degraded, small amounts of maltose and glucose accumulated in the culture supernatant. Maltotriose, however, appeared only temporarily. Outside the cells, the trisaccharide could not be split into glucose and maltose. Pullulan was hydrolyzed exclusively into a trisaccharide which during growth was immediately consumed. Hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and phosphoglucomutase could readily be demonstrated in cell extracts, but fructose-1,6-diphosphate aldolase was present with low activity only. The data suggest that intracellular glucose is metabolized mainly via the pentose phosphate pathway.Prof. Dr. Gerhard Drews gratefully dedicated to his 60th birthday     

虫生真菌蝉拟青霉的研究

观察了蝉拟青霉的无性世代,在24℃下载片培养结果,分生孢子经8hr萌发,24hr普遍形成菌丝,36hr出现产孢结构和产生次代分生孢子。该菌生长合适温度24—26℃,分生孢子萌芽要求相对湿度在90%以上。pH4—12范围均见生长,但以5—6为佳。对10种碳源和9种无机氮源利用检测结果,用葡萄糖作碳源孢子产量高,用果糖作碳源菌丝体产量高。不利用菊糖、L-山梨糖、L-鼠李糖。对KNO_3利用佳,但不能利用NaNO_2,和硫脲。该菌能较强抗紫外辐射。     

Developmental changes of antioxidative systems in tobacco leaves as affected by limited sucrose export in transgenic plants expressing yeast-invertase in the apoplastic space

It is generally believed that a restricted export of carbohydrates from source leaves causes oxidative stress because of an enhanced utilisation of O₂ instead of NADP⁺ as electron acceptor in photosynthesis. To test this hypothesis, developmental changes of antioxidative systems were investigated in wild-type and transgenic tobacco (Nicotiana tabacum L.) suffering from disturbed sink-source relations by expression of yeast invertase in the apoplastic space. Young expanding leaves of the wild type contained higher activities of Superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), glutathione reductase (EC 1.6.4.2) and a higher glutathione content than mature source leaves. The activity of monodehydroascorbate-radical reductase (EC 1.1.5.4) and the ascorbate content remained unaffected by the developmental stage in the wild type. In young expanding leaves of the transgenic plants the capacity of the antioxidative systems was similar to or higher than in corresponding leaves from the wild type. Source leaves of transgenic tobacco with an increased carbohydrate content showed a small chlorophyll loss, an increased malondialdehyde content, a selective loss of the activities of Cu/Zn-superoxide dismutase isoenzymes and a fourfold decrease in ascorbate compared with the wild type. There was no evidence that the protection from H₂O₂ was insufficient since source leaves of transgenic tobacco contained increased activities of catalase, ascorbate peroxidase, and monodehydroascorbate-radical reductase and an increased ascorbate-to-dehydroascorbate ratio compared with source leaves of the wild type. In severely chlorotic leaf sections of the transgenic plants, most components of the antioxidative system were lower than in green leaf sections, but the ascorbate-to-dehydroascorbate ratio was increased. These results suggest that carbohydrate-accumulating cells have an increased availability of reductant, which can increase the degree of reduction of the ascorbate system via glutathione-related systems or via the activity of monodehydroascorbate-radical reductase. At the same time, transgenic tobacco leaves seem to suffer from an increased oxidative stress, presumably as a result of a decreased consumption of O ₂ ^.- by Cu/Zn-superoxide dismutases in the chloroplasts. There was no evidence that carbohydrate-accumulating leaves acclimated to enhanced O ₂ ^.- production rates in the chloroplasts.     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.