Cytochemical and biochemical determination of acid phosphatase activity in yeasts]

Optimal conditions of the cytochemical assay for acid phosphatase in protoplasts and whole cells of S. cerevisiae have been described. Dimethyl sulfoxide was used to increase the permeability of the yeast cell envelope. In the yeast cells, grown up to the end of the exponential phase, acid phosphatase is shown to be located mainly in the central vacuole and on the cell envelope surface. A considerable activity of acid phosphatase is demonstrable on the surface of the plasma membrane and within adjacent vesicles that represent, presumably, part of the endoplasmic reticulum. Acid phosphatase can be considered as a marker enzyme for yeast cell vacuoles.     

Variation in the latency of acid phosphatase and ribonuclease throughout the growth cycle of Tetrahymena pyriformis

The latency of both acid phosphatase and ribonuclease was studied throughout the growth cycle of the ciliate protozoan Tetrahymena pyriformis and was found to be remarkably low in cells in the logarithmic phase of growth. The latency increased progressively throughout the log phase until it reached a maximum just after the cells had entered the stationary phase. The specific activity of acid phosphatase remained constant throughout the whole of the growth cycle while that of ribonuclease decreased as the cells began to leave log phase. The possibility is discussed that all rapidly dividing cells have a high proportion of their acid hydrolases outside the lysosomes and that these free hydrolases may have a function in cell division.     

Phosphorylation of Escherichia coli enolase

In vivo labeling of Escherichia coli JA200 pLC 11-8 resulted in 32P incorporation into enolase as demonstrated by immunoaffinity chromatography and electrophoresis followed by autoradiography. Complete acid hydrolysis, followed by thin layer chromatography was employed for determination of the phosphoamino acid residue. Comparison with phosphoamino acid standards resulted in the identification of a labeled residue corresponding to phosphoserine. In vitro labeling of cell extracts from glucose and acetate grown cells resulted in differential labeling of enolase. When specific radioactivities of in vivo labeled enolase were compared, 7 times more label was incorporated at late log phase in glucose grown cells than in late log acetate grown cells. At stationary phase, only 2.5 times more label was incorporated into glucose compared to acetate. When 32P-labeled enolase from glucose grown cells was subjected to treatment with potato acid phosphatase, dephosphorylation of the enzyme could be observed. Monitoring enzyme activity during the acid phosphatase treatment revealed a 70% decrease for the forward enzyme reaction, and a 3-fold increase, followed by a gradual decrease to almost zero, for the reverse enzyme reaction. Complete reversal of the changes in activity was possible by adding an aliquot of partially purified enolase kinase plus ATP.     

Effect of endocytosis upon acid phosphatase activity ofTetrahymena pyriformis

Summary Increased endocytosis inTetrahymena pyriformis, produced by presenting starved cells with either peptone-yeast extract medium or killed yeast cell suspension, results in increased cellular acid phosphatase activity.Tetrahymena, grown in peptone-yeast extract medium, showed increased acid phosphatase activity after phagocytosis of yeast cells. This increase was not apparent until about one hour after presentation and was maximal at about 2.5 hours.Tetrahymena, grown on yeast suspension, showed little increase in acid phosphatase activity on presentation with peptone-yeast extract medium. These results may indicate that endocytosis, of either particles or solutes, produces an adaptive increase in acid phosphatase activity (presumably lysosomal in nature) which is related to feeding.Histochemical examination failed to localise the increase in acid phosphatase activity cellularly, but small particles, of about 1 diameter, which showed acid phosphatase activity and were presumably lysosomes were noted. Closely orientated yeast cells showed varying intensities of lead deposition, from absence to intense staining. This suggests that newly ingested yeast cells may be ingested initially in a single phagosome and that thereafter one or more lysosomes may fuse with them.     

Comparison of the Protein Kinase and Acid Phosphatase Activities of Five Species of Leishmania1

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, historic-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [γ-³²P]ATP and Mg²⁺ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [γ-³²P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA^-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA⁺). The PNA^- and PNA⁺ forms of L. major both phosphorylated a 50-kDa protein when incubated with [γ-³²P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity. With the exception of L. braziliensis panamensis for which late log phase organisms contained 12-fold more tartrate-resistant acid phosphatase activity than did early log phase cells, stationary and log phase parasites contained approximately the same amount of acid phosphatase activity.     

