Nonspecific Acid Phosphatase Research Articles

Molecular analysis of the Salmonella typhimurium phoN gene, which encodes nonspecific acid phosphatase

The phoN gene of Salmonella typhimurium encodes nonspecific acid phosphatase (EC 3.1.3.2), which is regulated by a two-component regulatory system consisting of the phoP and phoQ genes. We cloned the phoN region into a plasmid vector by complementation of a phoN mutant strain and determined the nucleotide sequence of the phoN gene and its flanking regions. The phoN gene could encode a 26-kDa protein, which was identified by the maxicell method as the product of phoN. Results of the enzyme assay and Southern hybridization with chromosomal DNA of Escherichia coli K-12 suggests that there is no phoN gene in E. coli. The regulatory pattern of phoN in E. coli and Southern hybridization analysis of the E. coli chromosome with the S. typhimurium phoP gene suggest that E. coli K-12 also harbors the phoP and phoQ genes.

Catalytic histochemistry of acid and neutral hydrolases in plant seedlings

In contrast to human and animal tissues, little information is available on the activity, distribution and functional role of acid and neutral hydrolases in plant cells and tissues. Because it is known that these enzymes are relatively active during germination, they were analysed histochemically during this process using light microscope azo, azoindoxyl, indigogenic and tetrazolium methods. Proteases, glucosidases and glucuronidases could not be detected. Non-specific acid phosphatases were species-independent and showed considerable activities in aleuron and nutritional cells, in other cell types of cotyledon or endosperm tissue and in different types of embryonic cells. Acid glycosidases and non-specific esterases, in contrast, displayed a species-dependent activity and differences in localization. Of the glycosidases, alpha-D-galactosidase was the most active. Non-specific esterases, acid phosphatase and glucosaminidase were also present in the extracellular matrix. During germination, acid hydrolase activity either decreased or increased, depending on the seedling species and enzyme.

The use of phosphopeptides to distinguish between protein phosphatase and acid/alkaline phosphatase activities: opposite specificity toward phosphoseryl/phosphothreonyl substrates

The four main classes of protein phosphatases (PP-1, 2A, 2B and 2C), although differing in their ability to dephosphorylate phosphopeptide substrates, invariably display a marked preference toward phosphothreonyl peptides over their phosphoseryl counterparts. Conversely, all the acidic and alkaline phosphatases tested so far dephosphorylate phosphoseryl derivatives far more readily than phosphothreonyl ones. This opposite behaviour provides a criterion for discriminating between protein dephosphorylating activity due to authentic protein phosphatases as compared to nonspecific acid and/or alkaline phosphatases. In particular the phosphothreonyl peptides RRAT pVA and RRREEET pEEEAA appear to be especially suited for detecting the activity of PP-2C and PP-2A, since they are hardly dephosphorylated by acid and alkaline phosphatases. Conversely, the phosphoseryl peptides S pEEEEE and RRAS pVA can provide a sensitive evaluation of the majority of acid and alkaline phosphatases, while being refractory to protein phosphatases.

Purification, characterization, and subcellular localization of an acid phosphatase from black mustard cell-suspension cultures: Comparison with phospho enolpyruvate phosphatase

An acid phosphatase from Brassica nigra (black mustard) leaf petiole cell-suspension cultures has been purified 1633-fold to a final specific activity of 1225 (μmol orthophosphate produced/min)/mg protein and near homogeneity. The native protein was a glycosylated monomer having a molecular mass of 60 kDa and a p I of 4.5. The enzyme displayed a broad pH optimum of about pH 5.6 and was heat stable. The final preparation hydrolyzed a wide variety of phosphate esters. The highest specificity constants were obtained with 3-phosphoglycerate, 2,3-diphosphoglycerate, PP i and phospho enolpyruvate (PEP). The enzyme was activated 1.4-fold by 4 m m Mg 2+ or Mn 2+, but was strongly inhibited by Mo, P i, F, and several phosphorylated compounds. Subcellular localization experiments revealed that this nonspecific acid phosphatase is probably a secreted enzyme, localized in the cell wall. By contrast, B. nigra PEP phosphatase appeared to be localized in the cell vacuole. Peptide mapping via CNBr fragmentation was employed to investigate the structural relatedness of the two phosphatases. Their respective CNBr cleavage patterns were dissimilar, suggesting that B. nigra acid and PEP phosphatases are distinct polypeptides. Putative metabolic functions of these two phosphatases are discussed in relation to the biochemical adaptations of B. nigra cell-suspension cultures to nutritional phosphate deprivation.

