A method for enzyme- and immunohistochemical staining of large frozen specimens

Summary The extracellular presence of adenosine polyphosphatase was investigated in a number of rat tissues. The enzyme was demonstrated in basement membranes of epithelial cells of duodenum, urinary bladder, tongue, choroid plexus, submandibular salivary gland, lung and kidney, as well as in basement membranes of capillaries in these tissues. Furthermore adenosine polyphosphatase was demonstrated on collagen fibrils and in the cytoplasm of fibroblasts of all investigated tissues. It appears that the presence of adenosine polyphosphatase in basement membranes is a widespread phenomenon. Since extracellular ADP and ATP are known to promote respectively platelet aggregation and inflammation, the presence of extracellular ADP and ATP-hydrolyzing activity might contribute to inhibit these processes.     

Survey of the occurrence of adenosine polyphosphatase in extracellular matrix of rat tissues. A cytochemical investigation.

The extracellular presence of adenosine polyphosphatase was investigated in a number of rat tissues. The enzyme was demonstrated in basement membranes of epithelial cells of duodenum, urinary bladder, tongue, choroid plexus, submandibular salivary gland, lung and kidney, as well as in basement membranes of capillaries in these tissues. Furthermore adenosine polyphosphatase was demonstrated on collagen fibrils and in the cytoplasm of fibroblasts of all investigated tissues. It appears that the presence of adenosine polyphosphatase in basement membranes is a widespread phenomenon. Since extracellular ADP and ATP are known to promote respectively platelet aggregation and inflammation, the presence of extracellular ADP and ATP-hydrolyzing activity might contribute to inhibit these processes.     

Effect of detergents and proteolytic enzymes on membrane-bound phosphohydrolases in Escherichia coli cells with repressed and depressed biosynthesis of these enzymes]

Solubilization of protein membranes by detergents and protein liberation from the membranes induced by proteolytic enzymes results in a change of activity of membrane-bound phosphohydrolases--alkaline phosphatase and polyphosphatase. The activity of enzymes under conditions of repressed and derepressed biosynthesis of phosphohydrolases changes differently, thus indicating their different membrane environment in the two types of membranes. Some data were obtained on the localization of alkaline phosphatase in a hydrophobic region, possibly in lipid bilayer and polyphosphatase in the surface layers of the membrane.     

Interrelationship between metabolic and genetic regulation of alkaline phosphatase and poly- and pyrophosphatases]

The effects of orthophosphate and mutations in the regulatory genes of alkaline phosphatase on the activities of pyrophosphatase and polyphosphatase of E. coli were studied. It was shown that orthophosphate represses the synthesis of alkaline phosphatase as well as that of polyphosphatase without having any effect on pyrophosphatase. The genes phoR and phoS are involved in the formation of a repressory complex both for alkaline phosphatase and polyphosphatase. The gene phoT is probably involved in a partial repression of pyrophosphatase synthesis.     

Two Exopolyphosphatases of the Cytosol of the Yeast S. cerevisiae: Comparative Characteristics

In cell-free extracts of the yeast Saccharomyces cerevisiae that had been transferred from phosphate-deficient (–P) medium to complete (+P) medium (hypercompensation conditions), the specific and the total polyphosphatase activities increased (by 50 and 60%, respectively) compared to the control that was transferred from (+P) medium to (+P) medium. Specific and total polyphosphatase activities under hypercompensation conditions increased by 25 and 43% in cytosol, by 33 and 100% in vacuoles, and by 50 and 50% in the total membrane fraction, respectively. In contrast, the polyphosphatase activity in the cell envelope somewhat decreased compared to the control. Under the growth conditions indicated above, a novel high molecular weight polyphosphatase was revealed in the cytosol fraction along with the previously studied 40-kD polyphosphatase. Unlike the 40-kD polyphosphatase, which is most active with tripolyphosphate, this novel enzyme has a molecular mass of more than 440 kD and is most active with high molecular weight polyphosphates. This polyphosphatase is insensitive to antibodies that suppress the activity of the 40-kD polyphosphatase of the cytosol. In a number of properties, the high molecular weight polyphosphatase of the cytosol resembles the polyphosphatase of vacuoles, but it differs from the polyphosphatases of nuclei and mitochondria of S. cerevisiae. The ratio of the low and high molecular weight polyphosphatases depends on the culture growth conditions. Under hypercompensation conditions, the total activity of the high molecular weight polyphosphatase in the cytosol is five times higher than that of the 40-kD polyphosphatase. During growth without re-inoculation, the 40-kD polyphosphatase is predominant in the cytosol; its total activity in dependence on the growth stage is 3.5-12.5 times higher than the activity of the high molecular weight form.     

