Localization of alkaline phosphatase in cells of Bacillus intermedius

Alkaline phosphatase, an enzyme secreted by Bacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth of B. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na2HPO4 at a concentration of 0.01% diminished the activity of membrane-bound and extracellular phosphatases. CoCl2 at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.     

The cellular location of proteolytic enzymes ofBacillus intermedius

The activities of proteinases in the culture fluid and cellular fractions ofBacillus intermedius 3–19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. The production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2-to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of C0Cl₂ (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted.     

The influence of carbon sources and mononucleotides on the production of extracellular alkaline phosphatase by bacillus intermedium

The biosynthesis of extracellular alkaline phosphatase in the streptomycin-resistant strainsBacillus intermedius S3-19 and S7 in the presence in the medium of 5’-nucleoside monophosphates and different sources of carbon—glucose, sodium pyruvate, sodium lactate, or glycerol—was studied. It was established that, in the presence of mononucleotides, the content of extracellular alkaline phosphatase in both strains increased; the maximal effect was caused by 5’-AMP at a concentration of 20μg/ml. In medium with a low orthophosphate content, where active biosynthesis of alkaline phosphatase occurred, 1% glucose and 0.5% pyruvate stimulated this process 2.5–4 times, and 2% sodium lactate and sodium pyruvate, on the contrary, inhibited it by 20–40%. Analysis of the dynamics of growth and accumulation of extracellular phosphatase in the presence of different sources of carbon in the medium gives evidence of an interrelationship between the biosynthesis of alkaline phosphatase and carbon metabolism inBacillus intermedius.     

Hydrolytic Enzymes and Sporulation in Bacillus intermedius

The investigation of the activity of extracellular hydrolytic enzymes and sporulation in the bacterium Bacillus intermedius 3-19 showed that the activity of ribonuclease is maximal in the glucose-containing growth medium, in which sporulation is suppressed. At the sporulation stages II–IV, the synthesis of phosphatase was not regulated by the factors that influence this synthesis in the phase of growth retardation. Caseinolytic activity exhibited two peaks. The first peak was observed when thiol-dependent proteinase began accumulating in the medium. The second peak corresponded to the late stages of sporulation, i.e., the stages of spore maturation and the autolysis of sporangium. The regulatory relationship between proteinase synthesis and sporulation and the possible role of extracellular phosphatases and proteinases in the sporulation are discussed.     

Localization of proteolytic enzymes in cells of Bacillus intermedius

The activities of proteinases in the culture fluid and cellular fractions of Bacillus intermedius 3-19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. Production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2- to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of CoCl2 (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted.     

Phosphatase production byAspergillus ficuum

Summary Effects of nutritional and cultural conditions on cell growth and phosphatase production byAspergillus ficuum were studied.A. ficuum produced high levels of phosphatases when grown on a basal medium that contained a minimal amount (2 mg/100 ml) of phosphorus in an acidic growth medium. The organism produced a nonspecific acid phosphomonoesterase rather than phytin-specific phosphatase. The enzyme hydrolyzed a variety of phosphates and produced orthophosphate. The rate of phosphate hydrolysis was dependent on the pH of the reaction, where the pH optimum for acid phosphatase was 2.5 and that for phytase was 5.0. The organism slowly released the phosphatase, and the enzyme activity in the growth medium increased continually during a one-month growth period. For a high level of phosphatase production, low levels (1–5 mg%) of initial phosphorus were necessary and polyphosphates were the desired form rather than the monophosphate. The addition of surfactants, such as polyoxyethylene ethers and sodium oleate, to fungal culture medium markedly increased the level of phosphatase production.     