Ultrastructural Localization of ATPases in the Gastrodermis of Megalodiscus temperatus with Additional Observations on Schistosoma japonicum (Trematoda: Digenea)

Ultrastructural Localization of ATPases in the Gastrodermis of Megalodiscus temperatus with Additional Observations on Schistosoma japonicum (Trematoda: Digenea)

Histochemical demonstration of non-specific esterases and non-specific acid phosphatases using menadiol substrates

Although many synthetic substrates and methods are available for the histochemical detection of non-specific esterases and non-specific acid phosphatase, there are still further possibilities to investigate these hydrolases histochemically. This was shown for menadiol diacetate and menadiol diphosphate using tetrazolium salt, simultaneous azo-dye as well as metal salt methods in many rat tissues. In comparison, the azo-dye procedure with various Fast salts or hexazonium Pararosaniline or New Fuchsin delivered less satisfactory results; precisely localized stain in sufficient amounts was obtained for non-specific esterases using nitro BT, tetranitro BT or benzothiazolystyrylphthalhydrazidyl tetrazolium (BSPT) and for non-specific acid phosphatase with BSPT in the tetrazolium salt method or using cerium ions for phosphate trapping in the diaminobenzidine-nickel-hydrogen peroxide procedure.

Changes in intralysosomal environment in the uterine epithelium during early pregnancy in the rat

Non-specific acid phosphatase activity was studied in the rat uterine luminal and glandular epithelium at oestrous, late dioestrous, and 6th d of pregnancy using the substrates beta-glycerophosphate and p-nitrophenyl phosphate, at both pH = 5.0 and pH = 6.0. At late dioestrous and 6th d of pregnancy, a significantly larger lysosomal population in the luminal and glandular epithelium was operating at pH = 5.0 rather than pH = 6.0. At oestrous, the epithelial lysosomes showed no preference for an operational pH = 5.0 or 6.0. Uniquely, the luminal epithelial lysosomes showed a significant preference for the hydrolysis of p-nitrophenyl phosphate over beta-glycerophosphate at 6th d of pregnancy, compared to no substrate preference at oestrous and late dioestrous. The activity of 5-Nucleotidase was also estimated semi-quantitatively at oestrous, late dioestrous, and 6th d of pregnancy in the luminal and glandular epithelium. 5-Nucleotidase activity was greatest in the luminal and glandular epithelium at 6th d of pregnancy. These findings are considered with respect to the possible mechanisms involved in implementing the changes and the relevance to blastocyst implantation.

Effects of superior mesenteric and coeliac ganglionectomy on the small intestinal mucosa in the Hanford mini pig. I. Histological and enzyme-histochemical study

The effects of total superior mesenteric and coeliac ganglionectomy on the thickness of the mucosa, the cell composition of the epithelium and the enzyme activity of the absorptive cells was studied in 10 Hanford mini pigs 3 weeks and 6 months after ganglionectomy. The mucosal thickness increased after ganglionectomy by 10–33% ( P < 0.02) mainly due to increase in the villus height. Differential cell counts showed a postganglionectomy decrease in percentage of goblet cells of 20–40%. Absorptive cell counts increased significantly ( P < 0.05). Enterochromaffin cells (stained with the Masson-Fontana method) and 5-hydroxytryptamine (5-HT)-immunoreactive cells did not change significantly in the small intestine. The activity of l-leucine-amino-peptidase, non-specific alkaline phosphatase, adenosintriphosphatase, non-specific acid phosphatase, non-specific esterase and succinate dehydrogenase, as assessed by absorption photometry, increased by 2–18% ( P < 0.01) after ganglionectomy. Total ganglionectomy thus results in a rise in villus height and in an increase in the number of absorptive cells which, by their enzymatic activity, appear to be fully mature.

In vitro Effects of Phorbol and Phorbol Ester on the Proliferation and Enzyme Activities of Bone Marrow Stromal Cells in Rats

Stromal cells obtained from bone marrow of Wistar rats were cultured in liquid medium. The cultures were divided into control and experimental groups. After 24 h of culture, the medium was removed and replaced by a new one containing 10(-6) and 10(-9) M 4 beta-phorbol or phorbol 12,13-diacetate ester in the experimental cultures, or phorbol solvent alone in control cultures. After 7 days, the cultures were stained with Wright's stain or were subjected to cytochemical procedures for demonstration of nonspecific esterase, alkaline and acid phosphatases and diaphorase activities. The obtained results showed that phorbols stimulated proliferation: fibroblast colonies, fibroblasts, macrophages, immature reticular cells. The number of adipocytes was markedly lower in the experimental cultures. Phorbols stimulated activity of the investigated enzymes.

