Search PubMed⌕ Search

Biomedical subjects

A D Panek

Publications and source records attributed to A D Panek.

At least 37 records · Page 2Linked to original sources
PubMed1997

Trehalose accumulation in mutants of Saccharomyces cerevisiae deleted in the UDPG-dependent trehalose synthase-phosphatase complex.

In Saccharomyces cerevisiae, trehalose-6-phosphate synthase converts uridine-5'-diphosphoglucose and glucose 6-phosphate to trehalose 6-phosphate which is dephosphorylated by trehalose 6-phosphatase to trehalose. These two steps take place within a complex consisting of three proteins: trehalose-6-phosphate synthase encoded by the GGS1/TPS1 (= FDP1, = BYP1, = CIF1) gene, trehalose 6-phosphatase encoded by the TPS2 gene and by a third protein encoded by both the TSL1 and TPS3 genes. Using three different methods for trehalose determination, we observed trehalose accumulation in ggs1/tps1delta, tps2delta and tsl1delta mutants, and in the double mutants ggs1/tps1delta/tps2delta and also in ggs1/tps1delta deleted mutants suppressed for growth on glucose. All these mutants harbor MAL genes. Trehalose synthesis in these mutants is probably performed by the adenosine-5'-diphosphoglucose-dependent trehalose synthase, (ADPG-dependent trehalose synthase) which was detected in all strains tested. It is noteworthy that trehalose accumulation in these mutants was detected only in cells grown on weakly repressive carbon sources such as maltose and galactose or during the transition phase from fermentable to non-fermentable growth on glucose. alpha-Glucosidase activity was always present in high amounts. We also describe an adenosine-diphosphoglucosepyrophosphorylase (ADPG-pyrophosphorylase) activity in Saccharomyces cerevisiae which increased concomitantly with the accumulation of trehalose during the transition phase from fermentable to non-fermentable growth in MAL-constitutive (MAL2-8c) strains. The same was observed when MAL-induced (MAL1) strains were compared during growth on glucose and maltose. These results led us to conclude that maltose-induced trehalose accumulation is independent of the UDPG-dependent trehalose-6-phosphate synthase/phosphatase complex; that the ADPG-dependent trehalose synthase is responsible for maltose-induced trehalose accumulation probably by forming a complex with a specific trehalose-6-phosphatase activity and that ADPG synthesis is activated during trehalose accumulation under these conditions.

Cell-Free System↗
PubMed1997

Modulation of trehalase activity in Saccharomyces cerevisiae by an intrinsic protein.

The regulation of cytosolic trehalase activity in yeast has been described as cycles of activation by phosphorylation by cAMP protein kinase. In this paper, evidence is presented for another regulatory mechanism--the binding of an endogenous inhibitory protein. This negative modulator was isolated during the purification procedure of cytosolic cryptic trehalase from repressed wild-type cells of Saccharomyces cerevisiae. However, in derepressed cells the inhibitor was not found nor was it present in ras2 mutant cells submitted to a heat treatment. The trehalase inhibitory activity proved to be a calmodulin ligand protein and, therefore, involved in the modulation of trehalase activity by Ca2+ ions.

Fungal Proteins↗
PubMed1997

Ca2+/calmodulin-binding proteins in yeast. Catabolite repression and induction by carbon sources.

Soluble calmodulin-binding proteins from Saccharomyces carlsbergensis were analyzed in cells grown on glucose, maltose and galactose as carbon source. A large number of polypeptide chains showed affinity for calmodulin by affinity chromatography and overlay techniques. Amongst these, polypeptides of 115, 67 and 45 kDa were only detected during the second exponential phase of growth on glucose or non-fermentative carbon sources, suggesting that they might be subjected to catabolite repression. Polypeptides of 195 and 22 kDa were only observed in cells grown on maltose, whereas 88 kDa polypeptide was only observed in galactose-grown cells. Among the calmodulin -binding polypeptides, eight were phosphorylated in a Ca2+/calmodulin -dependent manner (220, 200, 175, 100, 62, 55, 31 and 16 kDa). Ca2+/calmodulin dependent [gamma-32P] incorporation was dramatically decreased in yeast cells submitted to a heat treatment.

Calmodulin↗
PubMed1997

Nitrogen starvation in a Saccharomyces cerevisiae strain deleted in the trehalose-6-phosphate synthase complex.