Purification and characterization of a soluble polyphosphatase from mitochondria of Saccharomyces cerevisiae

A polyphosphatase with the specific activity 2.2 U/mg was purified to apparent homogeneity from a soluble preparation of mitochondria of Saccharomyces cerevisiae. The polyphosphatase is a monomeric protein of approximately 41 kD. The purified enzyme hydrolyzes polyphosphates with an average chain length of 9 to 208 phosphate residues to the same extent, but its activity is approximately 2-fold higher with tripolyphosphate. ATP, PPi, and p-nitrophenyl phosphate are not substrates of this enzyme. The apparent Km values are 300, 18, and 0.25 microM obtained at hydrolysis of polyphosphates with a chain length of 3, 15, and 188 phosphate residues, respectively. Several divalent cations stimulated the enzyme activity 1.2-27-fold (Mg2+ = Co2+ = Mn2+ > Zn2+). Determination of the protein N-terminal sequence and its comparison with the EMBL data library indicates that the soluble polyphosphatase of mitochondria of S. cerevisiae is not encoded by the gene of the major yeast polyphosphatase PPX1.     

Some pathways of biosynthesis and degradation of polyphosphates from green algae Acetabularia mediterranea]

The activity of ATP: polyphosphate phosphotransferase was detected in free-cellular extracts of Acetabularia mediterranea. The enzyme activity in cells originally deficient in phosphorus and subsequently transferred into the phosphate-containing medium increases 5-10-fold as compared to normal. Polyphosphate degradation in A. mediterranea is probably produced by polyphosphatase, which was also detected in the free-cellular extract. It was shown that the polyphosphatase activity has two pH optima, i.e. 4.5 and 7.5, and is considerably increased when the cells are transferred into the phosphate-free medium. It is assumed that high-molecular polyphosphates involved in A. Mediterranea metabolism are responsible for regulation of orthophosphate and ATP level in the cells by ATP: polyphosphate phosphotransferase and polyphosphatase.     

Isolation and certain features of a polyphosphate phosphohydrolase deficient mutant of the fungus Neurospora crassa

A polyphosphatase deficient mutant of Neurospora crassa has been isolated. The criterion for selecting the mutant was the capacity of the fungus to assimilate polyphosphates as the source of exogenous phosphorus. The mutant like the parent strain ad-6, was an adenine auxotroph but differed from the parent strain by a lower growth rate though, at the stationary stage, its biomass reached the same level as in the strain ad-6. The character of changes in the activity of polyphosphatase in the course of growth was the same in the two cultures, but the activity of the enzyme in the mutant was considerably lower at all the growth stages. The content of polyphosphate fractions with the highest molecular weight increased twofold in the mutant culture. These data suggest that there is a close metabolic and topographic correlation between polyphosphatase and the highest molecular weight fractions of polyphosphates in N. crassa.     

Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains

Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S. cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction. Copyright 1998 John Wiley & Sons, Inc.     