A rapid method for determination of acid phosphatase activity of whole yeast cells

A simple and rapid procedure for determination of intracellular acid phosphatase activity without the need for disruption of cells is described. Candida lipolytica cell suspension was treated with 0.1% Triton X-100 for 30 min at room temperature and with intermittent shaking. The enzyme assay is carried out directly with the permeabilized cell suspension. Permeabilization of the yeast cells to p -nitrophenylphosphate by Triton X-100 provides almost 100% efficiency in determining the total acid phosphatase activity compared to results obtained with disrupted yeast cells.     

Phage formation in Staphylococcus muscae cultures; nucleic acid synthesis during virus formation

1. The total nucleic acid synthesized by normal and by infected S. muscae suspensions is approximately the same. This is true for either lag phase cells or log phase cells. 2. The amount of nucleic acid synthesized per cell in normal cultures increases during the lag period and remains fairly constant during log growth. 3. The amount of nucleic acid synthesized per cell by infected cells increases during the whole course of the infection. 4. Infected cells synthesize less RNA and more DNA than normal cells. The ratio of RNA/DNA is larger in lag phase cells than in log phase cells. 5. Normal cells release neither ribonucleic acid nor desoxyribonucleic acid into the medium. 6. Infected cells release both ribonucleic acid and desoxyribonucleic acid into the medium. The time and extent of release depend upon the physiological state of the cells. 7. Infected lag phase cells may or may not show an increased RNA content. They release RNA, but not DNA, into the medium well before observable cellular lysis and before any virus is liberated. At virus liberation, the cell RNA content falls to a value below that initially present, while DNA, which increased during infection falls to approximately the original value. 8. Infected log cells show a continuous loss of cell RNA and a loss of DNA a short time after infection. At the time of virus liberation the cell RNA value is well below that initially present and the cells begin to lyse.     

Ruptured fission yeast walls

Distributions of rupture sites of fission yeast cells ruptured by glass beads have been related to a new morphometric analysis. As shown previously (Johnson et al.,Cell Biophysics, 1995), ruptures were not randomly distributed nor was their distribution dictated by geometry, rather, ruptures at the extensile end were related to cell length just as the rate of extension is related to cell length. The extension patterns of early log, mid-log, late log, and stationary phase cells from suspension cultures were found to approximate the linear growth patterns of Kubitschek and Clay (1986). The median length of cells was found to decline through the log phase in an unbalanced manner.     

Comparison of the protein kinase and acid phosphatase activities of five species of Leishmania

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, histone-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [gamma-32P]ATP and Mg2+ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [gamma-32P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA+). The PNA- and PNA+ forms of L. major both phosphorylated a 50-kDa protein when incubated with [gamma-32P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ultrastructure of the cell surface and plasma membrane of exponential and stationary phase cells of Schizosaccharomyces pombe,grown in different media

In order to study the ultrastructure of the cell surface and plasma membrane of Schizosaccharomyces pombe as a function of growth conditions we investigated exponential and stationary phase cells grown in rich and minimal medium.Electron microscopic preparation techniques based on rapid cryofixation (without cryoprotectants) were used. The intramembraneous aspects of the plasma membrane were described by freeze fracturing. For the first time the dynamic surface structures could be directly analyzed by freeze drying in the scanning electron microscope and in thin section of freeze substituted samples. This preparation techniques reveal hair-like structures on the surface of yeast cells. The hairs of cells grown in the rich medium are longer than those grown in the minimal medium. A mutant defective in the structure of a cell surface galactomannoprotein (acid phosphatase) reveals (under conditions of maximal acid phosphatase expression) a cell surface structure that differs from the wild type. It is likely that the hairs represent the peripheral galactomannan layer or part of it.On the membrane fracture faces the number, shape, distribution and state of aggregation of the intramembraneous particles are different between membranes of growing and non-growing cells and between cells grown under different physiological conditions. In the minimal medium corresponding periodical structures on the plasmic and exoplasmic fracture faces were observed, which clearly differ between exponential and stationary phase cells. The number, length and depth of plasma membrane invaginations increase as the cells go from the exponential phase to the stationary phase. Short and flattened invaginations are filled with thin periodic structures.     