Little is known about the mechanisms by which nutrient limitation leads to G1 arrest in Saccharomyces cerevisiae. We have shown that mutant cells deleted in the trehalose-6-phosphate synthase gene able to grow on glucose, when starved for nitrogen, did not arrest in the G1 phase of the cell cycle. We attribute this effect to an increase in the activity of cAMP-dependent protein kinase. When grown on maltose without nitrogen they arrested properly in G1. Tests with a mutant cell deleted in the specific trehalose phosphatase proved that the presence of the trehalose precursor, trehalose-6-phosphate, was sufficient to trigger the negative sign during nitrogen starvation, leading the cells to arrest in the G1 phase of the cell cycle.

Cell Cycle↗
PubMed1996

Kinetics and energetics of trehalose transport in Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae are able to transport trehalose against a concentration gradient, without efflux or counterflow of the labeled substrate. Uptake was inhibited by uncouplers, acetic acid, and organic mercury compounds. The addition of trehalose resulted in alkalinization of the medium. The ratio of H+ depletion to trehalose uptake by yeast cells was approximately 1:1, which indicates the existence of a trehalose-H+ symporter in these cells. The optimum pH for this active H+-trehalose symport was 5.0, and both the Km and the Vmax were negatively affected by increasing or decreasing the extracellular pH from its optimum value. Kinetic studies showed the existence of at least two different trehalose transport activities in yeast cells: a high-affinity H+-trehalose symporter (Km = 4 mM), and a low-affinity transport activity (Km > 100 mM) that could be a facilitated diffusion process. The high-affinity H+-trehalose symporter was repressed by glucose, whereas the low-affinity uptake was constitutively expressed in S. cerevisiae.

Biological Transport, Active↗
PubMed1996

Regulation of UDPG-pyrophosphorylase isoforms in Saccharomyces cerevisiae and their roles in trehalose metabolism.

UDPG-pyrophosphorylase (EC 2.7.7.9) from Saccharomyces cerevisiae was studied and the presence of isoforms investigated. Its activity was monitored during growth of cultures in rich media containing glucose, galactose, sucrose, maltose or glycerol as carbon sources. The results suggest that UDPG-pyrophosphorylase is subject to both catabolite repression and catabolite inactivation. The inactivation process seems to be complex: in order to produce maximum inactivation, glucose and ammonium sulfate must be added together. Addition of glucose or ammonium sulfate separately produced little effect upon enzyme activity. Adsorption to and elution from a DEAE-Sephacel column of a crude protein extract prepared from yeast cells collected in stationary phase from a glucose medium showed three activity peaks, which we denominated isoform I, II, and III. Isoform I is constitutive, it was the only form present during exponential growth on glucose medium, and did not suffer any alteration after glucose exhaustion, heat shock or by growing cells on maltose. On the other hand, isoforms II and III were shown to be repressed by glucose, and induced by heat shock. Furthermore, isoform II of UDPG-pyrophosphorylase was present together with isoform I when yeast cells were grown on maltose. The presence of a MAL4C allele rendered isoform II constitutive. Interestingly, a gal3 mutant strain had low UDPG-pyrophosphorylase activity and isoforms I and II were not expressed. These results are discussed in relation to trehalose metabolism.

Hot Temperature↗
PubMed1996

Biotechnological applications of the disaccharide trehalose.

Trehalose is a disaccharide present in a variety of anhydrobiotic organisms which have the ability to promptly resume their metabolism after addition of water. It has been successfully used as a nontoxic cryoprotectant of enzymes, membranes, vaccines, animal and plant cells and organs for surgical transplants. It has been predicted that trehalose can also be used as an ingredient for dried and processed food. Therefore, the recent biotechnological applications of trehalose have imposed the standardization of methods for its production, as well as for its specific quantification.

Biotechnology↗
PubMed1996

A regulatory role for TSL1 on trehalose synthase activity.

