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M Gacto

Publications and source records attributed to M Gacto.

32 records · Page 2Linked to original sources
PubMed1995

Activation of cytoplasmic trehalase by cyclic-AMP-dependent and cyclic-AMP-independent signalling pathways in the yeast Candida utilis.

Derepressed cells of Candida utilis suspended in buffer exhibited both a transient cAMP-mediated signal and a marked activation of cytoplasmic trehalase when supplemented with glucose. Nitrogen sources or protein synthesis inhibitors, as well as protonophores or uncouplers, were also able to cause trehalase stimulation in derepressed cells even in the absence of the sugar. The increase in trehalase activity caused by nitrogen sources or protein synthesis inhibitors was not accompanied by changes in cAMP levels. Moreover, acridine orange inhibited both the cAMP signal and the glucose-induced activation of trehalase without affecting the increase in trehalase activity caused by nitrogen sources or protein synthesis inhibitors. These results suggest that cAMP is not involved as second messenger in the signal for trehalase stimulation induced by the latter compounds. By contrast, the addition of glucose to repressed cells suspended in buffer failed to cause the cAMP-mediated glucose signal and sugar-induced trehalase activation. No significant changes in either trehalase activity or cAMP concentration were observed upon addition to these cells of asparagine, cycloheximide, anisomycin or other agents, including protonophores and uncouplers. However, heat treatment of repressed cultures resulted in a moderate increase in trehalase activity with negligible change in cAMP levels, whereas such an effect was not observed in derepressed cultures. The thermally induced increase in trehalase activity was dependent on de novo protein synthesis and required the presence of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Candida↗
PubMed1995

Increased thermal stability of the enzyme content in permeabilized whole cells from the fission yeast Schizosaccharomyces pombe by exogenous trehalose and other compounds.

Cells of the fission yeast Schizosaccharomyces pombe were permeabilized by treatment with toluene-ethanol. The permeabilized cells lost the bulk of the internal trehalose pool while most of the alkaline phosphatase, invertase, alpha-glucosidase, or neutral trehalase activities located inside the cells remained unaffected. This system was used as an in situ assay to determine the involvement of trehalose in enzyme protection during thermal treatments. The addition of trehalose to suspensions of permeabilized cells resulted in a sugar-dependent thermoprotection of the internal marker enzymes. This approach demonstrates that in whole cells of the fission yeast trehalose plays a physiological role as a protective molecule against thermal denaturation of cellular enzymes.

Alkaline Phosphatase↗
PubMed1994

Cyclic AMP signalling pathway and trehalase activation in the fission yeast Schizosaccharomyces pombe.

The response of derepressed cells of Schizosaccharomyces pombe to the addition of glucose included a marked and reversible activation of neutral trehalase that was not produced in repressed cells. The protein synthesis inhibitor cycloheximide, the protonophore 2,4-dinitrophenol or the uncoupler sodium azide also enhanced trehalase activity in derepressed cells provided glucose was present in the incubation assays. However, only 2,4-dinitrophenol or cycloheximide was able to induce trehalase activation in repressed cells. Stimulation of trehalase by these compounds was preceded in all cases by a rapid increase in adenosine 3'-5'-cyclic monophosphate (cAMP) content. Since exogenous cAMP can activate trehalase both in repressed and derepressed growing cells, the results provide evidence for the existence of an induced cAMP signalling pathway in the fission yeast with several entries for trehalase activation. The correlation between cAMP increase and trehalase activation was not maintained when the enzyme was heat-shock-activated, supporting the concept that trehalase activity can be also enhanced in cells by another mechanism in which cAMP does not act as second messenger.

2,4-Dinitrophenol↗
PubMed1994

Sensitivity of fructose-1,6-bisphosphatase to glucose and cyclic AMP in the fission yeast Schizosaccharomyces pombe.

