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PubMed1996

Deletion of the ATH1 gene in Saccharomyces cerevisiae prevents growth on trehalose.

The biological function of the yeast trehalases (EC 3.2.1.28) consists of down-regulation of the concentration of trehalose via glucose formation by trehalose hydrolysis. While it is generally accepted that the cytosolic neutral trehalase (encoded by the NTH1 gene) is responsible for trehalose hydrolysis in intact cells, very little is known about a role of the vacuolar acid trehalase and the product of the recently described neutral trehalase gene YBRO106 (NTH2). We have analyzed the role of the acid trehalase in trehalose hydrolysis using the ATH1 deletion mutant (delta ath1) of Saccharomyces cerevisiae [M. Destruelle et al. (1995) Yeast 11, 1015-10251 deficient in acid trehalase activity under various nutritional conditions. In contrast to wild-type and a mutant deficient in the neutral trehalase (delta nth1), the delta ath1 mutant does not grow on trehalose as a carbon source. Experiments with diploid strains heterozygous for delta ath1 show a gene dosage effect for the ATH1 gene for growth on trehalose. The need for acid trehalase for growth on trehalose is supported by the finding that acid trehalase activity is induced during exponential growth of cells on trehalose while no such induction is measurable during growth on glucose. Our results show that the vacuolar acid trehalase Ath1p is necessary for the phenotype of growth on trehalose, i.e. trehalose utilization, in contrast to cytosolic neutral trehalase Nth1p which is necessary for intracellular degradation of trehalose. For explanation of the need for vacuolar acid trehalase and not cytosolic neutral trehalase for growth on trehalose, the participation of endocytosis for uptake of trehalose from medium to the vacuoles is discussed.

Carbon↗
PubMed2001

Multiple effects of protein phosphatase 2A on nutrient-induced signalling in the yeast Saccharomyces cerevisiae.

The trehalose-degrading enzyme trehalase is activated upon addition of glucose to derepressed cells or in response to nitrogen source addition to nitrogen-starved glucose-repressed yeast (Saccharomyces cerevisiae) cells. Trehalase activation is mediated by phosphorylation. Inactivation involves dephosphorylation, as trehalase protein levels do not change upon multiple activation/inactivation cycles. Purified trehalase can be inactivated by incubation with protein phosphatase 2A (PP2A) in vitro. To test whether PP2A was involved in trehalase inactivation in vivo, we overexpressed the yeast PP2A isoform Pph22. Unexpectedly, the moderate (approximately threefold) overexpression of Pph22 that we obtained increased basal trehalase activity and rendered this activity unresponsive to the addition of glucose or a nitrogen source. Concomitant with higher basal trehalase activity, cells overexpressing Pph22 did not store trehalose efficiently and were heat sensitive. After the addition of glucose or of a nitrogen source to starved cells, Pph22-overexpressing cells showed a delayed exit from stationary phase, a delayed induction of ribosomal gene expression and constitutive repression of stress-regulated element-controlled genes. Deletion of the SCH9 gene encoding a protein kinase involved in nutrient-induced signal transduction restored glucose-induced trehalase activation in Pph22-overexpressing cells. Taken together, our results indicate that yeast PP2A overexpression leads to the activation of nutrient-induced signal transduction pathways in the absence of nutrients.

Base Sequence↗
PubMed1990

Regulation of trehalose metabolism by Streptomyces griseus spores.

Spores of Streptomyces griseus contain trehalose and trehalase, but trehalose is not readily hydrolyzed until spore germination is initiated. Trehalase in crude extracts of spores, germinated spores, and mycelia of S. griseus had a pH optimum of approximately 6.2, had a Km value for trehalose of approximately 11 mM, and was most active in buffers having ionic strengths of 50 to 200 mM. Inhibitors or activators or trehalase activity were not detected in extracts of spores or mycelia. Several lines of evidence indicated that trehalose and trehalase are both located in the spore cytoplasm. Spores retained their trehalose and most of their trehalase activity following brief exposure to dilute acid. Protoplasts formed by enzymatic removal of the spore walls in buffer containing high concentrations of solutes also retained their trehalose and trehalase activity. Protoplasts formed in buffer containing lower levels of solutes contained low levels of trehalose. The mechanism by which trehalose metabolism is regulated in S. griseus spores is unresolved. A low level of hydration of the cytoplasm of the dormant spores and an increased level of hydration during germination may account for the apparent inactivity of trehalase in dormant spores and the rapid hydrolysis of trehalose upon initiation of germination.

