Secretion Of Phosphatase Research Articles

Homologous recombination partly restores the secretion defect of underglycosylated acid phosphatase in yeast.

The majority of secreted acid phosphatase in Saccharomyces cerevisiae is encoded by the PH05 gene. The secretion level of this acid phosphatase is directly determined by its level of glycosylation. Consequently, PHO5-11-encoded acid phosphatase which lacks 11 of 12 glycosylation sites is only poorly secreted. We have isolated and characterized both UV- and EMS-induced variants, which are partly able to restore the secretion of acid phosphatase. Our data indicate that the improved secretion is caused by mitotic intrachromosomal recombination between the PHO5-11 allele and the homologous tandemly repeated PHO3 sequences, resulting in the restoration of glycosylation sites in PHO5-11. Two different recombination mechanisms, unequal sister-chromatid exchange and sister-chromatid gene conversion, are responsible for these alterations of the PHO5-11 locus. Thus, recombination between mutant and wild-type sequences are able to restore the ability of mutant yeast cells to secrete acid phosphatase.

Effect of the microtubule inhibitor methyl benzimidazol-2-yl carbamate (MBC) on production and secretion of enzymes in Aspergillus nidulans

The effect of the antimicrotubular drug methyl benzimidazol-2-yl carbamate (MBC) on the production and secretion of acid phosphatase, α-galactosidase and β-galactosidase in Aspergillus nidulans was studied. A wild type and two benomyl resistant mutant ( benA10 and benC28 ) strains were used. All the strains secreted acid phosphatase and α-galactosidase into the culture medium, whereas β-galactosidase remained in the mycelium with a portion of its activity bound to the cell wall. When the wild type strain was incubated in the presence of a sublethal dose of MBC, a decrease of the activity of the enzymes studied was found. A reduction in the secretion of acid phosphatase and α-galactosidase into the culture medium was also observed. In addition, a decrease in the percentage of α- and β-galactosidase activities bound to the cell wall was detected. The MBC dose used for the wild type strain did not modify either the total enzyme activities or the secretion of the enzymes studied in the benomyl resistant mutant benA and benC strains. However, when those strains were grown in the presence of a sublethal dose of MBC, a decrease in the total enzyme activities as well as a reduction in acid phosphatase and α-galactosidase secretion was found. In addition, alterations in the percentage of enzyme activities bound to the cell wall were observed in both mutant strains. Results described in this work, clearly suggest that microtubules are involved in the polarized secretion of enzymes in A. nidulans .

Two isoforms of acid phosphatase secreted by tobacco protoplasts: differential effect of brefeldin A on their secretion

The effects of brefeldin A (BFA) on the secretion of acid phosphatase (APase) by tobacco protoplasts were investigated. Secretion of APase was inhibited by BFA in a dose-dependent manner, with a concomitant intracellular accumulation of the enzyme. The secreted APase was composed of two isoforms. BFA (10/ μg/ml) inhibited the secretion of one of the isoforms without inhibiting that of the other, and this phenomenon explains the partial inhibition of APase secretion as a whole. The inhibition of APase secretion was accompanied by changes in the morphology of the Golgi apparatus and also by an increment in massdensity of cells.

Expression, glycosylation and secretion of yeast acid phosphatase in hamster BHK cells.

The gene PHO5 coding for one of the repressible acid phosphatases of the yeast Saccharomyces cerevisiae has been expressed at high efficiency in the baby hamster kidney (BHK) cell line. The expression vector was constructed from PHO5 driven by the human beta-actin promoter and was transfected into BHK cells by the calcium phosphate method. The recombinant APase (r-APase) which was secreted in active form from the cells was estimated by SDS/polyacrylamide gel electrophoresis to have molecular mass M(r) = 62,000, indicating substitution of the polypeptide moiety by 2-3 asparagine-linked glycans. Analysis by sequential lectin affinity chromatography of glycopeptides obtained from r-APase with Pronase showed that the glycans are predominantly of the 2.2.4 triantennary and tetraantennary complex-type. These data suggest that the extensive glycosylation of yeast APase, which contains eight polymannose substituents, is not essential for secretion and expression of enzymatic activity of the transfected gene product.