Stationary phase cells from resting cultures of Schizosaccharomyces pombe, suspended in buffer, did not decrease fructose-1,6-bisphosphatase (FbPase) activity upon addition of glucose to the cell suspension. In contrast, the addition of glucose to cells from derepressed growing cultures resulted in a 4- to 5-fold reduction in FbPase activity within minutes after the addition of the sugar. This response was independent of protein synthesis, was accompanied by a rise in the cyclic AMP (cAMP) level and was concomitant with an increase in the activity of neutral trehalase. The addition of exogenous cAMP to these cells provoked a decrease in FbPase similar to that induced by glucose. The occurrence of a glucose-induced cAMP signal was not sufficient to trigger the decrease in FbPase activity, which appeared to require additional transduction elements not active in growth-arrested cells. In assays performed in vitro it was found that the enzyme activity was strongly inhibited by fructose-2,6-bisphosphate.

Culture Media↗
PubMed1991

Proteolytic activation of alpha,alpha-trehalose 6-phosphate synthase in Candida utilis.

Total trehalose 6-phosphate synthase activity increased in cell-free extracts from Candida utilis following short-term preincubation of the enzyme samples at 37 degrees C. This endogenous activation was prevented by the inhibitors of serine-type proteases, phenylmethylsulfonyl fluoride, antipain or chymostatin, but not by other protease inhibitors such as pepstatin. Fractionation of the cell extracts by Sephadex G-200 gel filtration revealed that the activity of one of the two synthase enzymes present in these cells was enhanced after the activation treatment. These observations indicate the existence of a proteolytically activatable enzyme form in the trehalose 6-phosphate synthase complex of this yeast in addition to the previously characterized enzyme, whose activity appears to be inactivated by reversible phosphorylation.

Candida↗
PubMed1991

On the role of trehalose in yeast cells subjected to hypersomotic shock.

Plate counts on both high and low water activity (aw) media were performed during the growth of Candida utilis in batch culture. The results revealed a marked discrepancy between the counts on the two media in the logarithmic phase. The discrepancy almost disappeared in stationary phase revealing a higher resistance of these cells, as compared to growing cells, to the severe dehydration imposed by the hyperosmotic shock upon transfer to low aw medium. Since the two types of cells differ in the level of endogenous trehalose the relation between plating discrepancy and trehalose content of the cells was investigated. Treatments that changed the intracellular trehalose concentration did not modify the plate counts on low aw medium. It was therefore concluded that the amount of trehalose into the cells is not the only factor conferring resistance against the hyperosmotic shock. Glycerol content did not correlate with resistance to the water stress either. Congruent with the former conclusion, other yeast species (Sporobolomyces salmonicolor, Schizosaccharomyces pombe) showed no correlation between changes in the trehalose content and susceptibility or resistance to the osmotic stress in low aw medium.

Candida↗
PubMed1989

Presence of two trehalose-6-phosphate synthase enzymes in Candida utilis.

Total trehalose-6-phosphate synthase activity decreased in cell extracts from Candida utilis under conditions inducing activation of the regulatory trehalase by protein kinase catalysed phosphorylation. The synthase activity was reactivated by treatment with alkaline phosphatase revealing the presence of an enzyme whose activity is inactivated by reversible phosphorylation. The occurrence in the trehalose-6-phosphate synthase complex of a second synthase enzyme whose activity is not controlled by phosphorylation and dephosphorylation was demonstrated following gel filtration of cell extracts. The activity of the isolated enzymes was differently modified in vitro by the presence of alkaline phosphatase, ATP, glucose or protein kinase.

Adenosine Triphosphate↗
PubMed1988

Differential location of regulatory and nonregulatory trehalases in Candida utilis cells.

The isolation of vacuoles by density gradient centrifugation of protoplast lysates from Candida utilis cells showed a high specific activity for nonregulatory trehalase in vacuoles whereas the regulatory trehalase activatable by phosphorylation behaves as a cytoplasmic enzyme. The vacuolar trehalase is a glycoprotein that can be precipitated by Con A-Sepharose. Treatment of this enzyme with endo H reduced its reactivity with the lectin without loss of enzyme activity and decreased its apparent molecular weight by gel filtration.

Candida↗
PubMed1987

Inhibition by alpha factor of the glucose-induced activation of regulatory trehalase in Saccharomyces cerevisiae.

