Formation of multiple forms of acid and alkaline phosphatases in relation to the culture age of Aspergillus niger

Acid phosphatase (EC 3.1.3.2 [EC] ) was extracted from mycelia ofAspergillus niger, then separated and purified into four fractions.These acid phosphatases, designated IA, IB, II and III, hadpH optima at 5.0, 4.5–5.0, 4.5 and 2.5, respectively.None required the presence of divalent cations, and all werestrongly inhibited by NaF. They were non-specific acid phosphatasesbut varied in their activities with various substrates. Thealkaline phosphatase (EG 3.1.3.1 [EC] ) of A. niger was also separatedinto two fractions, alkaline phosphatases I and II. Changes in the activity ratios of these acid and alkaline phosphataseswere studied during culture in a peptone medium. The activityof acid phosphatase II was higher than the others when the culturewas young. The activity of acid phosphatase III increased toa maximum in the actively growing phase, then decreased. Thatof acid phosphatase I became highest in the mature culture.In contrast, the activity of alkaline phosphatase I was higherthan the others in young cultures, while alkaline phosphataseII became dominant in the mature culture. Activities of the various acid and alkaline phosphatases indifferent regions of the growing colonies were also studied.The changing patterns of these enzymes in both liquid and surfacecultures were compared. When A. niger was cultured in a medium containing a low concentrationof phosphate, acid phosphatase activity greatly increased afterthe consumption of phosphate, but alkaline phosphatase activitydid not. ¹ The present experiments were carried out, for the most partat the Institute of Applied Microbiology of the University ofTokyo. (Received February 10, 1975; )     

Tentacles of in vitro-grown round-leaf sundew (<Emphasis Type="Italic">Drosera rotundifolia</Emphasis>L.) show induction of chitinase activity upon mimicking the presence of prey

Induction of plant-derived chitinases in the leaves of a carnivorous plant was demonstrated using aseptically grown round-leaf sundew (Drosera rotundifolia L.). The presence of insect prey was mimicked by placing the chemical inducers gelatine, salicylic acid and crustacean chitin on leaves. In addition, mechanical stirring of tentacles was performed. Chitinase activity was markedly increased in leaf exudates upon application of notably chitin. Application of gelatine increased the proteolytic activity of leaf exudates, indicating that the reaction of sundew leaves depends on the molecular nature of the inducer applied. In situ hybridization of sundew leaves with a Drosera chitinase probe showed chitinase gene expression in different cell types of non-treated leaves, but not in the secretory cells of the glandular heads. Upon induction, chitinase mRNA was also present in the secretory cells of the sundew leaf. The combined results indicate that chitinase is likely to be involved in the decomposition of insect prey by carnivorous plants. This adds a novel role to the already broad function of chitinases in the plant kingdom and may contribute to our understanding of the molecular mechanisms behind the ecological success of carnivorous plants in nutritionally poor environments.     

Some Properties of an Acid Phosphatase Isolated from the Seed Coats of Developing Pea Seeds

An acid phosphatase (EC 3 1 3 2)isolated from the seed-coatsof developing pea seeds was estimated to have MW 30000 by gelfiltration on Sephadex G-75 It was shown to act on a broad spectrumof physiological substrates, the most preferred being ß-glycerophosphate,3-phosphoglycerate and ADP, wich all showed rates of about halfthe maximum rate shown with p-nitrophenyl phosphate (p-NPP)Another model substrate frequently used in enzyme localizationstudies, -naphthyl acid phosphate, was hydrolysed at about 30% of the rate shown with p-NPP This acid phosphatase was enhancedor stabilized by the chelators EDTA and 1, 10-phenanthrolme,unaffected by Mg²⁺ and N-ethyl maleimide, but strongly inhibitedby Zn²⁺ and F^– Both oxidized and reduced glutathione werewithout effect at low concentration and slightly inhibitoryat high concentration (15 mm) Thiol groups are clearly not involvedin regulating the activity of this acid phosphatase, a featurewhich distinguishes it from acid phosphatases from several otherplant species. Pisum sativum L, pea, acid phosphatase, seed-coats, seed development     

Properties and biosynthesis of cell wall-bound acid phosphatase in Aspergillus nigerx

