Identification and Purification of a Derepressible Alkaline Phosphatase from Anacystis nidulans R2

We have examined the increase in alkaline phosphatase activity in the cyanobacterium Anacystis nidulans R2 upon phosphate deprivation. Much of the activity is released into the medium when A. nidulans is osmotically shocked, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. The polypeptide associated with phosphatase activity has been identified as a single species of M_r 160,000. Several lines of evidence demonstrate that this polypeptide is responsible for alkaline phosphatase activity: (a) It is absent when cells are grown in the presence of phosphate and specifically accumulates during phosphate deprivation. (b) It is the major periplasmic polypeptide extracted by osmotic shock. (c) It represents over 90% of the protein in a fraction of periplasmic polypeptides enriched for phosphatase activity. (d) Antibodies raised against the purified species of M_r 160,000 inhibit phosphatase activity by approximately 70%.     

The biosynthesis of alkaline phosphatase with a particulate fraction of Escherichia coli. The mechanism of inhibition by inorganic phosphate

1. By digitonin lysis of penicillin spheroplasts of Escherichia coli a particulate fraction P₁ was previously obtained that supported the sustained synthesis of alkaline phosphatase when supplied with amino acids, nucleotide triphosphates and other cofactors. This P₁ fraction, when subjected to mild ultrasonic treatment in the presence of sucrose and Mg²⁺, yielded the P₁(S) fraction, consisting of integrated particulate subcellular particles containing DNA and RNA. 2. The P₁(S) fraction from E. coli K10 wild type (R⁺₁R⁺₂P⁺) grown under repressed conditions supported the immediate synthesis of alkaline phosphatase in vitro. The synthesis occurred in phases. The first was followed by a lag, and then there was a linear rapid phase that continued for at least 3hr. Actinomycin D inhibited the appearance of the second phase. It was concluded that the particles are programmed to synthesize enzyme even when prepared from repressed cells, and therefore that synthesis of the specific messenger RNA for alkaline phosphatase in vivo was not inhibited when the bacteria were grown in an excess of inorganic phosphate. 3. Phosphate inhibited synthesis of enzyme to the same extent with the P₁(S) fractions of two constitutive strains as with the P₁(S) fraction of the wild-type strain. 4. Inorganic phosphate inhibited amino acid incorporation with the P₁(S) fraction and also inhibited enzyme synthesis in vitro. The effect on amino acid incorporation could be partially overcome by adding Mn²⁺ to the incubation mixtures. However, Mn²⁺ inhibited the synthesis of alkaline phosphatase. Also, inhibition of the incorporation of [³²P]CTP into RNA was overcome by Mn²⁺. The effect of phosphate on amino acid uptake was most probably due to a phosphorolysis of RNA by polynucleotide phosphorylase, also present in the P₁(S) fraction. This phosphorolysis may be responsible for the instability of messenger RNA in vitro and in vivo. 5. Phosphate also specifically inhibited the formation of alkaline phosphatase, since it did not affect markedly the induced formation of β-galactosidase by the same P₁(S) fraction. The specific effect is attributed to the prevention of formation of the enzymically active dimer from precursors, a Zn²⁺-dependent reaction. It is suggested that the repression of the synthesis of alkaline phosphatase in vivo in the wild-type strain was the sum of these two effects.     

Phosphate Stress in Cultures and Field Populations of the Dinoflagellate Prorocentrum minimum Detected by a Single-Cell Alkaline Phosphatase Assay

Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. When a phosphate group is cleaved from the ELF-97 reagent, the remaining molecule precipitates near the site of enzyme activity, thus fluorescently tagging cells with alkaline phosphatase activity. We characterized ELF-97 labeling in axenic cultures of a common dinoflagellate, Prorocentrum minimum, in order to understand ELF-97 labeling dynamics when phosphate nutrition varies. Enzyme activity, as detected by ELF-97 labeling, appears to be induced in late-log- or early-stationary-phase cultures if cells are grown in low-phosphate media and is lost when phosphate-stressed cells are refed with phosphate. ELF-97 appears to label an inducible intracellular alkaline phosphatase in P. minimum based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intracellular phosphatases. After laboratory cultures were characterized, ELF-97 was used to assay field populations of P. minimum in Narragansett Bay during two 1-week periods, and 12 to 100% of the P. minimum cells were labeled. The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies.     