The enzyme activity of the regulatory trehalase (alpha, alpha-trehalose glycohydrolase; EC 3.2.1.28) in stationary-phase cells of Saccharomyces cerevisiae increased upon addition of glucose to cell suspensions. Such increase was temporarily retarded in the presence of alpha factor in the medium. The transient inhibition required the joined action of the pheromone-like factor and the protease inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone. The inhibition of the glucose-induced activation of trehalase by alpha factor lends support to the involvement of adenosine 3',5'-cyclic monophosphate in the enzyme activation in vivo.

Cell Division↗
PubMed1986

Comparative study of two trehalases from Candida utilis.

Candida utilis ATCC 60459 contains two intracellular trehalase enzymes clearly distinguishable by molecular weight, behaviour in ion-exchange chromatography and kinetic properties. The high molecular weight trehalase (500 kDa trehalase) is specifically inhibited by acetate and accounts for less than 30% of the total trehalase activity found in cell extracts. The smaller trehalase (280 kDa trehalase) exists mostly as a cryptic enzyme whose activity can be postranslationally activated by cAMP-dependent phosphorylation. The enzyme activity of the 280 kDa trehalase is strongly inhibited by Zn2+ and markedly enhanced in the presence of Ca2+ and Mn2+. The activation by these cations, contrariwise to that induced by ATP and cAMP, does not imply a covalent modification of the 280 kDa enzyme. Several parameters have been determined for both enzymes. The 280 kDa enzyme has the properties shown by the so-called regulatory trehalases whereas the 500 kDa enzyme presents characteristics of a nonregulatory type of trehalase.

Adenosine Triphosphate↗
PubMed1978

Loss of cell-population-density-dependent incorporation of fucose into the lipid fraction of cultured human tumour cells.

The incorporation of radioactively labelled fucose into the lipid fraction of cultured normal human cells and several human tumour-cell lines was examined as a function of the cell population density. Normal cells exhibited a density-dependent pattern of incorporation, whereas in tumour cells the radioactivity incorporated was independent of the cell population density. An exception was found among the tumour cells, which suggests a possible correlation between the loss of this marker and the ability to produce tumours.

Cell Count↗
PubMed1976

Fucolipid metabolism as a function of cell population density in normal and murine sarcoma virus-transformed rat cells.

The incorporation of isotopically labeled fucose into the lipids of normal and murine sarcoma virus-transformed rat cells as a function of cell population density was examined. When normal cells were seeded at low cell density, the levels of the major fucolipids, i.e., fucolipids III and IV, were substantially reduced, but then they increased as the cells approached confluency. This variation in synthesis of fucolipids III and IV appeared to be primarily related to cell density and not to cell growth. Chase experiments revealed that the reduced level of fucolipids III and IV in sparse normal cells is due to decreased synthesis rather than to increased catabolism. In contrast to the observations with normal rat cells, the high level of fucolipid III and the low level of fucolipid IV in murine sarcoma virus-transformed rat cells was shown to be independent of cell population density.

Animals↗
PubMedDate not supplied

Lack of correlation between trehalase activation and trehalose-6 phosphate synthase deactivation in cAMP-altered mutants of Saccharomyces cerevisiae.

The rise in cAMP level that follows the addition of glucose or 2,4-dinitrophenol (DNP) to stationary-phase cells of Saccharomyces cerevisiae was accompanied by a marked activation of trehalase (3-fold increase) and a concomitant deactivation of trehalose-6 phosphate synthase (50% of the basal levels). In glucose-grown exponential cells, which are deficient in glucose-induced cAMP signalling, the addition of glucose also prompted a decrease in trehalose-6 phosphate synthase, but had no effect on trehalase activity. Mutants defective in the RAS-adenylate cyclase pathway (ras1 ras2 bcy1 strain), as well as mutants containing greatly reduced protein kinase activity either cAMP-dependent (tpkw1 BCY1 strains) or cAMP-independent (tpk1w1 bcy1 strains), were unable to show glucose- or DNP-induced trehalase activation but still displayed a clear decrease in trehalose-6 phosphate synthase activity upon addition of these compounds. These data suggest that the activity of trehalose-6 phosphate synthase, as opposed to that of trehalase, is not controlled by the cAMP signalling pathway "in vivo". Trehalose-6 phosphate synthase was competitively inhibited by glucose (Ki = 15 mM) and resulted unaffected by ATP in assays performed "in vitro".

Cyclic AMP↗