Disaccharides↗
PubMed2003

In silico and in vivo analysis reveal a novel gene in Saccharomyces cerevisiae trehalose metabolism.

BACKGROUND: The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p. RESULTS: In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain. CONCLUSIONS: These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

Gene Expression↗
PubMed1984

Induction of microvillar hydrolase activities by cell density and exogenous differentiation inducers in an established kidney epithelial cell line (LLC-PK1).

Several hydrolase activities characteristic of the apical brush border membrane of renal proximal tubule, leucine aminopeptidase, gamma-glutamyl transpeptidase, alkaline phosphatase, maltase, and trehalase, were identified in cultures of the LLC-PK1 kidney epithelial cell line. A coordinate increase in activities of these enzymes was observed upon development of a confluent cell density and functional membrane polarization. Further large progressive increases in individual hydrolase activities were induced after the addition of compounds known as differentiation inducers. Hexamethylene bisacetamide preferentially induced increased trehalase and maltase activities. Induced trehalase activity exhibited an increased Vmax but a similar Km compared with activity in control extracts. Induction required protein synthesis and was dependent on inducer concentration and exposure time. Treatment of confluent cultures with N,N'-dimethylformamide triggered an induction of maltase, trehalase, alkaline phosphatase, and gamma-glutamyl transpeptidase activities, whereas dimethylsulfoxide induced trehalase and gamma-glutamyl transpeptidase activities. Increased leucine aminopeptidase and maltase activities were observed after addition of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. Induction of trehalase activity by N,N'-dimethylformamide was reversible over a 4-day period after removal of inducer, but effects of hexamethylene bisacetamide were irreversible. These results suggest that the LLC-PK1 cell line reproducibly develops differentiation-specific characteristics under defined conditions in cell culture, which can be individually modulated by chemicals known as inducers of cell differentiation.

Acetamides↗
PubMed1979

Quantitative measurement of intestinal marker enzymes in intestinal metaplasia from human stomach with cancer.

Intestinal metaplasia in human stomach was distinguished macroscopically into sucrase-positive and trehalase-positive areas, and sucrase-positive and trehalase-negative areas, by location of these disaccharidase activities with TES-Tape. After location of these two areas with TES-Tape, tissues were taken from them for colorimetric measurement of sucrase, trehalase, leucine aminopeptidase (LAP), and alkaline phosphatase (ALP). Results showed that in the mucosa from sucrase-positive and trehalase-negative areas, trehalase activity was not detectable and the activities of sucrase, LAP, and ALP were lower than in sucrase-positive and trehalase-positive areas.

Alkaline Phosphatase↗
PubMed2006

Ammonium permease-based sensing mechanism for rapid ammonium activation of the protein kinase A pathway in yeast.

In the yeast Saccharomyces cerevisiae starvation for nitrogen on a glucose-containing medium causes entrance into G0 and downregulation of all targets of the PKA pathway. Re-addition of a nitrogen source in the presence of glucose causes rapid activation of trehalase and other PKA targets. Trehalase activation upon ammonium re-supplementation is dependent on PKA activity, but not on its regulatory subunit nor is it associated with an increase in cAMP. In nitrogen-starved cells, ammonium transport and activation of trehalase are most active in strains expressing either the Mep2 or Mep1 ammonium permease, as opposed to Mep3. The non-metabolizable ammonium analogue, methylamine, also triggers activation of trehalase when transported by Mep2 but not when taken up by diffusion. Inhibition of ammonium incorporation into metabolism did not prevent signalling. Extensive site-directed mutagenesis of Mep2 showed that transport and signalling were generally affected in a similar way, although they could be separated partially by specific mutations. Our results suggest an ammonium permease-based sensing mechanism for rapid activation of the PKA pathway. Mutagenesis of Asn246 to Ala in Mep2 abolished transport and signalling with methylamine but had no effect with ammonium. The plant AtAmt1;1, AtAmt1;2, AtAmt1;3 and AtAmt2 ammonium transporters sustained transport and trehalase activation to different extents. Specific mutations in Mep2 affected the activation of trehalase differently from induction of pseudohyphal differentiation. We also show that Mep permease involvement in PKA control is different from their role in haploid invasive growth, in which Mep1 sustains and Mep2 inhibits, in a way independent of the ammonium level in the medium.

Amino Acid Sequence↗
PubMed2003

Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae.