Dependence of the maximum temperature for growth of Saccharomyces cerevisiae on nutrient concentration

Anacystis nidulans (Synechococcus) was maintained in a medium of low phosphate concentration (0.1 mM) and grew with a normal doubling time of 5 hrs at 30°C. Such cultures ahd a normal pigment composition and alkaline phosphatase was detectable at low specific activities only.The onset of phosphate-limited growth occurred when the phosphate concentration in the medium fell to a value below 4 M (the limit of accurate determination by the assay method used) and resulted in increases in alkaline phosphatase activity, reaching a final 10 to 15 fold increase in specific activity after a period of several hours. Marked changes in the overall pigment composition occurred in this period of growth restriction. The addition of phosphate to such cultures resulted in a halt in synthesis of the enzyme and the restoration of normal pigmentation before growth resumed at the normal rate.Several organic phosphate esters could replace inorganic phosphate for growth and were also hydrolyzed by the partially purified enzyme, but growth rates were characteristically lower and the specific activity only 3 to 4 fold higher than in cultures grown in phosphate excess.Studies with the partially purified enzyme suggested that it differed in some of its properties from other alkaline phosphatases described in the literature.Abbreviations Used pNP pnitrophenol - pNPP pnitrophenylphosphate     

A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell

Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37 kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.     

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.     

Phosphate regulation of phosphatases in submerged cultures of Claviceps purpurea 129 producing clavine alkaloids

Summary Claviceps purpurea strain 129 was cultivated under submerged conditions in a sucrose-citrate medium containing high (36.8 mM) or low (1.84 mM) KH₂PO₄ concentrations. The permeabilized cells and culture supernatants contained alkaline and acid phosphatases. In the medium containing a high phosphate concentration, the synthesis of extracellular phosphatases was repressed, but that of cellular phosphatases was not. Extracellular phosphatases, especially alkaline phosphatases, were derepressed by transferring the mycelium into a phosphate-free medium. This derepression was inhibited by cycloheximide. In the presence of cycloheximide, the activities of the cellular phosphatases decreased markedly, indicating turnover of these enzymes. The cellular acid phosphatase was inhibited by phosphate (0.025 M–0.1 M) and NaF (0.01 M) while the cellular alkaline phosphatase was only inhibited by phosphate. Both cellular and extracellular alkaline phosphatases were more sensitive to repression by phosphate than the acid phosphatases. The alkaloid synthesizing enzymes were: a) present in mycelia grown in high levels of phosphate and b) activated by decreasing the intracellular phosphate level.     

Production of acid and alkaline phosphatases byMyxococcus coralloides

Acid and alkaline phosphatase ofMyxococcus coralloides were examined during vegetative growth in a liquid medium. Two extracellular phosphatases and two cell-bound phosphatases, acid and alkaline in both cases, were produced. The phosphatase production was unaltered by the presence of high concentrations of inorganic phosphate. Both enzymes were produced constitutively. These two hydrolases were released into the growth medium during the exponential growth phase (approximately 10% of total activity). The production of these enzymes was modified by the presence of organic acids and metal ions in the medium.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Response of root and sediment phosphatase activity to increased nutrients and salinity

Wetlands of northern Belize, distributed along a salinity gradient, are strongly phosphorus limited and dominated largely by three species of emergent macrophytes: Eleocharis cellulosa, Cladium jamaicense, and Typha domingensis. We assessed changes in root and sediment phosphatase activities of each species to simultaneous changes of nutrients (N, P) and salinity in a mesocosm experiment. Phosphorus and nitrogen treatment effects on both root and sediment phosphatase were highly significant for all the species, while salinity significantly affected root phosphatase activity in Cladium only. All species showed a significant negative correlation between root phosphatase activity and increasing tissue P content until a threshold of 0.2% P, 0.15% P and 0.12% P in Eleocharis, Cladium and Typha, respectively. There was also a significant negative correlation between soil available P and root and sediment phosphatases in all species. Activity of root phosphatases of Eleocharis and Typha were positively correlated with root tissue N. Both root and sediment phosphatases of all three species were positively correlated with soil available N. The strongest (positive) correlation was found between phoshatase activites and N/P ratios. The results confirmed that these systems are P-limited and that extracellular phosphatases respond to P enrichment by decreasing their activities. Expression of root phosphatase activity by dry root weight, sediment volume, or whole plant biomass gave very different relative results across nutrient treatments and species, suggesting that root phosphatase activities need to be interpreted in a wider context that considers root density.     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Ultrastructural localization of alkaline phosphatase in the cyanobacteriaCoccochloris peniocytis andAnabaena cylindrica

