Studies on host specificity in Paragonimus westermani: II. Histochemical and cytochemical characterization of metacercariae and worms from rats and dogs

Histochemical tests were done on newly excysted metacercariae and worms recovered from an abnormal host (rat) and the definitive host (dog) for some oxidoreductases, phosphatases and glycosidases. The results demonstrate that rat worms have enzymatic distribution and intensities more similar to those of metacercariae than to adult worms from dogs. Ultracytochemical examination of acid and alkaline phosphatase and Mg-ATPase activity was also carried out. Acid phosphatase activity occurred exceptionally in the excretory bladder and caeca of dog worms. No activity was observed in rat worms except for lysosomal granules in the tegument. Alkaline phosphatase activity was exhibited in the excretory bladder in both dog and rat worms. Mg-ATPase activity occurred in the tegument and parenchymal cells in dog worms and in the excretory bladder in rat worms. In metacercariae, little or no reaction for these enzymes was present except for Mg-ATPase activity on the excretory ducts. These observations, together with the histochemical results, indicate that metabolic activity in rat worms is higher than in metacercariae although it is strongly reduced compared with dog worms.     

Regional and Subcellular Distribution in Mammalian Brain of the Enzymes Producing Adenosine

The activities of 5'-nucleotidase, 2'-nucleotidase, alkaline phosphatase, and acid phosphatase were measured in rat and autopsied human brains. The four phosphatases in the rat brain showed little change in activity after death. The activities of adenosine-producing enzymes were compared in various parts of rat and human brains. When phosphatase activity was measured at pH 7.5, 5'-nucleotidase showed the highest activity in the most parts of the brain. The activity of 2'-nucleotidase and that of nonspecific phosphatase were almost the same at pH 7.5. However, higher phosphatase activity was observed in all parts of the brain when nonspecific phosphatase activity was measured at pH 10.0 or 5.5. High specific activity of 5'-nucleotidase in the brain was detected in the membranous components, especially in the synaptic membranes. The activity of 2'-nucleotidase was distributed in the soluble and synaptosomal fractions. The highest activity of both alkaline and acid phosphatases was recovered in the crude mitochondrial fraction, with the highest specific activity in the microsomal fraction. Phosphatase activity was distributed widely in the rat brain. The activity of 5'-nucleotidase was high in the medulla oblongata, thalamus, and hippocampus, but low in the peripheral nerve, spinal cord, and occipital lobe. The activity of 2'-nucleotidase was high in the vermis and frontal lobe. The highest acid and alkaline phosphatase activities were detected in the frontal lobe and in the olfactory bulb, respectively. The distribution of the four phosphatases in the autopsied human brain was similar to that in the rat brain. The highest 5'-nucleotidase activity was observed in the temporal lobe and thalamus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some properties of acid and alkaline phosphates from boar sperm plasma membranes

Boar sperm plasma membranes were purified by differential and sucrose density equilibrium centrifugation and were found to yield a single band at a density of 1.14 g/cm3. Both alkaline and acid phosphatase activities were enriched in this fraction. The alkaline phosphatase activity was optimal in 100 mM tris (hydroxymethyl) methylamine (Tris)-NaHCO3 at pH 9.9 with 0.05% Triton X-100 and 1 mM MgCl2. This activity was inhibited by ethylenediaminetetraacetic acid (EDTA), cadmium, zinc or heating at 60 degrees C for 30 min. Also, L-homoarginine caused approximately 70% inhibition and L-phenylalanine or L-leucine caused about 10 to 20% inhibition. Acid phosphatase activity was optimal in 100 mM sodium acetate at pH 5.1 with 0.05% Triton. Sodium dodecyl sulfate, potassium fluoride (KF) or sulfhydryl reagents inhibited the activity, while EDTA or heating at 60 degrees C had no effect. These data for enzymes from boar sperm plasma membranes can be used for future work on the quantitation of the enzymes, distinguishing these two phosphatases from other phosphohydrolases, purification of the enzymes and for comparison to phosphatases in other tissues.     

Phosphomonoesterases in the male genital system of some mammals.

The various male genital organs of the experimental animals used in this investigation (rat, guinea pig, rabbit, cat and dog) showed a widely different localizations of alkaline and acid phosphatases. Alkaline phosphatase was presented as secretory, stromal, nuclear and vascular, while with acid phosphatase, distinction was made only between secretory and nuclear phosphatases. Although the morphological distributions of both enzymes were sometimes overlapping, they were not identical.     

The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.     

Characterization of the pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate hydrolase activity in rat liver. Identity with alkaline phosphatase.