Plant cells selected for resistance to phosphate starvation show enhanced P use efficiency

In many organisms, phosphate starvation induces multigene systems that act to increase the availability and uptake of exogenous phosphates. Tissue-cultured tomato cells were plated onto solid media containing starvation levels of phosphate. While most cells died, we identified isolated clumps of callus capable of near-normal rates of growth. Starvation-resistant cells were used to start suspension cultures that were kept under phosphate starvation conditions. A selected cell line showed constitutively enhanced secretion of acid phosphatase and greatly increased rates of phosphate uptake. These pleiotropic effects suggest modification of a regulatory apparatus that controls coordinated changes in the expression of a multigene system. The somaclonal variant cell line grew normally under phosphate-sufficient conditions, but did significantly better than unselected cells under phosphate-limited conditions. In vitro selection may be a useful system for developing phosphate ultraefficient crop plants.

Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor.

We have shown that hybrid proteins composed of the yeast repressible acid phosphatase (PHO5) and bacterial beta-galactosidase (lacZ) interfere with secretion of native acid phosphatase (Wolfe, P. B. (1988) J. Biol. Chem. 263, 6908-6915). We now report that PHO5-LacZ hybrid proteins have a more general effect on secretion and prevent translocation of several secreted proteins. Translocation of both the mating pheromone alpha-factor and the vacuolar protease carboxypeptidase Y is partially blocked when PHO5-LacZ hybrids are expressed. Cell fractionation and protease sensitivity indicate that alpha-factor and carboxypeptidase Y accumulate in precursor form on the cytoplasmic surface of the endoplasmic reticulum. Indirect immunofluorescence with antibody directed against beta-galactosidase supports the localization of hybrid proteins to the endoplasmic reticulum. Analysis of the hybrid protein phenotype in vivo and in vitro suggests that the hybrid proteins deplete a soluble factor required for efficient translocation across the endoplasmic reticulum. First, a decrease in the expression of a hybrid protein in vivo decreases its effect on translocation. Second, an in vitro translation/translocation reaction, prepared from a hybrid-bearing strain, is deficient in its ability to translocate prepro-alpha-factor across yeast microsomal membranes. This deficiency is complemented by addition of cytosol prepared from wild type cells. Finally, the hybrid protein phenotype is shown to be independent of the requirement for SSA gene products.

Effects of seven different mutations in the pho1 gene on enzymatic activity, glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe

Structural gene mutants of the cell-surface glycoprotein acid phosphatase of Schizosaccharomyces pombe were analysed to define structural determinants that are responsible for enzymatic activity, N-glycosylation and secretion. All seven defined mutations cause a single amino acid substitution in the mature acid phosphatase protein and destroy the enzymatic activity. The mutational lesions are distributed throughout the pho1 gene. A ser to phe substitution at position 349 abolishes enzymatic activity only and does not affect glycosylation and secretion. Two mutations create a new N-glycosylation site by substitution of pro at position 56 by phe and ser, respectively. This new site is apparently used in the mutants. Their core-glycosylated acid phosphatase is slightly larger than that of the wild type. Overglycosylation seems not to affect secretion. Four different mutations (a gly to asp substitution at position 281 and ser to phe substitutions at positions 150, 271 and 277) cause intracellular accumulation of enzymatically inactive core-glycosylated acid phosphatase precursor. These mutational lesions apparently block transport of acid phosphatase from the endoplasmic reticulum to the Golgi apparatus.

Secretion of hepatic and intestinal alkaline phosphatases: Similarities and differences

The presence of plasma membrane-bound proteins in the serum of mammals has been noted for many years. Examples of such proteins include alkaline phosphatase (liver, bone, intestinal, and placental) [l], 5’-nucleotidase [2], and gamma-glutamyl transferase [3]. The mechanism of attachment of these proteins to the membrane is now fairly clear. Some proteins, e.g. gamma-glutamyl transferase, are attached to the plasma membrane by a hydrophobic transmembrane sequence [4]. Other proteins, including the alkaline phosphatases (AP), are bound by the fatty acid portion of a phosphatidylinositol-glycan anchor [5]. In spite of this information the mechanism for release of these enzymes from the membrane is not known. Moreover, because secretion is stimulated by factors unique to each organ, the mechanism(s) may differ from organ to organ, e.g. liver and intestine. A variety of suggestions have been offered to explain enzyme release, including detergent action, proteolysis, membrane fragmentation [6], or lipolysis [5]. Detergent action has been suggested most often for hepatocyte membrane proteins, because high concentrations of bile salts are present [7]. Normally the sinusoidal and canalicular alkaline phosphatases are secreted into the blood and bile respectively [S]. The enzyme in bile appears to be particulate [9], whereas that in the blood is largely soluble. The original interpretation of these data were that the sinusoidal enzyme was released by detergent action. However, recent work has shown that solublization with butanol was due not to detergent-like action, but to the activation of tissue phospholipase C that released fatty acids from the AP [lo]. Proteins attached to the membrane by a transmembrane segment could be released by the action of proteases in the membrane or in the surrounding fluid. Cell

