Acid phosphatase activity in the isolated brush border membrane of the tapeworm, Hymenolepis diminuta: partial characterization and differentiation from the alkaline phosphatase activity

The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzes p-nitrophenyl phosphate over a broad pH range. Acid phosphatase activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2-mercaptoethanol, and ethylenediaminetetra-acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X-100, while such treatment causes a significant loss of acid phosphatase activity. Both activities are nonspecific and hydrolyze a variety of phosphorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.     

Release of alkaline phosphatase activity by aqueous-ether treatment of whole cells of Serratia marcescens.

The effect of aqueous-ether treatment according to the method of Ribi et al. (1961) on the release of alkaline phosphatase from cells of two strains of Serratia marcescens was studied. By this method, lipopolysaccharide-protein (endotoxin) complexes associated with alkaline phosphatase activities were released from both strain 08 and strain Bizio. SDS-polyacrylamide gel electrophoresis followed by enzymatic assay showed the presence of two active components in each strain. Fractions released from strain 08 contained alkaline phosphatase A (140,000 dalton) and alkaline phosphatase B (110,000) daltons) while those from strain Bizio contained alkaline phosphatase A' (190,000 daltons) and alkaline phosphatase B (110,000 daltons). Although it is known that saline plays a role in the release of alkaline phosphatase activities from cell envelope of Gram-negative bacteria the presence of saline in the extracting medium affects only slightly the chemical composition and not at all on the enzymatic nature of the released components. By comparing the enzymatic profiles of the materials released by other techniques, such as polymyxin B treatment and osmotic shock, it appears that alkaline phosphatase activities released by aqueous-ether treatment of whole cells of S. marcescens originate from the periplasmic space.     

Identification of the phosphatase deinhibitor protein phosphatases in rabbit skeletal muscle.

In rabbit skeletal muscle the polycation-stimulated (PCS) protein phosphatases [Merlevede (1985) Adv. Protein Phosphatases 1, 1-18] are the only phosphatases displaying significant activity toward the deinhibitor protein. Among them, the PCSH protein phosphatase represents more than 80% of the measurable deinhibitor phosphatase activity associated with the PCS phosphatases. The deinhibitor phosphatase activity co-purifies with the PCSH phosphatase to apparent homogeneity. In the last purification step two forms of PCSH phosphatase were separated (PCSH1, containing 62, 55 and 34 kDa subunits, and PCSH2, containing 62 and 35 kDa subunits), both showing the same deinhibitor/phosphorylase phosphatase activity ratio. The activity of the PCSH phosphatase toward the deinhibitor is not stimulated by polycations such as protamine, histone H1 or polylysine, unlike the stimulation observed with phosphorylase as the substrate. The phosphorylase phosphatase activity of PCSH phosphatase is inhibited by ATP, PPi and Pi, whereas the deinhibitor phosphatase activity of the enzyme is much less sensitive to these agents.     

Subcellular distributions of rat CaM kinase phosphatase N and other members of the CaM kinase regulatory system

Ca2+/Calmodulin-dependent protein kinase (CaM kinase) regulatory system is composed of multifunctional CaM kinases such as CaM kinases IV and I, upstream CaM kinases such as CaM kinase kinases alpha and beta, which activate multifunctional CaM kinases, and CaM kinase phosphatases such as CaM kinase phosphatase and CaM kinase phosphatase N, which deactivate the activated multifunctional CaM kinases. To understand the combinations of CaM kinases I and IV, CaM kinase kinases alpha and beta, and CaM kinase phosphatases, the locations of the enzymes in the cell were examined by immunocytochemical studies of cultured cells. The results indicate that CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase occur in the cytoplasm and that CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N occur inside the cellular nucleus, suggesting that there are at least two different sets of CaM kinase regulatory systems, one consisting of CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase in the cytoplasm and the other consisting of CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N in the nucleus.     