Production of an exocellular acid phosphatase by the yeast Hansenula holstii NCYC 560

1. The yeast Hansenula holstii NCYC 560 produced invertase and an inducible acid phosphatase located betweent the cytoplasmic membrane and the yeast cell wall. 2. These enzymes were also found in the culture medium outside the cell boundaries. 3. The amount of cell wall mannan in cells grown in phosphate-limited medium decreased in comparison with that of cells grown in phospahte-rich medium. 4. It is proposed that the mannan in this yeast is a loose and highly permeable structure, allowing external enzymes to leave the cell boundaries.     

Fission yeast homologue of Tip41-like proteins regulates type 2A phosphatases and responses to nitrogen sources

A fission yeast (Schizosaccharomyces pombe) gene encoding a member of the TIP41-like protein family was identified and characterized. Deletion of the fission yeast tip41 gene leads to slower growth when ammonium chloride is the nitrogen source, but the growth rate is not affected when adenine is the nitrogen source. The tip41 mutant cells also enter the G1 phase of the cell cycle earlier than wild-type cells in response to nitrogen starvation. Overexpression of tip41(+) causes cell death, spherical cell morphology and blocks the shift to G1 phase upon nitrogen starvation. Overexpression of tip41(+) increases the activity of type 2A phosphatase. In a ppa2 deletion strain with reduced PP2A activity, overexpression of tip41(+) no longer blocks the shift to G1 upon nitrogen starvation. These results suggest that fission yeast Tip41 plays a role in cellular responses to nitrogen nutrient conditions at least partly through regulation of type 2A phosphatase activity.     

Microbial Utilization of Benzenesulfonic Acid

An enzyme which degrades yeast glucan and yeast cells in the logarithmic phase of growth (log yeast cells) and produces protoplasts from log yeast cells has been crystallized from the culture filtrate of a strain belonging to Fungi Imperfecti.

Analyses by ultracentrifugation and disc gel electrophoresis showed the crystalline enzyme to be homogeneous. Its molecular weight was found to be 24,500. The hydrolysis of laminarin, pachyman and yeast glucan was catalysed by the enzyme to produce a mixture of laminaridextrins. The conversion of log yeast cells to protoplasts was obtained by the addition of only this enzyme, the addition of mercaptoethanol or phosphomannanase to the enzyme promoted the conversion.     

Biochemical and genetic evidence that yeast extracellular protein phosphatase activity is due to acid phosphatase

In this paper evidences are presented strongly confirming that an extracellular 32P-phosphopeptide phosphatase activity of yeast is accounted for by acid phosphatase. Dephosphorylation of 32P phosphoseryl peptides was achieved with whole yeast cells, thus demonstrating extracellular location of protein phosphatase activity. The acid phosphatase and protein phosphatase activity copurified throughout purification procedure. Purified enzyme showed the same pH-profile and had the same Km value with phosphopeptide substrate as intact cells. Protein phosphatase activity is repressed by phosphate in the same manner as acid phosphatase activity, showing that not only repressible but also constitutive acid phosphatase displays protein phosphatase activity. Using mutant strains defective in acid phosphatase activity it was confirmed that acid phosphatase and protein phosphatase activities are the products of the same gene(s).     

Influence of growth temperature on the acid phosphatase activity in the yeast Yarrowia lipolytica

Abstract The adaptation of the yeast Yarrowia lipolytica 76-18 to growth temperature was studied by measuring the levels of secreted and intracellular acid phosphatase activities during growth at five temperatures from 8 to 36 °C. The intracellular acid phosphatase activity is maximal at a growth temperature of 20 °C. The level of the secreted phosphatase activity decreases as growth temperature increases from 15 to 36 °C. It is the growth temperature itself and not the growth rate that regulates these activities. The observed dependence of the acid phosphatase activity on the growth temperature indicates a possible participation of acid phosphatases in the temperature adaptation of yeast cells.     