Control and localization of the phosphatases in conidia of Neurospora crassa

Repressible acid, repressible alkaline, and constitutive alkaline phosphatases were studied with respect to their control and localization in conidia of Neurospora crassa. In contrast to constitutive alkaline phosphatase, the production and secretion of repressible phosphatases is regulated by phosphate level and pH of the culture medium. Phosphatase activity increased with conidial germination and was detectable partially in the growth medium after 5 h incubation. These enzymes were found to be located in different cell compartments. Part of the whole cell enzyme activity involved a soluble exoconidial fraction, and another part, a cell-bound enzyme that remained after successive washes. The cell-bound enzyme was sensitive to treatment with dilute acid and was thought to be located in the mural space. A third part of the enzyme activity was judged to be intracellular, as shown by treatments with surface-active agents and heat, which disrupted the conidia or destroyed the conidial permeability barriers. On the basis of these criteria, the constitutive alkaline phosphatase was considered to be more cryptic than the repressible phosphatases. The alkaline phosphatases were also active during heat treatment, suggesting they may be involved in the mechanism of secretion.Key words: Neurospora crassa, repressible acid phosphatase, repressible alkaline phosphatase, constitutive alkaline phosphatase, conidia.

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports

A cell culture system is characterized for monolayers of immature rat epididymal epithelial cells grown on permeable supports. Cover of the filters was achieved by days 4-5 and was maintained for 9-12 days. The secretion of acid phosphatase (ACP), alkaline phosphatase (AKP) and N-acetylglucosaminidase (NAG) into apical and basal compartments of culture chambers was monitored with time in culture for cells from the proximal and distal epididymis of 37-day-old animals. There was independent secretion of the three enzymes: secretion of NAG and AKP was mainly apical, that of ACP basal; daily secretion of ACP and AKP was constant throughout culture, that of NAG declined; there was greater secretion of NAG and AKP by cells from the proximal than the distal region. The initial high apical secretion of NAG is thought to reflect loss of enzyme from unattached cells, whereas the later AKP secretion is truly directional. Secretion was not influenced by the enzymes used in cell preparation. The cytotoxic agent Thimerosal inhibited secretion of all enzymes when placed beneath the cultures, indicating that secretion depended on viable cells, but initially stimulated release of AKP when applied above the cells possibly reflecting release from the cell membrane.

PHO5-LACZ hybrid proteins block translocation of native acid phosphatase in Saccharomyces cerevisiae.

A set of protein hybrids composed of variable portions of the amino-terminal residues of the yeast phosphate-repressible acid phosphatase (product of PHO5) and an active fragment of bacterial beta-galactosidase has been constructed. When these PHO5-LACZ hybrids are expressed in a yeast strain carrying an intact chromosomal PHO5 gene, they show a size-dependent interference with the secretion of native acid phosphatase. Hybrid proteins containing approximately 50 residues of acid phosphatase do not affect secretion of native acid phosphatase. Hybrids containing greater than 200 residues of acid phosphatase reduce the amount of secreted acid phosphatase more than by 50%. The interference with secretion is specific for acid phosphatase. The hybrids do not affect secretion of invertase, and do not confer a growth-deficient phenotype on yeast. Both the hybrid proteins and acid phosphatase accumulate in non-glycosylated, membrane-bound forms which are sensitive to proteolysis from the cytoplasmic side of the membrane. The hybrids and accumulated acid phosphatase co-migrate on Percoll density gradients with markers of the endoplasmic reticulum, but not with markers of the Golgi or secretory vesicles. These results suggest that PHO5-LACZ hybrid proteins specifically block secretion of native acid phosphatase by interfering with enzyme after targeting but before translocation across the endoplasmic reticulum.

The effect of indomethacin and aspirin on alkaline phosphatase secretion and [3H]thymidine incorporation by human osteoblasts.