Heterogeneous malignant non Hodgkin's lymphomas as a causative disorder in lethal midline granuloma

The present report describes the results of a combined morphological, enzyme- and immunohistochemical analysis of nine cases of malignant non Hodgkin's lymphomas (NHL) clinically presenting as lethal midline granuloma. In a previous report written before antibodies directed against B and T lymphocytes were available, a histiocytic origin of such neoplasms had been suggested. A panel of antibodies reactive with most B cells (L26, MB1, KiB3) and a majority of T cells (MT1, UCHL1) was applied on paraffin sections of formalin fixed tissues as well as antibodies directed against leukocyte common antigen (LCA), myeloid/histiocyte antigen (MAC 387), lysozyme, alpha-1-antitrypsin, alpha-1-antichymotrypsin, S-100 protein, prekeratin and immunoglobulin light chains. Enzyme histochemistry included tests for non-specific acid esterase, acid phosphatase, beta-glucuronidase and chloroacetate esterase. As a result, five T, two B and two unclassified (malignant histiocytosis probable) NHL were identified, indicating distinct heterogeneity of NHL as causative disorders in lethal midline granuloma.

Periplasmic nonspecific acid phosphatase II from Salmonella typhimurium LT2. Crystallization, detergent reactivation, and phosphotransferase activity.

The periplasmic nonspecific acid phosphatase II from Salmonella typhimurium was purified to homogeneity from a mutant strain that overproduces the enzyme (Uerkvitz, W., and Beck, C.F. (1981) J. Biol. Chem. 256, 382-389). It was shown that the enzyme transfers phosphate groups from organic phosphoric acid esters (donors) to water as well as to the 2'-, 3'-, or 5'-hydroxyls of nucleosides, nucleotides, and other compounds with free hydroxyl groups (acceptors). The enzyme was crystallized in two forms by precipitation with polyethylene glycol. Needles were formed in buffer containing Mg2+, whereas thin rectangular plates appeared in the presence of the non-ionic detergent n-octyl glucoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under partially or completely denaturating conditions revealed that the native enzyme is a tetramer consisting of identical 24-kDa monomers. Owing to surface inactivation, polyethylene glycol, non-ionic, or Zwitterionic detergents are indispensable for enzyme stability. The detergents are able to reactivate inactivated enzyme when present near or above their critical micelle concentration.

Cytochemical localization of phosphatase activity in vascular bundles and contiguous tissues of the leaf ofZea mays L.

The cytochemical localization of phosphatase activity has been carried out on small and intermediate vascular bundles and contiguous tissues of the leaf ofZea mays L. Similar localization patterns were obtained with the nucleoside triphosphates ATP, CTP, GTP, ITP, and UTP, and with ADP and β-GP. Reaction product (lead deposits) was observed on the plasma membrane of all cell types. It was invariably heavier on the plasma membranes of the bundle-sheath cells, vascular-parenchyma cells, and the thin-walled sieve tubes and their associated companion cells than on those of the mesophyll cells. Within the bundles, the heaviest lead deposits frequently were found on the plasma membranes of the thin-walled sieve tubes and the least amount (often lacking) on those of the thick-walled sieve tubes. Formation of reaction product was suppressed by NaF, vanadate, and molybdate but not by PCMBS (p-chloromercuribenzene sulfonic acid). The results of the substrate-specificity and inhibitor-sensitivity studies indicate that a nonspecific acid phosphatase was probably responsible for the deposition of the reaction product and not the plasma membrane H+-ATPase. These results, in addition to an evaluation of the pertinent literature, lead us to conclude that H+-ATPase activity has yet to be demonstrated unequivocally in association with the plasma membrane of phloem cells with lead precipitation procedures. Nevertheless, the differences in amounts of reaction product generally associated with the plasma membranes of the thick- and thin-walled sieve tubes of the maize leaf indicate that the two types of sieve tube differ from one another physiologically.