The tissue content of pyridoxal 5'-phosphate is controlled principally by the protein binding of this coenzyme and its hydrolysis by a cellular phosphatase. The present study identifies this enzyme and its intracellular location in rat liver. Pyridoxal-P is not hydrolyzed by the acid phosphatase of intact lysosomes. At pH 7.4 and 9.0, the subcellular distribution of pyridoxal-P phosphatase activity is similar to the for p-nitrophenyl-P, and the major portion of both activities is found in the plasma membrane fraction. The ratio of specific activities for pyridoxal-P and p-nitrophenyl-P hydrolysis remains relatively constant during the isolation of plasma membranes. These activities also behave concordantly with respect to pH rate profile, pH-Km profile, and response to chelating agents, Zn2+, Mg2+, and inhibitors. Kinetic studies indicate that pyridoxal-P binds to same enzyme sites as beta-glycerophosphate and phosphorylcholine. The data strongly favor alkaline phosphatase as the enzyme which functions in the control of pyridoxal-P and pyridoxamine-P metabolism in rat liver. Alkaline phosphatase was solubilized from isolated plasma membranes. The kinetic properties of the enzyme are not markedly altered by its dissociation from the membrane matrix. However, there are significant differences in its behavior toward Mg2+ which suggest a structural role for Mg2+ in liver alkaline phosphatase.     

Regional differences in the lipid composition and fluidity of rat colonic brush-border membranes

The lipid composition and fluidity of brush-border membranes prepared from rat proximal and distal colonocytes were determined. Fluidity, as assessed by steady-state fluorescence polarization techniques using the fluorophores 1,6-diphenyl-1,3,5-hexatriene, DL-2(9-anthroyl)stearic acid and DL-12(9-anthroyl)stearic acid, was decreased in distal compared to proximal plasma membranes. This pattern was similar to that previously described for both antipodal plasma membranes in rat enterocytes of the small intestine. The decrease in fluidity of the distal as compared to the proximal membranes resulted from an increase in cholesterol content, cholesterol/phospholipid molar ratio and degree of saturation of the fatty acid residues in the distal membranes. The specific activities of total alkaline phosphatase and cysteine-sensitive alkaline phosphatase, enzymes previously shown to be functionally dependent on the physical state of the colonic brush-border membrane's lipid, were also significantly lower in distal as compared to proximal clonic plasma membranes. These studies, therefore, demonstrate that differences in the lipid fluidity, lipid composition and certain enzymatic activities exist in brush-border membranes prepared from rat proximal and distal colonocytes. The regional variation in rat colonic luminal membrane lipid fluidity and composition may, at least partially, be responsible for differences in these enzymatic activities as well as in sodium and water absorption along the length of this organ.     

Alkaline fixation-resistant acid phosphatases in human tissues: histochemical evidence for a new type of acid phosphatase in endothelial, endometrial and neuronal sites

The effect of pH during formalin fixation on acid phosphatases in human tissues was studied. Lysosomal-type acid phosphatase was sensitive to alkaline fixation, being completely inactive after fixation at pH 9.0. Prostatic and tartrate-resistant osteoclastic/macrophagic types were alkaline fixation-resistant, as was an acid phosphatase localized in endothelium, endometrial stromal cells and intestinal nerves. The latter activity was further separable into fluoride- and tartrate-sensitive beta-glycerophosphatase and fluoride-sensitive, tartrate-resistant alpha-naphthyl phosphatase. The activities appeared to represent either different, tightly associated enzymes or separate activity centres of a single enzyme. Alkaline fixation-resistant alpha-naphthyl phosphatase at endothelial, endometrial and neuronal sites was also well demonstrated in unfixed or neutral formalin-fixed sections as tartrate-resistant activity similar to classical tartrate-resistant acid phosphatase, but these phosphatases appear to be antigenically different. Alkaline fixation-resistant acid phosphatase showed a restricted tissue distribution both in endothelium (mainly in vessels of abdominal organs) and at neuronal sites (only in intestinal nerves). Alkaline fixation-resistant acid phosphatase appears to represent a previously unknown or uncharacterized enzyme activity whose chemical properties could not be classified as any previously known type of acid or other phosphatases.     

Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics.

The inhibitory effect of a marine-sponge toxin, okadaic acid, was examined on type 1, type 2A, type 2B and type 2C protein phosphatases as well as on a polycation-modulated (PCM) phosphatase. Of the protein phosphatases examined, the catalytic subunit of type 2A phosphatase from rabbit skeletal muscle was most potently inhibited. For the phosphorylated myosin light-chain (PMLC) phosphatase activity of the enzyme, the concentration of okadaic acid required to obtain 50% inhibition (ID50) was about 1 nM. The PMLC phosphatase activities of type 1 and PCM phosphatase were also strongly inhibited (ID50 0.1-0.5 microM). The PMCL phosphatase activity of type 2B phosphatase (calcineurin) was inhibited to a lesser extent (ID50 4-5 microM). Similar results were obtained for the phosphorylase a phosphatase activity of type 1 and PCM phosphatases and for the p-nitrophenyl phosphate phosphatase activity of calcineurin. The following phosphatases were not affected by up to 10 microM-okadaic acid: type 2C phosphatase, phosphotyrosyl phosphatase, inositol 1,4,5-trisphosphate phosphatase, acid phosphatases and alkaline phosphatases. Thus okadaic acid had a relatively high specificity for type 2A, type 1 and PCM phosphatases. Kinetic studies showed that okadaic acid acts as a non-competitive or mixed inhibitor on the okadaic acid-sensitive enzymes.     