Acid phosphatase (EC 3.1.3.2 [EC] ) of Aspergillus niger myceliumwas distributed exclusively in the cell wall and soluble fractions,whereas alkaline phosphatase was distributed in the solubleand particulate fractions but only slightly in the cell wallfraction. Cell wall-bound acid phosphatase was released by fungal-walllytic enzymes such as snail gut juice. Cell wall-bound, released,and soluble acid phosphatases showed very similar enzymaticproperties except that the bound enzyme was more stable to heatand detergents. By DEAE-cellulose chromatography, the releasedacid phosphatase was found to correspond to acid phosphatasesI A, IB and II in the soluble fraction. When phosphate in the medium was consumed, the acid phosphataseactivity of the soluble fraction increased more rapidly thanthat of the cell wall fraction. When phosphate was added tothe derepressed culture, the acid phosphatase activity of thesoluble fraction decreased after a short lag period, while thatof the cell wall fraction continued to increase. When labeledamino acid was added to the derepressed culture, it was incorporatedinto the soluble acid phosphatase without a lag period, whileit was incorporated into the cell wall phosphatase after a lagperiod. From these observations, acid phosphatase was consideredto be synthesized first as the soluble form and then integratedinto the cell wall. ¹ The present experiments were carried out, for the most part,at the Institute of Applied Microbiology of the University ofTokyo. (Received January 19, 1976; )     

Effect of Phosphorus Nutrition on the Cellular Distribution of Acid Phosphatase in the Leaves of Lycopersicon esculentum L.

A histochemical study using light microscopy has been made ofthe distribution of acid phosphatase (EC 3.1.3.2 [EC] ) activity intransverse sections of fully expanded leaves of Lycopersiconesculentum grown in phosphate-deficient or sufficient media.Leaf tissues were prepared by two methods and were embeddedin paraffin wax. The location of acid phosphatase activity inleaf sections was determined by trapping orthophosphate releasedfrom p-nitrophenyl phosphate with lead acetate and subsequentlyconverting the lead phosphate to optically dense lead sulphide.In leaf sections from control tissue lead sulphide depositswere larpely confined to the spongy mesophyll cells. Whereasthe staining of the palisade cells was limited and of a granularnature, the staining of the spongy mesophyll cells was heavierand coincident with the outline of the individual cells. Moreover,the minor veins were more heavily stained than the surroundingmesophyll cells. Sections of phosphorus-deficient tissues wereheavily stained in both the palisade and spongy mesophyll layersand heavy deposits of lead sulphide were present in the regionsof the minor veins. It is suggested that the enhanced acid phosphataseactivity of the mesophyll cells in fully expanded leaves couldbe involved in the remobilization of phosphate within phosphorus-deficientplants, or be part of a phosphate transporting system, concentratingthe intracellular phosphate from the limiting supply in thesolution bathing the mesophyll cells. Lycopersicon esculentum L., tomato, acid phosphatase, phosphorus nutrition     

The Fine-Structural Localization of Phosphatases in Neurosecretory Cells Within the Ganglia of Certain Gastropod Snails

The neurosecretory cells in the cerebral ganglia of the snail,Planorbis trivolvis, have been examined for structural detailsand distribution of phosphatase activities. A more preliminarystudy on the localization of phosphatases in the neurosecretorycells of Helix aspersa has also been made. These neurons inboth species contain typical elementary neurosecretory granuleswhich appear to be elaborated or condensed in the Golgi saccules.Immature elementary granules are identifiable as "primary lysosomes"in that they contain acid phosphatase activity as well as nucleosidephosphatases. The mature elementary granules display no phosphataseactivity. Some saccules of the Golgi are also reactive for severalnucleoside phosphatases, including thiamine pyrophosphatase(TPPase). In Planorbis, TPPase is also present in the cisternaeof the endoplasmic reticulum. Larger, membrane bound, electron-densebodies (lipochondria) are found in close spatial associationwith the Golgi region. These also possess acid phosphatase;in addition, they contain nucleoside diphosphatases and (inPlanorbis) adenosine triphosphatase. Their content of acid phosphataseand the features of their fine structure indicate that theyare lysosomes akin to the dense bodies of vertebrate neurones. The significance and implications of these results are considered,as are other details of the neuronal and glial structure inPlanorbis.     