Yeast cells starved for inorganic phosphate on a glucose-containing medium arrest growth and enter the resting phase G0. We show that re-addition of phosphate rapidly affects well known protein kinase A targets: trehalase activation, trehalose mobilization, loss of heat resistance, repression of STRE-controlled genes and induction of ribosomal protein genes. Phosphate-induced activation of trehalase is independent of protein synthesis and of an increase in ATP. It is dependent on the presence of glucose, which can be detected independently by the G-protein coupled receptor Gpr1 and by the glucose-phosphorylation dependent system. Addition of phosphate does not trigger a cAMP signal. Despite this, lowering of protein kinase A activity by mutations in the TPK genes strongly reduces trehalase activation. Inactivation of phosphate transport by deletion of PHO84 abolishes phosphate signalling at standard concentrations, arguing against the existence of a transport-independent receptor. The non-metabolizable phosphate analogue arsenate also triggered signalling. Constitutive expression of the Pho84, Pho87, Pho89, Pho90 and Pho91 phosphate carriers indicated pronounced differences in their transport and signalling capacities in phosphate-starved cells. Pho90 and Pho91 sustained highest phosphate transport but did not sustain trehalase activation. Pho84 sustained both transport and rapid signalling, whereas Pho87 was poor in transport but positive for signalling. Pho89 displayed very low phosphate transport and was negative for signalling. Although the results confirmed that rapid signalling is independent of growth recovery, long-term mobilization of trehalose was much better correlated with growth recovery than with trehalase activation. These results demonstrate that phosphate acts as a nutrient signal for activation of the protein kinase A pathway in yeast in a glucose-dependent way and they indicate that the Pho84 and Pho87 carriers act as specific phosphate sensors for rapid phosphate signalling.

Adenosine Triphosphate↗
PubMed1998

Intracellular signal triggered by cholera toxin in Saccharomyces boulardii and Saccharomyces cerevisiae.

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S.cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.

Animals↗
PubMed1989

Intestinal disaccharidases of young turkeys: temporal development and influence of diet composition.

An experiment was done to determine maltase, sucrase, isomaltase, and trehalase activities in mucosa of different segments of small intestines of young turkeys as influenced by age and diet. Two-day-old poults were fed diets containing no added fat [44.6% starch, 2.2% ether extract by weight (HC)], 10% tallow (T), or 10% corn oil [(CO) 29.0% starch, 10.9% ether extract]. Diets HC, T, and CO were calculated to contain 2,705, 3,083, and 3,196 kcal ME/kg, respectively, and constant protein, TSAA, and lysine:ME ratios were maintained. Appreciable maltase and isomaltase specific activities (micromoles of substrate hydrolyzed per milligram protein per hour) were observed in 2-day-old poults, and activities of these enzymes increased in poults fed the HC diet through 7 and 14 days, respectively. At 2 days, specific activity of sucrase was low, and trehalase activity was not detected. Sucrase activity increased steadily through 28 days of age in poults fed the HC diet. Trehalase activity was detected at 7 days of age and reached a maximum by Day 21 after hatch. By Day 28, trehalase activity had disappeared from all segments except for the proximal jejunum. In 28-day-old poults fed the HC diet, specific activities of all disaccharidases were greatest in the jejunal segments; i.e., 21, 1.06, 7.24, and .034 mumol/mg protein/h for maltase, sucrase, isomaltase, and trehalase, respectively, in the proximal jejunum. Poults fed the T or CO diets had significantly lower disaccharidase activities than did those fed the HC diet, beginning at 7 days of age. Changes in specific activities of disaccharidases as related to age or diet or both were not always parallel, suggesting that each enzyme may be regulated by or affected by diet in a partly independent way.

Aging↗
PubMedDate not supplied

Selective release of apical membrane enzymes from cultured renal epithelia by phosphatidylinositol-specific phospholipase C.

