Localization of Acid and Alkaline Phosphatases in Staphylococcus aureus

The localization of acid and alkaline phosphatases in Staphylococcus aureus was studied by fractionation of cells after treatment with the L-11 enzyme and by electron microscopic histochemistry. The two enzyme activities were located in distinctly different positions at the surface of the cells. Acid phosphatase appeared to be localized around the cell membrane of the bacteria, because the enzyme was recovered exclusively in the membrane fraction and because deposition of lead phosphate was detected by electron microscopic histochemistry on the inner surface of the cell membrane of intact bacteria and spheroplasts. The highest specific activity of alkaline phosphatase was also associated with the membrane fraction. However, on electron microscopic histochemistry of intact cells, the deposition of lead phosphate was only seen on the outer surface of the cell wall.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.     

Localization of alkaline phosphatase inBacillus intermedius cells

Alkaline phosphatase, an enzyme secreted byBacillus 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 ofB. 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.     

Heat-stable alkaline phosphatase as a marker for human and monkey type-I pneumocytes

Summary The expression of the heat-stable isoenzyme of alkaline phosphatase in the human and monkey (Macaca mulatta, M. fascicularis) lung was investigated at the light- and electron-microscopic level, using cytochemical techniques and immunocytochemical procedures based on monoclonal and polyclonal antibodies against human term-placental alkaline phosphatase. Both in man and monkey, the enzyme was present in type-I pneumocytes. In the monkey, the enzyme was found in all type-I cells. In man, strong staining was observed only in some type-I cells and in certain cuboidal respiratory bronchiolar cells. Staining was localized on the apical and basal plasma membrane, in apical and basal caveolae, and in the underlying basement membrane. The level of heat-stable alkaline phosphatase expression in the human lung was 10-fold lower than in the monkeys studied. In human fetal lung, the onset of heat-stable alkaline phosphatase expression was associated with the development of the alveolar epithelium from 17–20 weeks gestation onward. It is concluded that: (1) heat-stable alkaline phosphatase is a specific constitutent of type-I pneumocytes in man and monkeys; and (2) its subcellular localization may explain its rapid appearance in the circulation under certain conditions.This work was supported by grants from the Fonds voor Kankeronderzoek van de Algemene Spaar- en Lijfrentekas, Nationale Loterij-FGWO (Grant No. 9.0005.84), the National Program for Reinforcement of the Scientific Research (PREST/UIA 04) and a research grant from the University of Antwerp     

Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane

Summary The virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls. Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium. We have examined the localization and orientation of this protein in the cell membrane. When the nucleotides encoding the first 30 to 41 amino acids from the N-terminus of the VirD4 protein were fused to the gene for alkaline phosphatase from which the signal sequence had been removed, alkaline phosphatase activity was detectable under appropriate conditions. Immunoblotting with VirD4-specific antiserum indicated that the VirD4 protein could be recovered exclusively from the membrane fraction of Agrobacterium cells. Moreover, when the membrane fraction was separated into inner and outer membrane fractions by sucrose density-gradient centrifugation, VirD4 protein was detected in the inner-membrane fraction and in fractions that sedimented between the inner and outer membrane fractions. By contrast, the VirD4/alkaline phosphatase fusion protein with the N-terminal sequence from VirD4 was detected only in the inner membrane fraction. Treatment of spheroplasts of Agrobacterium cells with proteinase K resulted in digestion of the VirD4 protein. These results indicate that the VirD4 protein is transported to the bacterial membrane and anchored on the inner membrane by its N-terminal region. In addition, the C-terminal portion of the VirD4 protein probably protrudes into the periplasmic space, perhaps in association with some unidentified cellular factor(s).Deceased June 5, 1988     

Ultrastructure of the ventriculus of the honey bee,Apis mellifera (L.): cytochemical localization of acid phosphatase,alkaline phosphatase,and nonspecific esterase