UDPG-dependent trehalose synthase activity was determined during growth on glucose medium in controls and yeast strains having deletions on components of the trehalose phosphate synthase complex. Deletion of TPS3 produced any alteration. In contrast, strains harboring deletions tsl1 delta or tsl1 delta/tps3 delta showed no activation of enzyme after glucose exhaustion. To evaluate the role played by TPS3 and TSL1 on trehalose synthase activity we have determined the effect of the addition of a cell free extract from a strain expressing only TPS3 and TSL1 to extracts of strains lacking TSL1, TPS3 or both. No effect was observed on trehalose synthase activity from tps3 delta mutant. The addition of the same extract to trehalose synthase from tsl1 delta or tsl1 delta/tps3 delta strains showed a two-fold activating effect, indicating that TPS3 and TSL1 regulated differently the UDPG-dependent trehalose synthase activity.

Glucose↗
PubMed1995

Trehalose metabolism--new horizons in technological applications.

Trehalose is responsible for the survival of anhydrobiotic organisms when under stress. Trehalose is a unique, non-reducing, extremely stable disaccharide which is able to protect proteins and membranes from damage caused by freezing, high temperatures and dehydration. Yeasts accumulate large amounts of trehalose and constitute excellent models for studying the response of eucaryotic cells to diverse stresses. The regulation of trehalose metabolism is reviewed and new technological applications for preservation of biological materials are discussed.

Animals↗
PubMed1995

Effect of trehalose during stress in a heat-shock resistant mutant of Saccharomyces cerevisiae.

Cells of a heat-shock resistant mutant were approximately 1000-times more resistant to lethal heat shock than those of the parental strain. We observed that exponentially growing cells of the mutant synthesized trehalose and showed increased osmotolerance, dehydration tolerance an ethanol tolerance, a fact not observed in wild type strains. The mutant synthesizes constitutively six proteins, among them two proteins of 56 and 63 kDa. Interestingly these molecular weights could correspond to the subunit of trehalose-6-phosphate synthase and to phosphoglucomutase II, respectively. Our results showed that glucose-growing cells of the hsr 1 mutant possessed high levels of activity of these enzymes when compared to the control strain.

Desiccation↗
PubMed1994

Trehalose metabolism in Saccharomyces cerevisiae during heat-shock.

When different strains of Saccharomyces cerevisiae grown at 23 degrees C were transferred to 36 degrees C, trehalose and glycogen were accumulated. Glycogen accumulation was less extensive and its synthesis started at least 15 min after initiation of trehalose synthesis. The steady-state intracellular concentration of trehalose increased simultaneously with the activities of the enzymes trehalose-6P synthase, UDPG-pyrophosphorylase, phosphoglucomutase and trehalase. A small but significant change was observed in hexokinase activity. Our results directly implicate isoform PII of hexokinase and the minor isoform of phosphoglucomutase in the pathway of trehalose formation during heat-shock. We also showed that the major isoform of phosphoglucomutase increased in activity but was not essential for trehalose accumulation. Studies with the glucose uptake system indicated that trehalose accumulation could be primarily determined by intracellular availability of substrates due to the increase in the rate of glucose uptake. The increased uptake appears to have two components: a kinetic effect of temperature upon glucose transporters and an increase in the numbers of molecules of the transporters, probably mediated by synthesis de novo.

Glucosyltransferases↗
PubMed1994

Trehalose inhibits ethanol effects on intact yeast cells and liposomes.

The effect of ethanol on stability of intact yeast cells has been investigated. Several strains with differences in trehalose metabolism were examined for their ability to survive in the presence of 10% (v/v) ethanol. A positive correlation was observed between cell viability and trehalose concentration. When leakage of electrolytes from the cells was recorded by observing changes in conductivity of the medium, we found that ethanol increases leakage, but the presence of trehalose reverses that effect. Similar studies were done with liposomes of similar composition to those seen in intact cells in log and stationary phases. In the presence of ethanol, carboxyfluorescein trapped in the liposomes leaked to the medium. When trehalose was added inside, outside or on both sides of the membrane, the ethanol-induced leakage was strongly inhibited. More leakage was observed in liposomes in gel phase state than in liquid-crystalline phase, suggesting that the thermotropic behavior of the lipids in the plasma membrane, together with trehalose, plays a role in enhancing ethanol tolerance.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Nylon-6 cylinders and a sponge-like derivative as supports for immobilizing trypsin.