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure

New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca- and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5'-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H2O2 which decomposes at 55 degrees-60 degrees C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H2O2 mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H2O2-DAB and especially the H2O2-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.

The high molecular weight and the low molecular weight acid phosphatases of the frog liver and their phosphotyrosine activity

1. 1. Two molecular weight classes of non-specific acid phosphatases (AcPases) (3.1.3.2) are present in the frog ( Rana esculenta) liver: a higher molecular weight (HMW) of M r 140,560 and a lower molecular weight (LMW) of M r 38,180 enzyme. 2. 2. The LMW AcPase was described earlier and the HMW AcPase of optimum pH 4.8 is shown to be a l(+)-tartrate sensitive, thermolabile, dimeric glycoenzyme slightly activated by DTT. 3. 3. The HMW and the LMW AcPases exhibit activity for phosphotyrosine which showed similar sensitivity to various effectors as the p-nitrophenyl phosphatase activity; however, both enzymes differed substantially in this respect suggesting that they might be involved in different metabolic steps.

Cytochemical localization of alkaline and acid phosphatase in human vanishing bone disease.

This report is the first cytochemical investigation of vanishing bone disease "Gorham's Disease" (Gorham and Stout 1955). The ultrastructural localization of non-specific alkaline phosphatase and of specific and non-specific acid phosphatase activity was studied in slices of tissue removed from a patient with this rare disorder. Sodium beta-glycerophosphate and phosphorylcholine chloride were used as substrates. Alkaline phosphatase was present around the plasma membranes of osteoblasts and associated with extracellular matrix vesicles in new woven bone. This is consistent with the proposed role for this enzyme (Robison 1923) and for matrix vesicles (Bonucci 1967) in the mineralization of bone (Bernard and Marvaso 1981). Concentrations of specific secretory acid phosphatase reaction product in the cytoplasm of degenerating osteoblasts may contribute to the imbalance between bone formation and resorption. Osteoclasts, while few in number, showed non-specific and specific acid phosphatase activity. The Golgi apparatus and heterophagic lysosomes of mononuclear phagocytes were rich in non-specific acid phosphatase. This was also present in the Golgi lamellae and lysosomes of endothelial cells. Acid phosphatase cytochemistry suggests that mononuclear phagocytes, multinuclear osteoclasts and the vascular endothelium are involved in bone resorption in this disease.

Lymphocyte emperipolesis into autologous monocytes in leukocyte cultures exposed to mitogenic lectins.

Lymphocyte-monocyte synergistic interaction in cooperative response to mitogens and antigens is well established. This paper describes a less known--antagonistic (effector-target)--lymphocyte-monocyte interaction that came into existence in a leukocyte culture after the commencement of cellular response to concanavalin A, phytohemagglutinin and Wistaria floribunda mitogen. An invasion of lymphocytes into monocytes and monocyte polykaryons has been found 24-48 h after exposure to mitogens. The invasion is not followed by lysosome fusion with lymphocyte-bearing vacuoles, but is associated with a sequential destruction of the vacuole wall, and eventual disintegration of some affected cells. The expression of pan-T-cell surface antigens, staining patterns for nonspecific esterase and acid phosphatase as well as ultrastructural features show that the lymphocytes entering into and those located within monocytes and polykaryons represent activated T-cells. The presence of developed Golgi complexes associated with coated and smooth vesicles, and lysosomal bodies with microvesicles, tubular arrays or dense cores suggest that these T-cells belong to subpopulations which possess cytolytic activities. The lymphocyte invasion is considered cytolytic emperipolesis directed towards some autologous cells of the mononuclear phagocyte series. Its extent depends upon the mitogen concentration, and density of cell population in the culture. It also shows individual variability. The relationship of cytolytic emperipolesis to phagocytosis, and its possible significance as a mechanism of cell-mediated elimination of undesirable cells is discussed.

Isolation and Characterization of Two Enzymes Capable of Hydrolyzing Fructose-1,6-Bisphosphatase from the Lichen Peltigera rufescens

Two enzymes capable of hydrolyzing fructose-1,6-bisphosphate (FBP) have been isolated from the foliose lichen Peltigera rufescens (Weis) Mudd. These enzymes can be separated using Sephadex G-100 and DEAE Sephacel chromatography. One enzyme has a pH optimum of 6.5, and a substrate affinity of 228 micromolar FBP. This enzyme does not require MgCl(2) for activity, and is inhibited by AMP. The second enzyme has a pH optimum of 9.0, with no activity below pH 7.5. This enzyme responds sigmoidally to Mg(2+), with half-saturation concentration of 2.0 millimolar MgCl(2), and demonstrates hyperbolic kinetics for FBP (K(m) = 39 micromolar). This enzyme is activated by 20 millimolar dithiothreitol, is inhibited by AMP, but is not affected by fructose-2-6-bisphosphate. It is hypothesized that the latter enzyme is involved in the photosynthetic process, while the former enzyme is a nonspecific acid phosphatase.