Summary Ventriculi (midguts) from 5-day- and 30-day-old honey bees, Apis mellifera (L.), were examined ultrastructurally and cytochemically. Midgut epithelia were composed of regenerative cells, endocrine cells, and pleomorphic columnar cells. Regions of the midgut were encountered in which the cytogeny of the columnar cells, the content of discharged vesicles, and the structure of the peritrophic membrane varied. In 5-day-old bees, regional variation in the ultrastructure of the cells indicated that absorption occurred primarily in the middle of the gut and that regulated enzyme secretion appeared to be confined to the posterior midgut. In 30-day-old bees, reduced pollen consumption was accompanied by diminished cell activity in the posterior midgut. Our ultrastructural data suggest that the honey bee, like other insects, may rely on countercurrent flow to distribute enzymes and nutrients efficiently throughout the ectoperitrophic and endoperitrophic compartments. Acid phosphatase and nonspecific esterase activity were localized cytochemically in primary and secondary lysosomes. Alkaline phosphatase activity was localized on the elongate microvilli of the striated border and within large electron-lucent microbodies. The association of alkaline phosphatase activity with the peroxisomal microbodies and their relation to phospholipid metabolism are discussed.     

Cell cycle variations in HeLa 65 plasma membrane alkaline phosphatase

HeLa plasma membranes from M, G₁, and S phase cells were isolated from growing synchronous cell cultures. It was found that the specific activity of plasma membrane alkaline phosphatase was over three times higher in the M phase cell than in the G₁ and S phase cell. However, sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis showed that the S phase plasma membrane contained 5.5 times more alkaline phosphatase protein than did the plasma membrane from mitotic cells, and 11.0 times more than the G₁ phase plasma membrane. This would indicate that the high specific activity in mitosis was due to modification of the alkaline phosphatase protein resulting in increased enzymatic activity.     

Alterations in Activity of Phosphatases during the Peridinium Bloom in Lake Kinneret

Acid and alkaline phosphatases have been isolated from Peridinium cinctum f. westii (Dinophyceae) during an algal bloom in Lake Kinneret. Acid phosphatase activity was fairly constant over the entire period of the bloom, although fluctuations in activity appeared to correlate with the chlorophyll content of the cells. Histochemical studies showed that the enzyme was localized inside the cell. Alkaline phosphatase activity was very low until May, a month after the peak of the bloom, when it increased sharply. Polyacrylamide gel electrophoresis revealed one or two bands of alkaline phosphatase that increased in intensity as the bloom progressed. However, the highest activity of the enzyme (in the last sample collected) corresponded to a new, very intense band on the gels. Similarly to acid phosphatase, alkaline phosphatase was also localized inside the cell. The appearance of alkaline phosphatase is probably related to the available phosphate concentration in the lake, although the influence of other factors that may contribute to the induction of the enzyme cannot be ruled out.     

Phosphatase activity in the heterotrophic dinoflagellate Pfiesteria shumwayae

The ELF-97 phosphatase substrate was used to examine phosphatase activity in four strains of the estuarine heterotrophic dinoflagellate, Pfiesteria shumwayae. Acid and alkaline phosphatase activities also were evaluated at different pH values using bulk colorimetric methods. Intracellular phosphatase activity was demonstrated in P. shumwayae cells that were actively feeding on a fish cell line and in food limited cells that had not fed on fish cells for 3 days. All strains, whether actively feeding or food limited showed similar phosphatase activities. P. shumwayae cells feeding on fish cells showed ELF-97 activity near, or surrounding, the food vacuole. Relatively small, spherical ELF-97 deposits were also observed in the cytoplasm and sometimes near the plasma membrane. ELF-97 fluorescence was highly variable among cells, likely reflecting different stages in digestion and related metabolic processes. The location of enzyme activity and supporting colorimetric measurements suggest that, as in other heterotrophic protists, acid phosphatases predominate in P. shumwayae and have a general catabolic function.     