Ectoenzymes in Prorocentrum minimum

Ectoenzymes, or enzymes associated with the cell-surface or periplasmic space, play an important role in organic matter cycling by rendering certain forms of dissolved organic matter bioavailable. Ectoenzyme activities may thereby help meet the nutritional demands of harmful algae such as Prorocentrum minimum. The activities of two ectoenzymes; leucine aminopeptidase and alkaline phosphatase, have been studied in axenic cultures of P. minimum. Leucine aminopeptidase releases non-polar amino acids such as leucine from the N-terminus of polypeptides, whereas alkaline phosphatase is an enzyme that is able to hydrolyze phosphate from phosphomonoesters. P. minimum alkaline phosphatase is the better studied of the two ectoenzymes and its characteristics are reviewed herein. Future research on P. minimum physiology will benefit from a growing suite of tools available for assessing the activity of alkaline phosphatase and other ectoenzymes in field populations and ultimately the work done with P. minimum will be useful for studies of other harmful species.     

Effects of sea urchin-based diets on serum lipid composition and on intestinal enzymes in rats

The dietary effects of two high protein diets from two species of sea urchin (Paracentrotus lividus and Echinus esculentus) as compared to a reference protein such as casein on serum lipid levels and on intestinal disaccharidases and alkaline phosphatase were studied. After 23 days, the containing the two sea urchins as diets compared to casein decreased the cholesterol level and significantly increased the HDL-cholesterol in serum. The consumption of Echinus esculentus meal produced a significant decrease in lactase activity. The intestinal alkaline phosphatase activity increased not significantly in animals fed on the sea urchin meal.     

Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeletal muscle

Summary A heat- and acid-stable proten inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does not occur when phosphorylated histone or phosphorylated protamine are used as substrates.     

Low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase in the developing chick brain: partial characterization and levels during development.

Low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase is largely expressed in chick brain tissue during development. The enzyme was purified from brain extract prepared from 19-day-old chick embryos and from adult chickens using ammonium sulfate fractionation, gel filtration on Sephadex G-75 and two DEAE-Cellulose ion-exchange chromatography steps. The purified enzymes from embryo and adult chick brains show identical molecular weight values (about 18-20 kDa) and biochemical and structural properties such as substrate specificity, sensitivity to inhibitors, and number of free reactive sulphydryl groups. These data suggest that they are the same enzyme protein. Although the total acid phosphatase activity does not change appreciably during development, the activity associated with the low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase markedly increases after birth and reaches the adult values within the first week of life. Taken together, our results suggest an involvement of the low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase in postnatal development and maturation of chick brain tissue. The variations in tyrosine phosphorylation profile of chick brain polypeptides analyzed by Western blotting at the same developmental stages are also reported.     

Regulation of synthase phosphatase and phosphorylase phosphatase in rat liver.

Using substrates purified from liver, the apparent Km values of synthase phosphatase ([UDPglucose--glycogen glucosyltransferase-D]phosphohydrolase, EC 3.1.3.42) and phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) were found to be 0.7 and 60 units/ml respectively. The maximal velocity of phosphorylase phosphatase was more than a 100 times that of synthase phosphatase. In adrenalectomized, fasted animals there was a complete loss of synthase phosphatase but only a slight decrease in phosphorylase phosphatase when activity was measured using endogenous substrates in a concentrated liver extract. When assayed under optimal conditions with purified substrates, both activities were present but had decreased to very low levels. Mixing experiments indicated that synthase D present in the extract of adrenalectomized fasted animals was altered such that it was no longer a substrate for synthase phosphatase from normal rats. Phosphorylase a substrate on the other hand was unaltered and readily converted. When glucose was given in vivo, no change in percent of synthase in the I form was seen in adrenalectomized rats but the percent of phosphorylase in the a form was reduced. Precipitation of protein from an extract of normal fed rats with ethanol produced a large activation of phosphorylase phosphatase activity with no corresponding increase in synthase phosphatase activity. Despite the low phosphorylase phosphatase present in extracts of adrenalectomized fasted animals, ethanol precipitation increased activity to the same high level as obtained in the normal fed rats. Synthase phosphatase and phosphorylase phosphatase activities were also decreased in normal fasted, diabetic fed and fasted, and adrenalectomized fed rats. Both enzymes recovered in the same manner temporally after oral glucose administration to adrenalectomized, fasted rats. These results suggest an integrated regulatory mechanism for the two phosphatase.     