Vesicular Transport of Extracellular Acid Phosphatases in Yeast Saccharomyces cerevisiae

A method for isolation of secretory vesicles from the yeast Saccharomyces cerevisiae based on the disintegration of protoplasts by osmotic shock followed by separation of the vesicles by centrifugation in a density gradient of Urografin was developed in this study. Two populations of the secretory vesicles that differ in density and shape were separated. Acid phosphatases (EC 3.1.3.2) were used as markers of the secretory vesicles. It was shown that the constitutive acid phosphatase (PHO3 gene product) is mainly transported to the cell surface by a lower density population of vesicles, while the repressible acid phosphatase (a heteromer encoded by PHO5, PHO10, and PHO11 genes) by a vesicle population of higher density. These data provide evidence that at least two pathways of transport of yeast secretory proteins from the place of their synthesis and maturation to the cell surface may exist. To reveal the probable reasons for transport of Pho3p and Pho5p/Pho10p/Pho11p enzymes by two different kinds of vesicles, we isolated vesicles from strains that synthesize the homomeric forms of the repressible acid phosphatase. It was demonstrated that glycoproteins encoded by the PHO10 and/or PHO11 genes could be responsible for the choice of one of the alternative transport pathways of the repressible acid phosphatase. A high correlation coefficient between bud formation and secretion of Pho5p phosphatase and the absence of correlation between bud formation and secretion of minor phosphatases Pho10p and Pho11p suggests different functional roles of the polypeptides that constitute the native repressible acid phosphatase.     

Alkaline phosphatase activity in Protogonyaulax tamarensis

A non-toxic strain of the marine dinoflagellate Protogonyaulaxtamarensis (= Gonyaulax tamarensis has been isolated from abloom in the Adriatic Sea, off the Emilia-Romagna coast. Culturesof the cells were grown in the laboratory in enriched seawaterat various initial ambient orthophosphate (P_i concentrations,ranging from 0.3 to 40.5 µM. The growth rate varied from0.3 to 0.8 divisions day^–1 depending on the P_i concentration.Alkaline phosphatase activity was inversely proportional toambient P levels. From measurements of kinetic parameters, thebinding of the artificial substrate p-nitrophenylphosphate tothe P.tamarensis alkaline phosphatase was quite strong (K_m=50µM). Maximal activity was observed at pH 8.4, althoughthe pH-activity curve was broad, in contrast to that of otheralkaline phosphatases. Protogonyaulax tamarensis alkaline phosphatase,measured over a 24h period, exhibited an apparent diurnal fluctuationin activity, in common with the enzyme from other dinoflagellates.     

Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants

Abstract: A new ELF (enzyme labelled fluorescence) assay was applied to detect phosphatase activity in glandular structures of 47 carnivorous plant species, especially Lentibulariaceae, in order to understand their digestive activities. We address the following questions: (1) Are phosphatases produced by the plants and/or by inhabitants of the traps? (2) Which type of hairs/glands is involved in the production of phosphatases? (3) Is this phosphatase production a common feature among carnivorous plants or is it restricted to evolutionarily advanced species? Our results showed activity of the phosphatases in glandular structures of the majority of the plants tested, both from the greenhouse and from sterile culture. In addition, extracellular phosphatases can also be produced by trap inhabitants. In Utricularia, activity of phosphatase was detected in internal glands of 27 species from both primitive and advanced sections and different ecological groups. Further positive reactions were found in Genlisea, Pinguicula, Aldrovanda, Dionaea, Drosera, Drosophyllum, Nepenthes, and Cephalotus. In Utricularia and Genlisea, enzymatic secretion was independent of stimulation by prey. Byblis and Roridula are usually considered as “proto‐carnivores”, lacking digestive enzymes. However, we found high activity of phosphatases in both species. Thus, they should be classified as true carnivores. We suggest that the inflorescence of Byblis and some Pinguicula species might also be an additional “carnivorous organ”, which can trap a prey, digest it, and finally absorb available nutrients.     

Identification of Antheridic Acid as an Antheridiogen in Anemia rotundifolia and Anemia flexuosa

Antheridic acid was identified by retention time and full massspectra from GCMS analysis as an antheridiogen in Anemia rotundifoliaand A. flexuosa. In the dark spore germination assay, antheridicacid was active down to 10^–10 and 5 ? 10^–12g.ml^–1in A. rotundifolia and A.flexuosa, respectively. In the antheridiumformation assay, antheridic acid was active down to 10^–10g.ml^–1 in both A. rotundifolia and A.flexuosa (Received April 14, 1987; Accepted July 8, 1987)     

Gibberellic Acid Controls the Secretion of Acid Phosphatase in Aleurone Layers and Isolated Aleurone Protoplasts of Avena fatua

The role of gibberellic acid (GA₃) in controlling the secretion(across the plasma membrane) and release (through the cell wall)of acid phosphatase (E.C. 3.1.3.2 [EC] .) from Avena aleurone layershas been investigated. Evidence from this comparative studywith intact aleurone layers and isolated aleurone protoplastsreveals that the secretion of acid phosphatase is under GA₃control. The mechanism underlying secretion and release of theenzyme from aleurone cells is discussed. Key words: Avena fatua, Acid phosphatase, Aleurone protoplasts, Gibberellic acid, Secretion     