Since it has been suggested that long-term treatment with indomethacin and other non-steroidal anti-inflammatory drugs (NSAIDs) may result in destructive changes in the hip joint, we have examined the effect of indomethacin and aspirin on the secretory and mitotic activity of human osteoblasts in culture. Both indomethacin and aspirin inhibited the secretion of alkaline phosphatase and the uptake of [3H]thymidine by osteoblasts in a dose-dependent fashion at therapeutic concentrations. Both drugs also inhibited insulin- and 1,25-dihydroxycholecalciferol-stimulated alkaline phosphatase production and [3H]thymidine uptake by human osteoblasts. It is concluded that indomethacin and aspirin, and possibly other NSAIDs, may have an inhibitory effect on osteoblast function.

Regulation of synthesis and secretion of acid and alkaline phosphatases in Neurospora crassa

We show that N. crassa represses the production of acid phosphatase at pH higher than 8.0, irrespective of the carbon source used, whereas production was stimulated by sucrose at slightly acidic pH. The same profile of acid phosphatase production was observed in the pho-2A, pho-3A, nuc-1A, nuc-2A and pregc mutant strains. We also show that acid phosphatase synthesized by the pregc mutant strain grown on high phosphate medium has pronounced differences when compared to the enzyme synthesized by the wild-type strain grown on low phosphate medium in terms of heat stability, steady-state kinetic properties and DEAE-cellulose chromatography. In addition, the synthesis and/or secretion of only phosphate-repressible alkaline phosphatase is affected by mutations in acu-1, and acu-5 and acu-7 genes. These results, which indicate distinct pathways for the synthesis and secretion of acid and alkaline phosphatases in N. crassa, contradict the dosage titration model proposed by Metzenberg et al. (1974) whereby the synthesis of these enzymes should occur through a single hierarchical regulatory circuit as a response to phosphate starvation.

Expression of ?-amylase and other gibberellin-regulated genes in aleurone tissue of developing wheat grains

Aleurone tissue from freshly harvested immature wheat grains (Triticum aestivum L. cv. Sappo) which is normally unresponsive to gibberellic acid can be made responsive by subjecting the tissue to a pre-incubation treatment in a simple buffered medium prior to the addition of the growth substance. The effectiveness of this treatment is dependent on grain age, with grains less than 15-20 days post anthesis failing to become converted to a responsive state whilst tissue from grains older than this become increasingly susceptible. Tissue from grains of a certain age (approx. 25-28 days post anthesis) produce small amounts of α-amylase following this treatment even in the absence of exogenously applied growth substance. Using different (32)-labelled complementary-DNA probes for α-amylase in wheat it was demonstrated that the failure of freshly harvested tissue to produce α-amylase was correlated with the absence of the appropriate mRNA species. Inability to accumulate α-amylase mRNA in response to gibberellic acid was removed by the pre-iccubation treatment and also by enforced drying. The gibberellin-regulated expression of other unidentified genes also responds to pre-incubation or drying. Induction of gibberellin-responsiveness in immature aleurone cells did not extend to the secretion of acid phosphatase, protease and ribonuclease.

The effect of myo-inositol deficiency on phosphatases of yeast.

Activities of several phosphohydrolases are significantly enhanced when cells of the inositol-requiring yeast, Saccharomyces uvarum ATCC 9080, are deprived of inositol. This effect is most pronounced for the external acid phosphatase and cannot be explained simply by limitation of cellular growth, because starvation for vitamins or sulphate has no effect on acid phosphatase activities. Excessive secretion of acid phosphatase by spheroplasts prepared from inositol-deficient cells is greatly reduced when the spheroplast medium is supplemented with inositol and is immediately suppressed by the addition of cycloheximide. These results together with data obtained from experiments with whole cells, employing cycloheximide and actinomycin D, point to a regulatory effect of inositol limitation at the level of transcription. The external enzymes beta-D-fructofuranosidase, alpha-D-galactosidase and L-asparaginase, and the vacuolar enzyme carboxypeptidase Y are not affected by inositol deficiency indicating that inositol deficiency has no general effect on protein secretion.

Secretion of alkaline phosphatase and phsopholipase C in Pseudomonas aeruginosa is specific and does not involve an increase in outer membrane permeability

Secretion of alkaline phosphatase and phsopholipase C in Pseudomonas aeruginosa is specific and does not involve an increase in outer membrane permeability

Clomiphene Citrate Administration to Normogonadotropic Subfertile Men: Blood Hormone Changes and Activation of Acid Phosphatase in Seminal Fluid