Co-purification of type I alkaline phosphatase and type I phosphoprotein phosphatase from various animal tissues

A purification procedure, which included ethanol treatment as a step for dissociating the large molecular forms of type I phosphoprotein phosphatase, was employed for the studies of the alkaline phosphatase and phosphoprotein phosphatase activities in bovine brain, heart, spleen, kidney, and uterus, rabbit skeletal muscle and liver, and lobster tail muscle. The results indicate that the major phosphoprotein phosphatase (phosphorylase a as a substrate) and alkaline phosphatase (p-nitrophenyl phosphate as a substrate; Mg²⁺ and dithiothreitol as activators) activities in the extracts of all tissues studied were copurified as an entity of M_r = 35,000. The purified enzymes from different tissues exhibit similar physical and catalytic properties with respect to either the phosphoprotein phosphatase or the alkaline phosphatase activity. The present findings indicate that (a) the M_r = 35,000 species, which represents a catalytic entity of the large molecular forms of type I phosphoprotein phosphatase, is widespread in animal tissues, indicating that it is a multifunctional phosphatase; (b) the association of type I alkaline phosphatase activity with type I phosphoprotein phosphatase is a general phenomenon.     

Identification of the Pseudornonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity

Summary Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg²⁺, the K_m values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K _inf1 ^sups values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.     

Characterization of ascorbyl esters hydrolysis in fish

The hydrolysis of ascorbate mono-, tri- and polyphosphates by trout intestinal alkaline phosphatase was examined. K_m values were established as 1.19, 4.1 and 3.7 mM, respectively. The enzyme catalyzed ascorbate triphosphate hydrolysis with 60% efficiency of that for ascorbate monophosphate. With the K_m value of 1.19 mM for ascorbate monophosphate the trout enzyme exhibits similar affinity with this substrate as with p-nitrophenyl phosphate (1.00–1.67 mM). Two K_m values for micro- and millimolar ranges of ascorbate monophosphate concentrations ranges were calculated as: 27.9 μM and 1.19 mM, respectively. Specific intestinal alkaline phosphatase inhibitor L-phenylalanine (100 mM), inhibited reaction rate by 20% in 10 min. We conclude that alkaline phosphatase, which is in a great abundance in the trout intestine, serves as ascorbate esters hydrolase, thus releasing active ascorbic acid into circulation.     

Kinetics of cytochrome c oxidase from yeast. Membrane-facilitated electrostatic binding of cytochrome c showing a specific interaction with cytochrome c oxidase and inhibition by ATP (With an appendix on the occurrence of two-dimensional diffusion of cytochrome c on the membrane surface)

The steady-state kinetics of purified yeast cytochrome c oxidase were investigated at low ionic strength where the electrostatic interaction with cytochrome c is maximized. In 10 mM cacodylate/Tris (pH 6.5) the oxidation kinetics of yeast iso-1-cytochrome c were sigmoidal with a Hill coefficient of 2.35, suggesting cooperative binding. The half-saturation point was 1.14 μM. Horse cytochrome c exhibited Michaelis-Menten kinetics with a higher affinity (K_m = 0.35 μM) and a 100% higher maximal velocity.In 67 mM phosphate the Hill coefficient for yeast cytochrome c decreased to 1.42, and the species differences in Hill coefficients were lessened. Under the latter conditions, a yeast enzyme preparation partially depleted of phospholipids was activated on addition of diphosphatidylglycerol liposomes. When the enzyme was incorporated into sonicated yeast promitochondrial particles the apparent K_m for horse cytochrome c was considerably lower than the value for the isolated enzyme.ATP was found to inhibit both the isolated oxidase and the membrane-bound enzyme. With the isolated enzyme in 10 mM cacodylate/Tris, 3 mM ATP increased the half-saturation point with yeast cytochrome c 3-fold, without altering the maximal velocity or the Hill coefficient. 67 mM phosphate abolished the inhibition of the isolated oxidase by ATP.The increase in affinity for cytochrome c produced by binding the oxidase to the membrane was not observed in the presence of 3 mM ATP, with the result that the membrane-bound enzyme was more sensitive to inhibition by ATP. ADP was a less effective inhibitor than ATP, and did not prevent the inhibition by ATP.It is proposed that non-specific electrostatic binding of cytochrome c to phospholipid membranes, followed by rapid lateral diffusion, is responsible for the dependence of the affinity on the amount and nature of the phospholipids and on the ionic strength.ATP may interfere with the membrane-facilitated binding of cytochrome c by a specific electrostatic interaction with the membrane or by binding to cytochrome c.     