Histochemical localization of acid and alkaline phosphatases and glucose-6-phosphatase of the hepatopancreas of the crab, Scylla serrata (Forskål)

Simultaneous histochemical localization of non-specific monophosphate esterases, acid and alkaline phosphatases, as well as a specific monophosphate esterase, glucose-6-phosphatase has been made on the hepatopancreas of the marine crab, Scylla serrata (Forskål). Maximum activity of the 3 enzymes was observed in the juvenile and mature absorptive cells. Lining cells of the main hepatopancreatic duct exhibited a moderate activity of the 3 enzymes whereas the embryonic and fibrillar cells and connective tissue of the gland showed negative reactions for the 3 enzymes. The secretory cells showed a positive reaction for these enzymes only at the brush border.Bilateral eyestalk removed evoked a rise in the activity of the 3 enzymes within 2–4 h. The same effect was observed after injection of eyestalk extract to both normal and eyestalkless animals followed by restoration to the normal level after 24 h.The present observations indicate that glucose-6-phosphatase and acid phosphatase may be under the direct influence of eyestalk hormone(s) while alkaline phosphatase activity appears to be related to changes in the substrate. The physiological significance of the various cell types and enzymes is discussed.     

Para-nitrophenyl Phosphate as a Substrate for Some Acid Phosphatases in Roots of Vicia faba

A cytochemical and biochemical study of acid phosphatases inroots of Vicia faba using p-nitrophenylphosphate(p-NPP) as substratehas shown the lack of specificity of the substrate which canbe acted upon by a K⁺-activated acyl phosphatase, nucleotidepyrophosphatase, glucox-6-phosphatase, 5' (3')-ribonucleotidephosphohydrolase, nucleotide phosph-transferase and those acidphosphatases demonstrable with ß-glycerophosphate.It is suggested that care should be taken in the interpretationof both biochemical and cytochemical studies employing sucha non-specific substrate as P-NPP. Vicia faba, broad bean, root, acid phosphatases     

Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium.

A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions. These studies revealed the presence of three major enzymes, all of which were subsequently purified: a cyclic 2' ,3'-nucleotide phosphodiesterase (EC 3.1.4.d), an acid hexose phosphatase (EC 3.1.3.2), and a nonspecific acid phosphatase (EC 3.1.3.2). A fourth enzyme hydrolyzed bis-(p-nitrophenyl)phosphate but none of the other substrates tested. No evidence was found for the existence of an alkaline phosphatase (EC 3.1.3.1) or a specific 5'-nucleotidase (EC 3.1.3.5) in S. typhimurium LT2. All three phosphatases could be measured efficiently in intact cells, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures. The nonspecific acid phosphatase, which was purified to apparent homogeneity, yielded a single polypeptide band on both sodium dodecyl sulfate and acidic urea gel electrophoretic systems.     

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.     

Functional Significance of Acid Phosphatase Distribution During Embryo Development in Pisum sativum L

The distribution of P₁, ester P and acid-insoluble nucleic acidP has been studied in relation to acid phosphatase activity(EC 3. 1. 3. 2) in the component parts of developing pea seeds(Pisum sativum L.). Despite the favourable pH of the liquidcontents of the embryo sac (pH 5.5), only very low acid phosphataseactivity was detected in this fluid (c. 0.01 units per seed).Potential substrates for phosphatase action were in fact absentfrom the secretion, the only form of P present being P_i, inconcentrations up to 8 mM. The data support the hypothesis thatthe high acid phosphatase activities which develop in the seed-coatsare involved in regulating the supply of P as P₁ to the developingembryo. Pisum sativum L., pea, embryo development, acid phosphatase, phosphorus, seed-coats, seed development     

Histochemical Localization of Enzymes in the Cucurbitaceae Acid Phosphatases: Acid Phosphatases

Histochemical localization of acid phosphatases was successfullycarried out on the steins of Cucumis, Cucwbita and Colocynthis—threeof the most important genera of the Cucurbitaceae whose speciesare cultivated for their fruits in the tropics. The localizationsshowed generally widespread activity and similarity in distributionfor this enzyme in the tissues of these plants. For each plantacid phosphatase activity decreased with age. The possible significanceof these localizations has been discussed in the light of thedistributional evidence and the roles usually ascribed to thisenzyme. Conventional histochemical techniques for the localizationof acid phosphatase have also been evaluated.     