Cytochemical localization of alkaline phosphatase in the cell membrane ofDictyostelium discoideum amebae

Summary We demonstrated that alkaline phosphatase was localized on the cell membrane ofDictyostelium discoideum amebae and on isolated plasma membranes. The enzyme activity was specifically inhibited by 0.01 M KCN or cysteine. The same method could also be applied to baker's yeast and MDCK cells (dog kidney cells in vitro).     

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.     

Localization of leucine aminopeptidase and vitamin B-12 binding protein in rabbit peripheral blood polymorphonuclear leukocytes

Polymorphonuclear leukocytes were isolated from the peripheral blood of rabbits by Ficoll-Hypaque centrifugation followed by dextran sedimentation. The granulocytes were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. Leucine aminopeptidase, when assayed with L-leucine-7-amido-4-methyl-coumarin as substrate, showed a similar distribution to N-acetyl-ß-glucosaminidase and thus is localized to the tertiary granules. There was no leucine aminopeptidase associated with the plasma membrane of these cells. Further experiments with purified plasma membranes and inhibitor studies using diazotized sulphanilic acid further confirmed that leucine aminopeptidase had a purely intracellular localization. Vitamin B-12 binding protein showed a similar localization to alkaline phosphatase indicating that, as in human polymorphonuclear leukocytes, vitamin B-12 binding protein is located to the specific granules.     

Electron microscopic cytochemical localization of nucleoside phosphatases in normal and chronic granulocytic leukaemic human neutrophils

Summary Using electron microscope cytochemistry and cells separated on Ficoll-Hypaque, Mg²⁺-dependent ATPase, ADPase and 5-nucleotidase were predominantly localized as ectoenzymes on normal human granulocytes. Large deposits of ATPase final reaction product and more finely granular deposits of 5-nucleotidase final reaction product were firmly attached to the outer surface of cell plasma membranes. The final reaction product from ecto-ADPase was, however, only loosely associated with the plasma membrane. In addition, finer deposits of ADPase final reaction product were seen in specific granules and in background cytoplasm. No nucleotidase phosphatase activity was localized to the alkaline phosphatase-containing granules (phosphasomes) recently described by Rustinet al.In granulocytes from patients with chronic granulocytic leukaemia, ecto-ATPase had a patchy distribution on the plasma membranes. There was considerable heterogeneity between cells with regard to ADPase and 5-nucleotidase localization. In some cells, ADPase was seen only as an ectoenzyme and in a few it was present in specific granules, but in others it was seen at both sites, while in some cells no activity was detected. 5-Nucleotidase localization was normal in some cells but lacking from many. No correlation was found between enzyme heterogeneity and the degree of morphological cell maturity.     

Subcellular localization and dynamics of components of the respiratory burst oxidase

Membrane and cytosolic factors cooperate to generate NADPH-oxidase. The study of the syndrome of NADPH-oxidase deficiencies, chronic granulomatous disease, has enabled the identification of two membrane factors: a flavin adenine dinucleotide flavoprotein and ab cytochrome. The nature of the cytosolic components is still unknown, but a 47-kD protein, whose phosphorylation occurs in parallel with the generation of a respiratory burst in intact cells, seems to be one of the cytosolic factors. The subcellular localization of the membrane-bound NADPH-oxidase components has been studied in neutrophils: In unstimulated cells, only a minute fraction of the NADPH-oxidase components is localized in the plasma membrane, whereas 80% is localized in the membrane of the specific granules and the majority of the rest is in a newly described membrane-bound compartment, the secretory granules, identified by latent alkaline phosphatase. During stimulation, these NADPH-oxidase components are translocated to the plasma membrane as a result of fusion of granule membrane with plasma membrane. Only the NADPH-oxidase components present in the plasma membrane are incorporated in the respiratory burst oxidase generated in intact cells.     