Stable acid phosphatase: II. Effects of pH and inhibitors

Summary Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl phosphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at nearneutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membrances. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.Supported by the Medical Research Council of Great Britain     

Purification and characterization of a Mn2+/phospholipid-dependent protein phosphatase from pig brain membranes

A Mn²⁺/phospholipid-dependent protein phosphatase has been identified and characterized from brain membranes. The phosphatase contains three subunits with molecular weights of 64,000, 54,000, and 35,000 in a 1:1:1 molar ratio. On gel filtration, the enzyme has an apparent molecular weight of 180,000. The phosphatase was active on many substrates, including p-nitrophenyl phosphate, phosphotyrosine, phosphothreonine, phosphorylase a, myelin basic protein, histones, type 1 phosphatase inhibitor-2, microtubule protein, and synapsin I. To dephosphorylate phosphoproteins, the phosphatase was dependent on such acidic phospholipids as phosphatidylinositol and phosphatidylserine but not on neutral phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The phospholipid-mediated activation of the phosphatase was time and dose dependent and could be reversed by Triton X-100 or gel filtration. Kinetic study further indicates that phospholipid was able to increase theV _max of the phosphatase but had no effect on theK _m value for substrates, suggesting a direct interaction of phospholipids with the phosphatase. Conversely, in order to dephosphorylate phosphoamino acids such as phosphotyrosine and phosphothreonine, this phosphatase was entirely dependent on Mn²⁺. Phospholipids had no effect on the dephosphorylation of phosphoamino acids, whereas Mn²⁺ had no effect on the dephosphorylation of phosphoproteins. It is concluded that this Mn²⁺/phospholipid-dependent membrane phosphatase has two distinct activation mechanisms. The enzyme requires Mn²⁺ to dephosphorylate micromolecules, whereas acidic phospholipids are needed to dephosphorylate macromolecules. This suggests that Mn²⁺ and phospholipids may play a role in regulating the substrate specificity of this multisubstrate membrane phosphatase.     

Potentiation of the effect of steroid hormones (progesterone and testosterone) inducing the reinitiation of meiosis in Xenopus oocytes in vitro, by inhibitors of phosphatase activity

The induction of meiosis reinitiation by steroid hormones (progesterone and testosterone) in Xenopus laevis oocytes was studied in vitro in presence of inhibitors of phosphatase activity such as beta-glycerophosphate, considered as a competitive inhibitor, and the two ions, Zn++ and MoO--4. Kinetics of the germinal vesicle breakdown indicating the reinitiation of meiosis, have shown that while these phosphatase inhibitors were not active by themselves under the present experimental conditions, they enhanced the process elicited by progesterone or testosterone.     

Molecular nature of three liver alkaline phosphatases detected by drug administration in vivo: differences between soluble and membranous enzymes

1. Activities of alkaline phosphatase, liver-membranous, liver-soluble and serum-soluble, were dramatically induced in dogs by treatment with both phenobarbital and brovanexine. The treatment induced a 17-fold increase in membranous, a 155-fold increase in soluble, and a 105-fold increase in serum alkaline phosphatases. 2. There was no difference in the enzymatic behavior of the three forms of alkaline phosphatase, on heat stability, amino acid inhibition and optimum pH. 3. When the three alkaline phosphatases were treated initially with n-butanol, their apparent molecular size was identical. After treatment with phosphatidylinositol-specific phospholipase C, the liver-soluble and serum-soluble alkaline phosphatase were of the same molecular size. Liver-membranous alkaline phosphatase, however, was larger in molecular size than the other two forms, suggesting a difference between soluble and membranous alkaline phosphatase forms. 4. In terms of the sugar moiety of the three alkaline phosphatase forms, the membranous enzyme showed more of the higher affinity fraction and less of the lower affinity fraction of concanavalin A, compared with the soluble enzymes. 5. Consequently, it is possible that the membranous enzyme may be solubilized by an enzyme such as phosphatidylinositol-specific phospholipase C and modify further the sugar moiety of alkaline phosphatase molecules, resulting in serum alkaline phosphatase transfer from the soluble enzyme in liver.     