Auxin induces exocytosis of acid phosphatase in coleoptiles from Zea Mays

Auxin-stimulated elongation growth of maize coleoptiles has been suggested to be associated with enhanced exocytotic activity. However, the problem in plants is one of finding a soluble parameter, which can be used as a direct measure of exocytosis (H. D. Blackbourn and N. H. Battey [1993]. Physiol. Plant. 89: 27–32). In yeast, acid phosphatase (EC 3.1.3.2) is used as a marker for secretory activity (E. Harsay and A. Bretscher [1995]. J. Cell Biol. 131: 297–310). Therefore, extracellular acid phosphatase activities in maize tissues were investigated. Coleoptile (7.36 nkat mg^-1) and mesocotyl (8.9) showed higher specific extracellular acid phosphatase activities than primary leaf (6.0), root (4.9) and root tip (2.7). In coleoptiles extracellular acid phosphatase activity was 6.7% of total homogenate activity (mesocotyls 10.6%). Auxin (30 μM IAA) increased the extracellular acid phosphatase activity of coleoptiles (146% of control). This effect was tissue-specific; extracellular acid phosphatase activity of mesocotyls was not enhanced by IAA. The stimulating effect of auxin on extracellular acid phosphatase activity in coleoptiles was reversed by the protonophore nigericin (0.3 μM). Furthermore, localization of an acid phosphatase activity in Golgi vesicles was shown by co-migration of the Golgi marker latent IDPase (EC 3.6.1.6) and acid phosphatase activity (65% of total microsomal activity) on isopycnic continuous sucrose density gradients. Tonoplast-enriched membrane frctions (24% of microsomal acid phosphatase) and plasma membrane-enriched fractions (11%) contained lower amounts of acid phosphatase. The data presented suggest that acid phosphatase activity is a useful marker for hormone-induced secretory activity in plant cells.     

Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole

Genistein and bromotetramisole(Br-t) strongly activate cystic fibrosis transmembrane conductanceregulator (CFTR; ABCC7) chloride channels on Chinese hamster ovarycells and human airway epithelial cells. We have examined the possiblerole of phosphatases in stimulation by these drugs using patch-clampand biochemical methods. Genistein inhibited the spontaneous rundown ofchannel activity that occurs after membrane patches are excised fromcAMP-stimulated cells but had no effect on purified protein phosphatasetype 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [³²P]PO₄ release from prelabeled casein,recombinant GST-R domain fusion protein, or immunoprecipitatedfull-length CFTR. Br-t also slowed rundown of CFTR channels, but, inmarked contrast to genistein, it did inhibit all four proteinphosphatases tested. Half-maximal inhibition of PP2A and PP2C wasobserved with 0.5 and 1.5 mM Br-t, respectively. Protein phosphataseswere also sensitive to (+)-p-Br-t, a stereoisomer of Br-tthat does not inhibit alkaline phosphatases. Br-t appeared to actexclusively through phosphatases since it did not affect CFTR channelsin patches that had low apparent endogenous phosphatase activity (i.e.,those lacking spontaneous rundown). We conclude that genistein and Br-tact through different mechanisms. Genistein stimulates CFTR withoutinhibiting phosphatases, whereas Br-t acts by inhibiting amembrane-associated protein phosphatase (probably PP2C) that presumablyallows basal phosphorylation to accumulate.

     

Protein Dysfunction and Inhibition of Amino Acid Incorporation in Root Segments from Wheat and Mung Bean Seedlings caused by some Amino Acid Analogues

The effects of para-fluorophenylalanine (p-FPA) and azetidine-2-carboxylicacid (AZ) on acid phosphatase and peroxidase activity, and onL-leucine incorporation, in root segments of Triticum aestivumL. cv. Fenman and Vigna radial a (L.) Wilczek, were studied.Incubation with 50 mmol^–3 AZ significantly reduced phosphataseand peroxidase activities in wheat roots, but with 20 mol m^–3p-FPA, only the peroxidase activity was reduced. In mung beanroots, phosphatase activity was inhibited by both AZ and p-FPA.Effects of the ortho- and meta- isomers of FPA on wheat rootphosphatase and peroxidase, and on mung bean phosphatase, werealso examined. Leucine uptake and incorporation were not inhibitedby 5 h pre-incubation with either p-FPA or AZ, but were inhibitedafter 24 h of pre-incubation. The results support the view that,in the shorter term, the analogues inhibit enzyme activity bybecoming incorporated to produce non-functional protein and,in the longer term, metabolism is further affected by inhibitionof protein synthesis. Key words: Fluorophenylalanin, Azetidine-2-carboxylic acid, Triticum aestivum, Vigna radiata