1. Two types of nylon-6 supports (small cylinders and a sponge-like derivative) were prepared for immobilizing enzymes. Nylon-6 beads were solubilized by immersion in 80% formic acid and then reprecipitated using two different types of non-solvent solutions (distilled water or a 1:1 acetone:water solution) giving rise to a sponge-like derivative and to a colloidal suspension, respectively. The latter was molded into a thin thread which was cut into small cylinders. 2. Trypsin (EC 3.4.21.4) was covalently bound to glutaraldehyde-activated nylon-6 cylinders as well as to the sponge-like derivative. The maximum (100%) apparent initial enzymatic activity was found for the trypsin bound to small cylinders, while the initial activity of trypsin bound to the sponge-like material was 61% in comparison with that of trypsin-small cylinders, under the same conditions of enzyme immobilization reaction (1 g of nylon support and 5 ml of 1.3 mg/ml trypsin in 0.1 M sodium phosphate buffer, pH 8.5, at 10 degrees C for 18 h) and of enzymatic reaction (1 g of trypsin-nylon in a batch reactor, 2 ml of 0.7% w/v azocasein solution in 50 mM borate buffer, pH 8.5, at 37 degrees C, with shaking, for 1 h). However, the decrease of activity after enzyme immobilization was more conspicuous for the trypsin-small cylinders than for the trypsin-sponge. The former retained approximately 25% of its initial activity, while the latter retained approximately 67% of its initial activity, after seven cycles of utilization for 1 h, pH 8.5, at 37 degrees C and 8 days of storage, pH 8.5, at 4 degrees C in the presence of azocasein. 3. Scanning electron microscopy was performed to visualize the surface of the support after each step of the immobilization process. The electron micrographs show that the two types of nylon supports had a rough surface, which became rougher and full of craters after treatment with 5 N HCl. On the other hand, the partially hydrolyzed nylon surface acquired the appearance of Swiss cheese after treatment with 2.5% glutaraldehyde. After reaction with the enzyme molecules the surface became rougher again.

Caprolactam↗
PubMed1994

On the hsp26 of Saccharomyces cerevisiae.

The function of the small size hsps in Saccharomyces cerevisiae has yet to be convincingly established. In this paper we present some aspects of the physiology of hsp26. Several mutant strains were analyzed with respect to the expression of the HSP26 gene using anti-hsp26 antibody for identification. The bcy1 mutant which lacks the regulating subunit of protein kinase A failed to produce full expression of HSP26 under heat shock whereas a ras2 mutation which lowers significantly the level of cAMP, produced no detectable effect. During normal growth hsp26 protein is induced during diauxie and its synthesis continues during the second exponential phase. Both BCY1 and CYR1 genes seen to be required for induction during the transition phase albeit not directly but rather interacting with some other regulatory component. The structure of hsp26 is discussed by homology with other small hsps.

Amino Acid Sequence↗
PubMed1994

Periplasmic trehalase from Escherichia coli--characterization and immobilization on spherisorb.

1. Trehalase was partially purified from Escherichia coli and characterized. The Km for trehalose was 0.78 mM, the pH optimum 5.5 and the temperature optimum 30 degrees C. 2. Trehalase represented approximately 50% of the total protein released by osmotic shock. The preparation was free of nonspecific carbohydrate hydrolases, which act on sucrose, galactose and maltose, permitting trehalose determination in biological samples, such as insect hemolymph and free cell extracts among others. 3. The enzyme was stable in 50 mM maleate buffer, pH 6.2, at -8 degrees C for at least 6 months and could be used to determine trehalose in the range of 6 to 30 nmol. 4. Immobilization of the enzyme was achieved by covalent linkage to spherisorb-5NH2 (spherical silica gel). Retention of total catalytic activity averaged 32%. 5. The reactor, stored for one month at -5 degrees C, retained 98% of its initial immobilized activity. 6. This immobilized form of the enzyme could be used routinely for specific determinations of trehalose.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

The interaction of Saccharomyces cerevisiae trehalase with membranes.

Plasma membranes isolated from cells of Saccharomyces cerevisiae previously submitted to a heat-shock showed a 10-fold increase in membrane-bound trehalase activity. Trehalase was purified to a high specific activity and was shown to be inhibited by glucose 6-phosphate and by the addition of a neutral phospholipid-like surfactant. Purified trehalase binds spontaneously to egg phosphatidylcholine small unilamellar vesicles, when in its active, phosphorylated form. When the enzyme was treated with alkaline phosphatase no binding was observed. The significance of this reversible binding for the control of trehalose metabolism in yeast cells is still unknown.

Cell Membrane↗