Summary Histochemical techniques applied at the ultrastructural level have established the periplasmic space as the site of cell bound alkaline phosphatase activity inAnabaena cylindrica andCoccochloris peniocytis. For localization of activity unfixed cells were reacted with calcium nitrate, which acts as the initial capture reagent. After this deposition, the cells were suspended in 2% lead nitrate to convert the calcium phosphate to more electron dense lead phosphate. The majority of cell bound activity appeared to be associated with layer 3 of the cell wall. InA. cylindrica a secondary site of cell bound activity appeared to be in the sheath. Placement in a phosphate free medium caused a substantial increase in the enzyme activity ofA. cylindrica while the activity present in log phase cells ofC. peniocytis was similar to that found in phosphate starved cells.C. peniocytis also secretes the enzyme into the surrounding medium.     

Histochemical study of a number of hydrolases,including a new acid phosphatase,in various tissues of men and mice

Summary Two acid phosphatases have been demonstrated histochemically in mouse ventral prostate, seminal vesicles, coagulating glands, and liver and in human prostate. The first is the lysosomal acid phosphatase demonstrable by the Gomori technique. The second differs from thisβ-glycerophosphatase in that it splits naphthol AS phosphates but notβ-glycerophosphate; it has a different histochemical pH optimum and it is not inhibited by MoO₄ or NaF. The enzyme does not represent the “tail” of alkaline phosphatase activity as it is not inhibited by inhibitors of alkaline phosphatase and it has a different localization in liver and in human prostate. The enzyme may be membrane-bound but a lysosomal localization has still to be confirmed.     

Enzymatic properties of lipase and characteristics production byLactobacillus delbrueckii subsp.bulgaricus

Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl₂ to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.     

Alkaline and acid phosphatases from the extensively halotolerant bacteriumHalomonas elongata

Alkaline and acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2, respectively) ofHalomonas elongata were cytochemically localized on the cell envelope. These enzymes were then isolated and partially purified by sonication, ammonium sulfate precipitation, and column chromatography from cells grown in alanine defined medium at 0.05, 1.37, and 3.4M NaCl. Enzyme assays were conducted at pH 5.0 and 9.0 with varying concentrations of NaCl, KCl, and LiCl in the assay buffer. Results showed higher acid phosphatase activity compared with that of alkaline phosphatase; and all enzyme activities were optimal at NaCl concentrations similar to the medium NaCl concentrations for the cells grown at 1.37 and 3.4M. However, minimum enzyme activities were observed for cells grown at the low salt concentration (0.05M). Although samples showed strong activities at some KCl concentrations, generally the enzyme activities decreased significantly when KCl or LiCl was substituted for NaCl. Polyacrylamide gel electrophoresis followed by histochemical staining for the phosphatases showed only one band for both enzymes for each cell sample grown at the different NaCl concentrations.     

Study of the cell walls of yeasts Rhodotorula. VI'nfluence of 2-hydroxybiphenyl on phosphatase activities of Rh. rubra (author's transl)]

The inhibition of acid phosphatase activity observed after culture of Rh. rubra in phosphate rich mediums is raised by the culture of this yeast in presence of 2-hydroxybiphenyl (OHph2). The cell wall alkaline phosphatase activity was inhibited by this derivative; When cultivated with OHph2 an intra and a more extracellular acid phosphatase activity appeared. The comparative studies of the two extracellular acid phosphatases secreted in the medium with or without the OHph2 show they have similar characteristics. They are eluated at the same time from Sephadex G-200, DEAE- and CM-cellulose columns, and have the same Km. They are both glycoproteins, with the sugars forming the polyose fragment identical, but the enzyme secreted in the medium containing the OHph2 contains less sugar than the one secreted in the medium without OHph2. The appearance of this acid phosphatase activity was attributed to the alteration of the membrane glycosylating systems or to the important ultra structure modifications of the cell wall of Rh. rubra when this yeast is cultivated with OHph2.