Induction of acid phosphatase in cotton seedlings: Enzyme purification,subunit structure and kinetic properties

About a 16-fold rise in acid phosphatase (EC 3.1.3.2) activity was observed during the early stages of germination of cotton embryos. Administration of cyclobeximide to the germinating embryos significantly blocked the enhancement of acid phosphatase activity. This indicated that translational activity was essential for the induction of enzyme activity. Conclusive proof for the de novo synthesis of the enzyme was obtained by showing the incorporation of ³⁵S from ³⁵SO²⁻₄ into the cysteine residues of the purified acid phosphatase. The enzyme was purified (1046-fold) to electrophoretic homogeneity by ammonium sulphate fractionation, CM-Sephadex C-50 and affinity chromatography on concanavalin A-Agarose. PAGE gave two isozyme bands. The M, of the phosphatase was 200 k as determined by molecular sieving on Sephadex G-200. SDS-PAGE of acid phosphatase revealed a single band of M 55 k. Thus the native enzyme is a tetramer of four identical subunits. The K_m of the enzyme with p-nitrophenyl phosphate was 0.5 mM. Optimal enzyme activity was observed at pH 5.0, using p-nitrophenyl phosphate as substrate. The enzyme activity remained linear for 105 min at 37° and was proportional to the concentration of protein within the range 0.6–2.4 μg.     

Alkaline phosphatase in the integument of Ceratitis capitata: Developmental profile and functional properties

Electrophoretic analysis of alkaline phosphatase from the integument during development, reveals two bands of enzyme activity. One corresponding to phosphatase activity during pupation and just prior to eclosion and the other during the middle of the pupal stages. On the contrary in the haemolymph there is one band on enzyme activity through all the developmental stages. The haemolymph alkaline phosphatase band does not comigrate with any integumental enzyme band. The developmental profile of the integumental alkaline phosphatase activity has also been compared to that of the haemolymph. It was found that the pattern of activity is completely different. In the integument, two peaks of enzyme activity were found: one just prior to pupation and the other during eclosion. These two peaks do not coincide to that of haemolymph alkaline phosphatase activity. The pH optimum for both enzyme forms of third instar larvae, although broad especially for haemolymph form, was clearly in the alkaline range, with a peak at pH 8.5–9.0. The two isozymes have different affinities for the substrate tyrosine-O-phosphate. Tyrosine-O-phosphate is the preferred substrate for the integumental enzyme form with a K_m of 0.4 mM. We suggest that alkaline phosphates from the integument is specific for the hydrolysis of tyrosine-O-phosphate.     

Extracytoplasmic phosphatases of selected species of Saccharomyces, Kluyveromyces and Rhodotorula

Phosphatase activities of yeasts belonging to the genera Saccharomyces, Kluyveromyces and Rhodotorula were studied. Rhodotorula rubra exhibited activities at acid, neutral and alkaline pH; the other yeasts only had activity at acid pH. Growing yeasts in a constant pH (4.5) medium decreased phosphatase activities in Saccharomyces and Kluyveromyces, while neutral activity was enhanced in Rhodotorula rubra which excreted more enzyme under these conditions. Washing cells with sucrose solutions lowered phosphatase activities in all yeasts, due to enzyme liberation. Acid phosphatase activities in isolated and purified cell walls were very small. Phosphatases thus appear not to be strongly bound to yeast cell walls.     

Use of yeast spores as a biocatalyst

Vegetative cells of the yeast Saccharomyces cerevisiae 4011 efficiently sporulated at pH 7.7–8.0 in the presence of 1.0–3.0% of potassium acetate. Spores were prepared by lysing them with a lytic enzyme, zymolyase. Alkaline phosphatase (an enzyme selected as a model) in spores exhibited higher stability toward heat and pH than it did in vegetative cells, and was immobilized in a polyacrylamide gel lattice without any appreciable loss of activity. The activity of alkaline phosphatase in spores and immobilized spores was stably maintained during repeated use for the enzyme reactions. These results indicated the usefulness of yeast spores as a biocatalyst.     