A protein phosphotyrosine phosphatase distinct from alkaline phosphatase with activity against the insulin receptor

Rat liver plasma membranes were found to have a relatively high ratio of acid to alkaline phosphatase activity when compared to rabbit liver and human placental membranes, respectively. The rat liver plasma membranes contained PPTl phosphatase activity against the soluble autophosphorylated insulin receptor beta-subunit. The PPT phosphatase activity of the membranes, using 32P-histone 2b as a substrate, was inhibited by 100 microM Zn+2, insensitive to 10 mM EDTA, and displayed maximal activity at neutral pH. Dephosphorylation of the insulin receptor beta-subunit by rat liver membranes was inhibited by Zn+2, and stimulated by EDTA. These results prove that the plasma membrane of a physiologically relevant insulin target tissue contains a PPT phosphatase, distinct from alkaline phosphatase, which catalyzes the dephosphorylation of the insulin receptor beta-subunit.     

Phosphatidic acid phosphatase and phospholipdase A activities in plasma membranes from fusing muscle cells.

Plasma membrane from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distributions of the plasma membrane markers, (Na+ + k+)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.     

Appearance of different phosphatase forms and phosphorus partitioning in nodules of chickpea (Cicer arietinum L.) during development

Acid and alkaline phosphatase and phytase activities were determined in the bacteroid free fractions of chickpea (Cicer arietinum L.) nodules at 15 days intervals, from 40 days after sowing (DAS) to 85 DAS. In general, the activities and specific activity of both the acid and alkaline phosphatases declined at 55 DAS. Out of the various substrates studied, ATP was the best substrate for both phosphatases. Activities of phosphatases with glucose-6-phosphate and fructose-6-phosphate were low in comparison to these with fructose 1,6 bisphosphate. The efficiency of acid phosphatase for utilizing fructose 1,6 bis phosphate as a substrate increased with nodule development. A fructose 1,6 bis phosphate specific acid phosphatase with elution volume to void volume (Ve/Vo) ratio of around 2.0 was observed in mature nodules (80 DAS). Acid phosphatase at 40 DAS was resolved into two peaks which were eluted at Ve/Vo of about 1.5 and 1.8. However, at 60 DAS the peak with Ve/Vo of 1.5 could not be detected. With ATP as substrate, a high (Ve/Vo of 1.2) and low MM form (Ve/Vo of 2.1) alkaline phosphatases were observed at 40 DAS however at 60 DAS stage only one peak with Ve/Vo of 1.7 was detected. Although, a low activity of acid phytase was observed in nodules at all stages of development but neither alkaline phytase nor phytic acid could be detected. It appears that the nodules acquire inorganic phosphate from the roots. The higher content of water soluble organic phosphorus in mature nodules could be due to the low activities of phosphatases at maturity.     

Increase of PTP levels in vascular injury and in cultured aortic smooth muscle cells treated with specific growth factors

Migration and proliferation of vascular smooth muscle cells are key events in injury-induced neointima formation. Several growth factors and ANG II are thought to be involved in neointima formation. A recent report indicated that vascular injury is associated with increased mRNA levels of protein tyrosine phosphatase (PTP)-1B (PTP-1B). In the present study, we tested the following hypotheses: 1) rat carotid artery injury induces the expression of PTP-1B, Src homology-2 domain phosphatase (SHP-2), and PTP-proline, glutamate, serine, and threonine sequence (PEST) protein; and 2) polypeptide growth factors as well as ANG II increase the levels of tyrosine phosphatases in cultured rat aortic smooth muscle cells. We found that vascular injury induced by balloon catheter increases the protein levels of aforementioned phosphatases and that these effects occur in a PTP specific, as well as temporally and regionally specific, manner. Moreover, treatment of cultured primary rat aortic smooth muscle cells with PDGF or bFGF, but not with IGF1, EGF, or ANG II, increases PTP-1B, SHP-2, and PTP-PEST protein levels. These results suggest that increased PDGF and bFGF levels, occurring after vascular injury, may induce expression of several PTPs.     