Glycerol, a metabolic end product of Trichomonas vaginalis and Tritrichomonas foetus

Glycerol was demonstrated as an end product of anaerobic glucose metabolism in Trichomonas vaginalis and Tritrichomonas foetus, produced in addition to acetate, H 2, CO 2, and lactate or succinate. In T. vaginalis strain C-1, glycerol amounted to 16% of the fermentation products and was formed at an average rate of 38 nmol min −1 (mg protein) −1. Corresponding figures for T. foetus strain KV 1 were 7% and 4.8 nmol min −1(mg protein) −1. The amounts of glycerol detected compensated almost exactly for the deficits in fermentation products recognized earlier, thus complete redox balances can now be provided for both organisms. The metronidazole-resistant T. foetus strain KV 1-1MR-100 excreted only negligible amounts of glycerol and carried out an ethanol-CO 2 fermentation. Aerobiosis hardly affected glycerol formation in T. vaginalis strains C-1 and NYH 286, but almost completely abolished it in T. foetus strain KV 1. An NADP-dependent glycerol 3-phosphate dehydrogenase and a Mg 2+-dependent glycerol 3-phosphatase were detected in the cytosol of both species. The phosphatase is distinct from the particle-bound nonspecific acid phosphatase. Glycerol kinase activity was not detected in either organism. Enhanced pCO 2 did not affect the ration of fermentation products in T. vaginalis strain C-1, but significantly increased the amount of succinate, and decreased the amounts of acetate, H 2, and CO 2, formed by T. foetus.

Subcellular distribution of acid NADPase activity within the parenchymal cells of rat liver.

We examined the distribution of acid NADPase activity in rat hepatocytes by cytochemistry and subcellular fractionation. Cytochemical studies revealed strong NADPase activity in many lysosomes and Golgi saccules situated between the medial and trans aspect of the stack. Light, spotty deposits of reaction product also were observed frequently within elements of the Golgi-associated endoplasmic reticulum lysosome (GERL) system. Biochemical studies with liver homogenates indicated that NADPase activity was distributed across various subcellular fractions in a pattern resembling typical nonspecific acid phosphatase activity, as monitored using beta-glycerophosphate as substrate. Thus, by differential centrifugation, most cellular NADPase activity was found in the large granule (ML) fraction (61%). The microsomal (P) fraction showed about 17% whereas the nuclear (N) and cytosol (S) fractions each accounted for about 11% of the remaining NADPase activity, respectively. Separation of the ML fraction into endosomes and secondary lysosomes revealed most of the acid NADPase activity associated with lysosomes. An alternate one-step method for isolation of fractions indicated that 3% of the recovered acid NADPase activity, but 55% of the Golgi marker enzyme galactosyl transferase, was associated with a purified intact Golgi fraction. Cytochemical studies with ML and intact Golgi fractions indicated that bona fide reaction product was localized to lysosomes and to the same saccules that were reactive in vivo. We propose that saccules making up the trans half of the Golgi stack may represent a site for accumulation of a biogenetic precursor of the lysosomal acid NADPase in hepatocytes.

Leu-M1-positive small cell carcinoma

A case of metastatic small cell carcinoma of the lung is reported; initially, on the basis of morphology, phenotyping with monoclonal antibodies, and cytochemistry, the carcinoma was interpreted as a hematopoietic neoplasm. Noncohesive blast-like cells observed in bone marrow and lymph node biopsy specimens stained with the monoclonal antibodies Leu-M1 and OKIa1 and were also positive for nonspecific esterase and acid phosphatase. Although these findings suggested a monocytic origin for the neoplastic cells, further analysis, including ultrastructural examination, disclosed metastatic small cell carcinoma. This case illustrates the need for caution in the interpretation of staining with monoclonal antibodies because of the potentially wide range of normal and abnormal cells and tissues that may react with monoclonal reagents.