Relationship between phosphates and alkaline phosphatase of Anabaena flos-aquae in continuous culture

Summary Anabaena flos-aquae was grown in chemostats with phosphate-limiting growth and dilution rate of 0.015–0.03 h^-1. The yields of cells were dependent on dilution rate and a two-fold increase obtained by growth in the presence of 15 mM KNO₃. Alkaline phosphatase activity varied 20-fold, lowest activity with excess phosphate light-limited cells and the highest activity with cells grown in the presence of 15 mM KNO₃. There was no correlation between hot water soluble phosphate of cells and alkaline phosphatase activity.     

Identification of nutrient-dependent changes in extracellular pH and acid phosphatase secretion in Aspergillus nidulans

The present study was designed to identify nutrient-dependent changes in extracellular pH and acid phosphatase secretion in the biA1 palC4 mutant strain of Aspergillus nidulans. The palC4 mutant was selected as lacking alkaline phosphatase, but having substantially increased acid phosphatase activity when grown on solid minimal medium under phosphate starvation, pH 6.5. Gene palC was identified as a putative member of a conserved signaling cascade involved in ambient alkaline sensing whose sole function is to promote the proteolytic activation of PacC at alkaline pH. We showed that both poor growth and conidiation of the palC4 mutant strain on solid medium, alkaline pH, were relative to its hypersensitivity to Tris (hydroxymethyl) aminomethane buffer. Also, the secretion of acid phosphatase was repressed when both the wild-type and palC4 mutant strains were grown in low-phosphate yeast extract liquid medium, pH 5.0, indicating that the secretion of this enzyme is not necessary to regenerate inorganic phosphate from the organic phosphate pool present in yeast extract.     

Biosynthesis of glycoproteins in the pathogenic fungus Candida albicans: Activation of dolichol phosphate mannose synthase by cAMP-mediated protein phosphorylation

Following incubation with ATP and a cAMP-dependent protein kinase under optimal conditions of lipid acceptor, phospholipid and metal ion requirements, the transfer activity of partially purified dolichol phosphate mannose synthase (DPMS) increased about 60% and this activation correlated with a 50% increase in V_max with no alteration in the apparent K_m for GDP-Manose. Phosphorylation with [γ-³²P]ATP resulted in the labeling of several polypeptides, one of which exhibited the molecular weight of the enzyme (30 kDa) and was also recognized using a specific anti-DPMS monoclonal antibody. This and the fact that the phosphate label could be removed by an alkaline phosphatase indicate that Candida DPMS may be regulated by phosphorylation–dephosphorylation, a mechanism that has been proposed for the enzyme in other organisms.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme a reductase and acylcoenzyme a: Cholesterol acyltransferase activities by phoshorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA: cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and ‘reactivation’ of enzyme activity. Cytosol caused a 78% increase in acyl-CoA: cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl₂ and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A: cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA: cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500×g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl₂, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA: cholesterol acyltransferase activity is observed.     

Purification and Characterization of Phosphoenolpyruvate Carboxylase from Developing Seeds of Brassica

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified to apparent homogeneity with about 29% recovery from developing seeds of Brassica using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sepharose CL-6S. The purified enzyme with mol wt of about 400 kD exhibited maximum activity at pH 8.0. The enzyme had an absolute requirement for a divalent cation which was satisfied by Mg²⁺. The enzyme showed typical hyperbolic kinetics with PEP and HCO^?3 with Km of 0.125 and 0.104 mM, respectively. Glu-6-P could activate the enzyme, whereas other phosphate esters such as fru-1, 6-P₂, L-glycerophosphate and 3-PGA did not have any effect on the enzyme activity. Noneof the amino acids at 5 mM concentration had any significant effect on the enzyme activity. Nucleotide monophosphates and diphosphates did not inhibit the enzyme significantly, whereas ATP inhibited the enzyme activity. Oxaloacetate and malate inhibited the enzyme non-competitively with respect to PEP with Ki values of 0.127 and 1.25 mM, respectively. The enzyme activity in vivo seems to be regulated ’Tlainly by availability of its substrate and activation by glu-6-P, both of which are supplied through glycolysis.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value