Schistosoma mansoni: distribution and characteristics of alkaline and acid phosphatase

The biochemical distribution and characteristics of the alkaline and acid phosphatase activities in both sexes of Schistosoma mansoni are reported. Alkaline and acid phosphatase activities were found in the epidermis as well as in the internal tissues. Alkaline activity is mostly located within the epidermis in both sexes. The acid activity is high in the epidermis of males and more or less equally distributed in females.Starch gel electrophoresis of worm homogenates revealed two sites of activity for both phosphatases. One is an anodic band which is a soluble phosphatase, the other, a membrane bound phosphatase, which remains at the origin under the electrophoretic conditions used. These findings are discussed in relation to previous results and to the possible roles played by these enzymes in adult S. mansoni.     

Histochemical changes in acid and alkaline phosphatase activities in the growing follicles and corpora lutea of the rat ovary

Changes in acid and alkaline phosphatase activities are studied histochemically in different components of the growing follicles and corpora lutea of different generations. Theca cells of the growing follicles showed strong acid phosphatase activity as compared to that observed in the oocyte and granulosa cells. Alkaline phosphatase activity is localized only in the theca interna cells of growing follicles. However, after ovulation with the formation of corpora lutea, the granulosa lutein cells also showed this activity. Changes in the histochemical distribution of the acid and alkaline phosphatases in the follicles and corpora lutea are discussed in relation to folliculogenesis and corpus luteum and regression.     

Phosphatase activity in Trypanosoma cruzi. Phosphate removal from ATP, phosphorylated proteins and other phosphate compounds

T. cruzi epimastigotes have a lysosomal acid phosphatase (pH 4.0) and acid and alkaline phosphatases (pH 5.5 and 8.0) localized in the cytosolic fraction. The levels of the lysosomal acid phosphatase increase with the age of the cultures, but the cytosolic phosphatases decline after the logarithmic phase of growth. The lysosomal phosphatase preferentially hydrolyses low mol. wt phosphate esters; whereas, the cytosolic alkaline phosphatases primarily act on phosphorylated proteins, and both the cytosolic acid and alkaline phosphatases on uridine nucleotide derivatives. The parasite also contains a microsomal glucose 6-phosphatase, and ATPases (Mg2+ and Ca2+-activated) derived from plasma membranes and mitochondria.     

Acid phosphatases from beet root (Beta vulgaris) plasma membranes

Several acid phosphatases (EC 3.1.3.2) were found in beet root ( Beta vulgaris L.) plasma membranes. Two of them were partially purified by an extraction of plasma membranes with octylglucoside and successive gel-filtration and anion-exchange chromatographies. With p -nitrophenyl-phosphate (pNPP) as substrate, most of the phosphatase activity was found in a fraction containing an 82-kDa protein. This phosphatase showed an optimum pH of 5.4 and was inhibited by Cu²⁺, Zn²⁺, molybdate or vanadate. The other phosphatase had a lower specific activity with p NPP, but was able to dephosphorylate phospho-myelin basic protein (phospho-MBP). This phosphatase presented two polypeptides with molecular masses of 36 and 65 kDa and was 83% inhibited by 2 n M okadaic acid, which suggests it is a PP2A protein phosphatase. As the phosphatase activity was high in soluble (non-membrane) fractions, the possibility that phosphatases in plasma membranes were soluble contaminants was assessed. Following the method of Bérczi and Møller (Plant Physiol. 116:1029, 1998), it was found that about 45% of both acid and protein phosphatase activities could be due to soluble enzymes trapped inside membrane vesicles.     

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium

The alkaline phosphatases present on isolated brush border and basal lateral membranes of rat duodenal epitheilum were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6–9.8, similar substrate k_m's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin or papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000–150,000, and both enzymes show an R_f value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The V_max of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 μmoles/mg-h as opposed to 14 μmoles/mg-h. The differences in V_max are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.     

Comparative studies of acid and alkaline phosphatase activities in nonvascular smooth muscle membranes

Acid and alkaline phosphosphatase activities of subcellular fractions isolated from rat gastric muscle and vas deferens by differential centrifugation, sucrose density gradient and cation-induced aggregation methods were studied using p-nitrophenyl phosphate as the substrate. Alkaline phosphatase and a large portion of acid phosphatase activities were found to be of plasmalemmal origin. Acid and alkaline phosphatase activities were different in the effect of Mg2+, fluoride, vanadate, EDTA and resistance to heat inactivation suggesting that these two phosphatase activities were not expressed by the same enzyme.     

The cell surface associated phosphatase activity of Mycobacterium bovis BCG is not regulated by environmental inorganic phosphate

Non-specific phosphomonoesterase activities (alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2)) were examined at the cell surface of Mycobacterium bovis BCG. Using p-nitrophenylphosphate as the substrate, peaks of phosphatase activity were detected at pH 6.0, pH 10.0 and pH 12.0, suggesting the presence of one acid phosphatase and two alkaline phosphatases with distinct optimum pH values. Contrary to the situation observed in several other microorganisms, the expression of these enzymes is not regulated by the environmental inorganic phosphate concentration.