Membrane proteins can be attached to the plasma membrane in several ways. Recently, a mechanism has been described, by which a number of cell surface proteins are anchored to the exoplasmic side of the plasma membrane by covalent linkage to glycosyl-phosphatidylinositol (GPI). The growth properties of renal epithelial cells in tissue culture enable free access to apical cell surface and brush border membrane proteins. To study the nature of membrane anchoring of apical plasma membrane enzymes in cultured renal epithelial cells, confluent LLC-PK1, OK, NRK, and MDCK epithelia were treated in tissue culture dishes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and the PI-PLC-specific release into the tissue culture medium of the apical membrane enzymes alkaline phosphatase (AP), gamma-glutamyl transpeptidase, leucine aminopeptidase, trehalase, and maltase was determined. Of the five enzymes tested, AP and trehalase, already described as GPI-anchored membrane proteins, were specifically released by PI-PLC from intact cell monolayers. Of the four cell lines investigated, LLC-PK1 cells express AP and trehalase which were released by PI-PLC. In OK cells, which lack AP activity, only trehalase was found to have PI-PLC-releaseable enzyme activity. MDCK cells, on the other hand, express AP activity, releaseable by PI-PLC, but no trehalase activity. In studies on the time course of synthesis and reinsertion of AP into the apical membrane of LLC-PK1 cells after removal by PI-PLC, a 60% recovery of AP activity was obtained only after 7 days. Analysis of protein release by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of culture supernatants after surface labeling with biotin and subsequent Western blotting with streptavidin revealed four protein bands at approximately 130, 90, 30, and 20 kD in LLC-PK1 cells and five GPI-anchored proteins at 110, 85, 65, 40, and 26 kD in OK cultures. The finding of a PI-PLC-specific release of apical membrane enzymes from renal tubular cell lines of different species (pig, opossum, rat, and dog) and of different nephron origin indicates a high conservation of the GPI anchor of renal brush border membrane proteins and further proves the high degree of differentiation retained by the cell lines in tissue culture. In addition, this method may provide a possible tool for isolating GPI-anchored apical membrane proteins from intact epithelial monolayer cultures.

Animals↗
PubMed1994

Sequence around the centromere of Saccharomyces cerevisiae chromosome II: similarity of CEN2 to CEN4.

We report the sequence of a 12 kilobase region spanning the centromere of Saccharomyces cerevisiae chromosome II. The sequence from the left arm includes genes for histones H2A and H2B. The sequence from the right arm includes a gene that probably encodes a novel trehalase, as well as the COQ1 gene (for an enzyme involved in coenzyme Q biosynthesis), and an open reading frame with significant similarity to bacterial genes of unknown function. The trehalase gene (YBR0106) on chromosome II is located beside the centromere and transcribed towards it. This is identical to the arrangement of the neutral trehalase gene (NTH1) beside the centromere of chromosome IV. The centromere regions of chromosomes II and IV may therefore have arisen through a duplication of the centromere region of an ancestral chromosome. The YBR0106 and NTH1 proteins are 77% identical in predicted amino acid sequence, but there is no pronounced sequence similarity between the two centromeres (CEN2 and CEN4) outside of the universally conserved CDE I and CDE III elements. The genes flanking the centromere and trehalase genes differ between the two chromosomes, so the similarity between chromosomes II and IV may be less extensive than that recently reported between chromosomes III and XIV.

Alkyl and Aryl Transferases↗
PubMed2005

Extracellular trehalose utilization by Saccharomyces cerevisiae.

Two haploid strains of Saccharomyces cerevisiae viz. MATalpha and MATa were grown in glucose and trehalose medium and growth patterns were compared. Both strains show similar growth, except for an extended lag phase in trehalose grown cells. In both trehalose grown strains increase in activities of both extracellular trehalase activities and simultaneous decrease in extracellular trehalose level was seen. This coincided with a sharp increase in extracellular glucose level and beginning of log phase of growth. Alcohol production was also observed. Secreted trehalase activity was detected, in addition to periplasmic activity. It appeared that extracellular trehalose was hydrolyzed into glucose by extracellular trehalase activity. This glucose was utilized by the cells for growth. The alcohol formation was due to the fermentation of glucose. Addition of extracellular trehalase caused reduction in the lag phase when grown in trehalose medium, supporting our hypothesis of extracellular utilization of trehalose.

Culture Media↗
PubMed2003

Enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu.

In our previous studies, it was found that Saccharomycopsis fibuligera sdu cells could accumulate 18.0% (gg(-1)) trehalose from soluble starch in SSY medium. However, the yeast strain contained high activities of acid and neutral trehalases, which were reported to mobilize trehalose accumulated by the cells during fermentation. In order to enhance the yield of trehalose, it is necessary to remove the trehalase activities from the cells. By mutagenesis of ethylmethanesulfonate, one mutant that assimilated trehalose very slowly, but grew on other carbon sources as fast as its parent strain, was isolated. In Biostat B2 2-1 fermentation, trehalose accumulation of the mutant was much higher than that of the wild type when grown in YPS medium containing starch. The activities of acid and neutral trehalases of this mutant were much lower than those of the wild type, respectively. We think the reduction of acid and neutral trehalase activities is considered to be responsible for the increased yield of trehalose accumulated by the mutant.