Recognition of stem cells in the colonial internae of the Rhizocephala Peltogasterella gracilis and Sacculina polygenea at the parasitic life cycle stage

The colonial internae of two decapod parasites Peltogasterella gracilis and Sacculina polygenea (Crustacea: Cirripedia: Rhizocephala) were studied in vitro by histological, and histochemical methods. We found stem cells, characterized by basophilic cytoplasm, a large nucleus and nucleolus, and a high alkaline phosphatase activity. The cytochemical manifestation of alkaline phosphatase activity in totipotent stem cells of the colonial rhizocephala is the first evidence of commonality in the functional characteristics of embryonic stem cells in vertebrate and invertebrate animals of such different taxa as the mammals and Crustacea.     

Stable acid phosphatase: II. Effects of pH and inhibitors

Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl posphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at near-neutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membranes. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.     

Antibodies against neuroactive amino acids and neuropeptides. II. Simultaneous immunoenzymatic double staining with labeled primary antibodies of the same species and a combination of the ABC method and the hapten-anti-hapten bridge (HAB) technique.

In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed.     

Earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge

Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences: (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis.E.L. Cooper has been supported by The Alexander von Humboldt Foundation, a GAAC grant from the Federal Republic of Germany and two grants from NATO, a Cooperative Research grant (971128) and an Advanced Research Workshop grant (976680)     

Partial purification and characterization of phosphotyrosyl-protein phosphatase(s) from human erythrocyte cytosol

Phosphotyrosyl-protein phosphatase activity of human erythrocyte cytosol can be resolved into two fractions by DEAE-cellulose chromatography followed by P-cellulose chromatography. Both 32P-Tyr-phosphatases are able to dephosphorylate 32P-Tyr of poly (Glu-Tyr) 4:1 but not angiotensin II and synthetic peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Gly, previously phosphorylated on tyrosine residues by rat spleen tyrosine-protein kinase. Both 32P-Tyr-phosphatase activities distinctly differ from either 32P-Ser-casein phosphatase activity or "acid" and "alkaline" p-nitrophenylphosphatase activities with regard to catalytic and physico-chemical properties such as substrate specificity, chromatographic behaviour, response to various effectors.     

In-gel protein phosphatase assay using fluorogenic substrates

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.     

Tyrosine kinase inhibitors reverse butyrate stimulation of human Caco-2 intestinal epithelial cell alkaline phosphatase but not butyrate promotion of dipeptidyl dipeptidase

Short chain fatty acids such as sodium butyrate are concentrated in the colonic lumen and may protect against colon carcinogenesis by maintaining colonocytic differentiation, but the mechanisms by which they act are not fully understood. It has recently been suggested that short chain fatty acids modulate cellular tyrosine kinase activity in addition to altering chromatin structure via regulation of histone acetylation and DNA methylation. Therefore, the authors evaluated the influence of tyrosine kinase inhibition on the effects of 10 mM butyrate on human Caco-2 intestinal epithelial differentiation, using alkaline phosphatase and dipeptidyl dipeptidase specific activity as markers of differentiation, and two tyrosine kinase inhibitors, of different mechanisms of action and different effects on Caco-2 brush border enzyme specific activity, to block tyrosine kinase activity. As expected, butyrate stimulated both alkaline phosphatase and dipeptidyl dipeptidase specific activity. The tyrosine kinase inhibitors prevented, and indeed one inhibitor reversed the effects of butyrate on alkaline phosphatase specific activity. However, tyrosine kinase inhibition did not prevent butyrate stimulation of dipeptidyl dipeptidase specific activity. Different pathways are likely to regulate the effects of butyrate on expression of these two brush border enzymes. Butyrate stimulation of alkaline phosphatase, but not dipeptidyl dipeptidase, may involve tyrosine phosphorylation signaling.