Alkaline phosphatase which lacks its own signal sequence becomes enzymatically active when fused to N-terminal sequences of Escherichia coli haemolysin (HlyA)

Summary Fusion of the alkaline phosphatase gene (phoA) which lacks its own signal peptide sequence to the N-terminal region of hlyA, the structural gene for Escherichia coli haemolysin, leads to active alkaline phosphatase (AP). AP activity depends on the length of the N-terminal region of hlyA. An optimum is reached when 100–200 amino acids of HlyA are fused to PhoA but fusion of as little as 13 amino acids of HlyA to PhoA is sufficient to yield appreciable AP activity. When cells are treated with lysozyme most of the AP activity is found associated with the membrane fraction but a substantial amount is also found in the soluble fraction, most of which may represent, a periplasmic pool of AP. The soluble portion of AP activity is significantly increased when the cells are disrupted by ultrasonication, which indicates that the fusion proteins are only loosely associated with the membrane and that large parts are already located on the outside of the cytoplasmic membrane. The expected fusion proteins were identified in the soluble and the membrane fractions and their amounts in these fractions correlated well with AP activity.     

Enzyme analysis and subcellular fractionation of human peripheral blood lymphocytes with special reference to the localization of putative plasma membrane enzymes

Human lymphocytes were isolated from defibrinated blood by Ficoll-Hypaque centrifugation with erythrocyte hypotonic lysis. Homogenates of mixed lymphocytes were subjected to analytical subcellular fractionation by sucrose gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their marker enzymes: cytosol (lactate dehydrogenase), plasma membrane (5′-nucleotidase), endoplasmic reticulum (neutral α-glucosidase), mitochondria (malate dehydrogenase), lysosomes (N-acetyl-β-glucosaminidase), peroxisomes (catalase). γ-Glutamyl transferase was exclusively localized to the plasma membrane. Leucine amino-peptidase, especially when assayed in the presence of Co²⁺, was also partially localized to the plasma membrane. Experiments with diazotized sulphanilic acid, a non-permeant enzyme inhibitor, showed that these plasma membrane enzymes are present on the cell surface. No detectable alkaline phosphatase was found in the lymphocytes. Acid phosphatase and β-glucuronidase were localized to lysosomes and there was some evidence for lysosomal heterogeneity. Leucine amino peptidase, optimal at pH 8.0, showed a partial localization to intracellular vesicles, possibly lysosomes, especially when assayed in the presence of EDTA. These studies provide a technique for determining the intracellular distribution of hitherto unassigned lymphocyte constituents and serve as a basis for investigating the cell pathology of lymphocytic disorders.     

Localization of some enzymes in the main venom glands of viperid snakes

Several secretory and nonsecretory enzymes were localized histochemically in the main venom gland of 13 viperid snakes. All secretory cells show the intracellular oxidative enzymes succinate dehydrogenase and monoamine oxidase. The granular reactions obtained for both enzymes resemble mitochondria in distribution. Distinctive cells with a very high succinate dehydrogenase activity are dispersed among the secretory cells of all species except Atractaspis. Nonspecific acid phosphatase activity is found in the supranuclear region of the secretory cells in species that do not secrete this enzyme and throughout the cytoplasm in snakes that secrete the enzyme. Nonspecific alkaline phosphatase activity occurs in the secretory cells of those snakes whose venom shows this activity. Leucine amino peptidase (aryl amidase) activity is found in the venom and in the secretory cells of all the species. In Vipera palaestinae both the venom and the secretory cells of the main venom gland contain nonspecific esterase, L-amino acid oxidase and phosphodiesterase activities. The localization of phosphodiesterase and L-amino acid oxidase do not show major differences between glands at different intervals from an initial milking. Adenosine-monophosphate phosphatase activity is localized in the supranuclear region of the secretory cells in the glands of Vipera palaestinae and Aspis cerastes. Its activity is found in the venom of Aspis only.     