Bioreactors↗
PubMed1992

On the mechanism by which a heat shock induces trehalose accumulation in Saccharomyces cerevisiae.

When the temperature of exponential-phase cultures of Saccharomyces cerevisiae was abruptly raised from 28 to 40 degrees C, trehalose immediately accumulated, whereas the activities of trehalase and trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase complex increased after a lag period of about 10 min. Heat shock also induced a sudden rise in intracellular glucose, simultaneously with a decrease in the concentration of hexose phosphate and fructose 2,6-bisphosphate. The increase of trehalose-metabolizing enzymes, but not the accumulation of glucose and trehalose, was prevented by cycloheximide. Investigation of the kinetic properties of partially purified enzymes showed that both non-activated and cyclic AMP-dependent-protein-kinase-activated forms of trehalase are almost inactive in the absence of Ca2+ and that the concentration of free Ca2+ required for half-maximal activity increased with increasing temperature, being approx. 1 microM at 30 degrees C and 20 microM at 40 degrees C for the activated form of trehalase. In contrast, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase were three times more active at 40 degrees C. It is proposed that the rapid accumulation of trehalose induced by heat shock may be in part explained by changes in the kinetic properties of trehalase and trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase.

Enzyme Activation↗
PubMed1996

Glucose-triggered signalling in Saccharomyces cerevisiae: different requirements for sugar phosphorylation between cells grown on glucose and those grown on non-fermentable carbon sources.

Addition of glucose or fructose to cells of the yeast Saccharomyces cerevisiae grown on a nonfermentable carbon source triggers within a few minutes posttranslational activation of trehalase, repression of the CTT1 (catalase) and SSA3 (Hsp70) genes, and induction of the ribosomal protein genes RPL1, RPL25 and RPS33. By using appropriate sugar kinase mutants, it was shown that rapid glucose- or fructose-induced activation of trehalase requires phosphorylation of the sugar. On the other hand, partial induction of RPL1, RPL25 and RPS33 as well as partial repression of CTT1 and SSA3 were observed in the absence of sugar phosphorylation. In glucose-grown nitrogen-starved yeast cells readdition of a nitrogen source triggers activation of trehalase in a glucose- or fructose-dependent way, but with no apparent requirements for phosphorylation of the sugar. Repression of CTT1 and SSA3 under the same conditions was also largely dependent on the presence of the sugar and also in these cases there was a strong effect when the sugar could not be phosphorylated. Nitrogen induction of RPL1, RPL25 and RPS33 was much less dependent on the presence of the sugar, and only phosphorylated sugar caused a further increase in expression. These results show that two glucose-dependent signalling pathways, which can be distinguished on the basis of their requirement for glucose phosphorylation, appear to be involved in activation of trehalase, repression of CTT1 and SSA3 and induction of ribosomal protein genes. They also show that nutrient-induced repression of CTT1 and SSA3 is not a response to improvement of the growth conditions because the addition of nonmetabolizable sugar does not ameliorate the growth conditions. Similarly, the upshift in ribosomal protein synthesis cannot be a response to increased availability of energy or biosynthetic capacity derived from glucose, but it is apparently triggered to a significant extent by specific detection of glucose as such.

Carbohydrate Metabolism↗
PubMed1971

Isolation, mapping, and characterization of trehalaseless mutants of Neurospora crassa.

Mutant strains of Neurospora crassa that lack trehalase and are unable to grow on trehalose were isolated, and the gene (tre) was positioned on the right arm of linkage group I. Maltase and beta-galactosidase activities are almost identical in tre(-) strains, whereas that of invertase was reduced by more than half and those of acid phosphatase and amylase were somewhat increased. Heterocaryons between standard and trehalaseless strains yield less than one-tenth the activity of the former. In addition, strains with duplications heterozygous for trehalase produce less than 1% of the activity of the standard strain. An inhibitor of trehalase has been found in tre(-) strains; its sensitivity to heat and proteolysis, and its nondialyzability suggest that this substance is a protein. The mig gene, which determines the rate of migration of trehalase on acrylamide gels, has been shown to be less than 1 map unit away from the tre gene.

Acid Phosphatase↗
PubMed1988

Changes in activities of several enzymes involved in carbohydrate metabolism during the cell cycle of Saccharomyces cerevisiae.

Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.

Alcohol Dehydrogenase↗