Clomiphene citrate was administered as a 50 mg oral daily dose to 44 normogonadotrophic (serum FSH 2–10 mIU/ml) subfertile men for 3 months. The treatment resulted in significant increases in FSH and LH concentrations, whereas prolactin remained unchanged. Serum testosterone and oestradiol both increased highly significantly. The increased testosterone levels suggest that the elevated LH levels had not led to “down regulation” of Leydig cell LH/hCG receptors, neither had the greatly increased estradiol led to depletion of these receptors. This is suggested to be a result of the blocking of testicular oestradiol receptors by the estrogen antagonist, clomiphene. Sperm count increased highly significantly during the treatment. The spermatic fluid concentrations of zinc and magnesium ions were also increased, whereas fructose remained unchanged. The katalytic activity of acid phosphatase in spermatic fluid increased highly significantly, whereas the concentration of the main prostate-specific acid phosphatase, as measured by a specific radioimmunological method, remained unchanged. Therefore, the increased Zn and Mg ion concentrations may be responsible for activation of acid phosphatase (s) in semen, or the treatment led to increased secretion of other prostatic acid phosphatase(s) than the main enzyme. However, it is clear that the secretion of the main prostatic acid phosphatase into semen is under different control than that of Zn++ and Mg++.

A HISTOLOGICAL AND HISTOCHEMICAL STUDY OF DIGESTION IN THE BIVALVEARCTICA ISLANDICAL

1. The histology and enzyme histochemistry of the stomach and digestive gland of Arctica islandica were examined. Several enzymological and morphological features contribute to a well-developed system of extracellular digestion in this species.2. Extracellular exopeptidases and alkaline phosphatases are largely derived from primary and secondary ducts. The epithelium of these ducts also secretes esterases and acid phosphatases. In all cases, secretory activity of the ducts overshadows that of the stomach wall. Other digestive enzymes, including acid endopeptidases, are supplied by fragmenting digestive cells. Starvation has little apparent effect on most enzyme activity, but does depress secretion of alkaline phosphatases and intracellular production of acid phosphatases. These enzymes can be induced under appropriate feeding conditions.3. The primary ducts in A. islandica are organized into many ciliated folds, rather than separate rejection and acceptance tracts characteristic of anisomyarian bivalves. ...

ACCUMULATION OF WATER SOLUBLE PHOSPHORUS AND HYDROLYSIS OF POLYPHOSPHATES BY CLADOPHORA GLOMERATA (CHLOROPHYCEAE)1,2

ABSTRACTConcentrations of hot‐water extractable phosphorus from most samples of Cladophora glomerata (L.) Kütz. were relatively high (0.06–0.68%) and correlated closely with total dissolved P in ambient Lake Michigan water. Cladophora was able to hydrolyze polyphosphates by enzymes found in intracellular, extracellular and cell wall fractions. The intracellular phosphatase activity is pH dependent with the optimal hydrolysis rate at pH 7.8. Secretion of phosphatases is affected by pH, with maximum rate at 7, but affected little by light intensity. Magnesium is the most effective metallic cofactor required for maximal rates of intracellular phosphatase activities.

The structure and function of the adhesive organ in strigeid trematodes

A short account of the biology and life-cycle of Diplostomum spathaceum is given.The morphology of the forebody is described in some detail. The lappets are well developed and function as organs of attachment as well as being reservoirs for the secretion from the forebody gland cells. The adhesive organ is variable in form and the muscles facilitating the movements are described in detail. Pressure changes in the excretory system are thought to assist in evaginating the adhesive organ.The histochemical tests gave the following results. The phosphatases present in the adhesive organ and the forebody gland cells are of the acid variety, the alkaline phosphatase is only present in the cuticle, traces of it occur in the lappets and forebody gland cells. No leucine aminopeptidase activity was observed. The esterase present is either a pseudocholinesterase or a non-specific B-esterase. The presence of RNA is confined to areas of enzyme formation.The in vitro studies confirmed the absence of alkaline phosphatase and the presence of acid phosphatase and esterase in the forebody. They also showed a secretion of acid phosphatase and esterase to the exterior of the parasite.Diplostomum spathaceum has very little effect on the host mucosa, but some lysis of the host tissues in contact with the forebody and especially the lappets, is observed. The response of the host to the parasite is likewise very small.The source of nutrients for D. spathaceum is believed to be the contents of the host alimentary tract, host mucus secretions and to a small extent disintegrated host tissues.I am very grateful to Dr D. A. Erasmus for advice and encouragement during this study and to Professor J. Brough for his interest and for provision of excellent working facilities. The work was conducted during the tenure of a Rotary Foundation Scholarship for International Understanding and a grant from Nylands Nation (Helsingfors, Finland).