The intramitochondrial location of cytochrome c peroxidase in wild-type and petite Saccharomyces cerevisiae

Cytochemical and ultrastructural analysis of wild-type cells of Saccharomyces cerevisiac, grown aerobically in a glucose-limited chemostat, shows that cytochrome c peroxidase is localized between the membranes of the cristae, that is, in the intracristal space. This enzyme is thus positioned appropriately within the organelle to act as an alternate terminal oxidase for the respiratory chain. The proximity of the peroxidase to major sites of generation of its two substrates may account for the small leakage of hydrogen peroxide from yeast mitochondria, as compared with the larger outflow from mammalian mitochondria.In the cytoplasmic petite mutant, gross distortion of promitochondrial membrane arrangement is evident. Nevertheless, cytochrome c peroxidase activity is present in the same amounts as is found in wildtype cell, and is localized predominantly within annuli of membrane which constitute the promitochondria in these cells.No unequivocal evidence was obtained for the localization of catalase in microbodies or other organelles in either wild-type or petite cells.     

Modulation of the beta-adrenergic-response in cultured rat heart cells

Summary Subcellular fractionation studies in resting human neutrophils indicated a bimodal distribution for cytochrome b. A. major peak of cytochrome b co-sedimented with gelatinase under different experimental conditions. This localization was partially overlapped with specific granules (using lysozyme and lactoferrin as specific granule markers), but clearly resolved from azurophilic granules, plasma membrane, mitochondria, as well as from a novel alkaline phosphatase-rich intracellular organelle. A minor localization of cytochrome b was found in fractions enriched in both the plasma membrane marker 5-nucleotidase and alkaline phosphatase. A significant portion of ubiquinone cell content co-fractionated with the gelatinase-containing granules. After phorbol myristate acetate (PMA)-cell stimulation, cytochrome b was mobilized to fractions showing respiratory burst activity and enriched in 5-nucleotidase activity. This mobilization paralleled secretion of gelatinase and lysozyme to the extracellular medium. Furthermore, neutrophil stimulation with fluoride in the absence of cytochalasin B induced release of gelatinase and generation of superoxide anion with only minimal release of lysozyme. Preincubation of cells with the anion channel blocker 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) prevented lysozyme release, but had only a minor effect on the release of gelatinase and did not inhibit the superoxide anion generation elicited by N-formyl-methionyl-leucyl-phenylalanine or PMA. These results suggest a main location of cytochrome b in mobilizable gelatinase-containing granules, which can constitute a subpopulation of specific granules. Furthermore, these findings show that the gelatinase-containing granule is functionally involved in the respiratory burst in neutrophils and that membrane fusion between plasma membrane and the gelatinase-containing granule occurs during activation of cells.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - FMLP N-formyl-methionyl-leucyl-phenylalanine - PMA 4-phorbol, 12-myristate, 13-acetate     

Mycelial forms of <Emphasis Type="Italic">Pseudallescheria boydii</Emphasis> present ectophosphatase activities

Phosphatase activities were characterized in intact mycelial forms of Pseudallescheria boydii, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 41.41 ± 2.33 nmol p-NP per h per mg dry weight, linearly with increasing time and with increasing cell density. MgCl₂, MnCl₂ and ZnCl₂ were able to increase the (p-NPP) hydrolysis while CdCl₂ and CuCl₂ inhibited it. The (p-NPP) hydrolysis was enhanced by increasing pH values (2.5-8.5) over an approximately 5-fold range. High sensitivity to specific inhibitors of alkaline and acid phosphatases suggests the presence of both acid and alkaline phosphatase activities on P. boydii mycelia surface. Cytochemical localization of the acid and alkaline phosphatase showed electron-dense cerium phosphate deposits on the cell wall, as visualized by electron microscopy. The product of p-NPP hydrolysis, inorganic phosphate (Pi), and different inhibitors for phosphatase activities inhibited p-NPP hydrolysis in a dose-dependent manner, but only the inhibition promoted by sodium orthovanadate and ammonium molybdate is irreversible. Intact mycelial forms of P. boydii are also able to hydrolyze phosphoaminoacids with different specificity.