A novel pathway for transport and metabolism of a fluorescent phosphatidic acid analog in yeast

Phosphatidic acid is a central intermediate of biosynthetic lipid metabolism as well as an important signaling molecule in the cell. These studies assess the internalization, or retrograde transport , and metabolism of phosphatidic acid in yeast using a fluorescent analog. An analog of phosphatidic acid fluorescently labeled at the sn -2 position with N-4-nitrobenz-2-oxa-1, 3-diazole-aminocaproic acid (NBD-phosphatidic acid) was introduced to yeast cells by spontaneous transfer from phospholipid vesicles. Transport and metabolism of the NBD-phosphatidic acid were then monitored by fluorescence spectrophotometry, fluorescence microscopy and routine biochemical methods. Primary metabolites of the NBD-phosphatidic acid in yeast were found to be NBD-diacylgycerol and NBD-phosphatidylinositol. Experiments in cells possessing different levels of phosphatidate phosphatase activity suggest that conversion of the NBD-phosphatidic acid to NBD-diacylglycerol is not a pre-requisite for internalization in yeast. Internalization is sensitive to decreased temperature, but neither ATP depletion nor a sec6-4 mutation, which interrupts endocytosis, has an affect. Thus, internalization of NBD-phosphatidic acid apparently occurs via a non-endocytic route. These characteristics of retrograde transport of NBD-phosphatidic acid in yeast differ significantly from transport of other NBD-phospholipids in yeast as well as NBD-phosphatidic acid transport in mammalian fibroblasts.     

Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.

Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell.     

Location of Acid Phosphatase and β-Fructofuranosidase Within Yeast Cell Envelopes

After 16 hr of incubation in a low-phosphate, aerated medium, bakers' yeast was obtained with a high titer of acid phosphatase (EC 3.1.3.2) and beta-fructofuranosidase (EC 3.2.1.26). All of the beta-fructofuranosidase and 75% of the acid phosphatase were easily released by mechanical disruption in a French pressure cell. The cell wall suffered a limited number of cracks, but this was sufficient for the co-release of these enzymes. Both enzymes were subject to autolytic release, although correlation was inconclusive because of the relative instability of acid phosphatase. The data are consistent with the bulk of the two enzymes being located in the periplasmic space. Ethylacetate treatments yielded ghosts with high beta-fructofuranosidase but low acid phosphatase activities. The surviving acid phosphatase was not representative of that in live cells. It was resistant to release by mechanical disruption and showed a high susceptibility to heat inactivation. The beta-fructofuranosidase in live cells and in ethylacetatetreated cells exhibited polydispersity in heat inactivation susceptibility; but the kinetics were indistinguishable, and facile release by mechanical disruption was shown in both cases.     

Effects of ice-seeding temperature and intracellular trehalose contents on survival of frozen Saccharomyces cerevisiae cells

Freezing tolerance is an important characteristic for baker’s yeast, Saccharomyces cerevisiae, as it is used to make frozen dough. The ability of yeast cells to survive freezing is thought to depend on various factors. The purpose of this work was to study the viability of yeast cells during the freezing process. We examined factors potentially affecting their survival, including the growth phase, ice-seeding temperature, intracellular trehalose content, freezing period, and duration of supercooling. The results showed that the ice-seeding temperature significantly affected cell viability. In the stationary phase, trehalose accumulation did not affect the viability of yeast cells after brief freezing, although it did significantly affect the viability after prolonged freezing. In the log phase, the ice-seeding temperature was more important for cell survival than the presence of trehalose during prolonged freezing. The importance of increasing the extracellular ice-seeding temperature was verified by comparing frozen yeast survival rates in a freezing test with ice-seeding temperatures of −5 °C and −15 °C. We also found that the cell survival rates began to increase at 3 h of supercooling. The yeast cells may adapt to subzero temperatures and/or acquire tolerance to freezing stress during the supercooling.