A Modified Adenosine Polyphosphatase Histochemical Method to Demonstrate Langerhans Cells in Fragile Epidermis

Adenosine polyphosphatase enzymes provide useful markers for epidermal Langerhans cells. Established adenosine polyphosphatase histochemical methods were refined and applied to demonstrate Langerhans cells in thin sheets of murine dorsal epidermis. The skin was supported during staining by attaching the keratinized surface to polyallyl diglycol carbonate “plastic” slides with cyanoacrylate adhesive and flattening it with pressure from a glass slide on the dermal surface. Optimal specific staining of dendritic Langerhans cells occurred after fixation of ethylenediaminetetraacetic acid-separated epidermal sheets in cacodylate buffered formaldehyde for 20 min and incubation, in the presence of magnesium and lead ions, with 9.36 × 10^-4 M adenosine diphosphate (ADP) for 45 min. Better definition of the cells was obtained with ADP as a substrate than with any concentration of adenosine triphosphate.     

A modified adenosine polyphosphatase histochemical method to demonstrate Langerhans cells in fragile epidermis

Adenosine polyphosphatase enzymes provide useful markers for epidermal Langerhans cells. Established adenosine polyphosphatase histochemical methods were refined and applied to demonstrate Langerhans cells in thin sheets of murine dorsal epidermis. The skin was supported during staining by attaching the keratinized surface to polyallyl diglycol carbonate "plastic" slides with cyanoacrylate adhesive and flattening it with pressure from a glass slide on the dermal surface. Optimal specific staining of dendritic Langerhans cells occurred after fixation of ethylenediaminetetraacetic acid-separated epidermal sheets in cacodylate buffered formaldehyde for 20 min and incubation, in the presence of magnesium and lead ions, with 9.36 X 10(-4) M adenosine diphosphate (ADP) for 45 min. Better definition of the cells was obtained with ADP as a substrate than with any concentration of adenosine triphosphate.     

RIBONUCLEIC ACID-POLYPHOSPHATE FROM ALGAE: III. HYDROLYSIS STUDIES

RNA-polyphosphate was isolated from synchronous Chlorella cells.After each of a series of hydrolytic treatments, RNA-polyphosphatewas chromatographically analyzed by means of a two-column ion-exchangesystem. Alkaline hydrolysates contained primarily ribonu-cleotides,pyrophosphate, and tripolyphosphate. Acid hydrolysates containedribonucleotides, purine bases, ribonucleosides, orthophosphate,and an unknown, inorganic, phosphorus-containing compound (X-P).Treatments with pancreatic ribonuclease, spleen phosphodiesterase,and yeast polyphosphatase left large amounts of RNA-polyphosphatefragments. Treatment with venom phosphodiesterase yielded ahigh molecular weight inorganic polyphosphate fraction freefrom RNA. Such material was hydrolyzed to pyro- and tripolyphosphateby potassium hydroxide, to orthophosphate and an unknown compoundX-P by perchloric acid, and to ortho-, pyro-, and tripolyphosphateby hydroxylamine under ester hydrolysis conditions. Syntheticinorganic polyphosphate was stable to potassium hydroxide andhydroxylamine under the same conditions and yielded only orthophosphateupon perchloric acid hydrolysis. Both natural and syntheticpolyphosphates were hydrolyzed to low molecular weight fragmentsby yeast polyphosphatase. The evidence at present indicatesthat in Chlorella polyphosphate is not a simple phosphate anhydridechain. (Received June 14, 1965; )     

Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting

Yeast endosomes, like those in animal cells, invaginate their membranes to form internal vesicles. The resulting multivesicular bodies fuse with the vacuole, the lysosome equivalent, delivering the internal vesicles for degradation. We have partially purified internal vesicles and analysed their content. Besides the known component carboxypeptidase S (Cps1p), we identified a polyphosphatase (Phm5p), a presumptive haem oxygenase (Ylr205p/Hmx1p) and a protein of unknown function (Yjl151p/Sna3p). All are membrane proteins, and appear to be cargo molecules rather than part of the vesicle-forming machinery. We show that both Phm5p and Cps1p are ubiquitylated, and that in a doa4 mutant, which has reduced levels of free ubiquitin, Cps1p, Phm5p and Hmx1p are mis-sorted to the vacuolar membrane. Mutation of Lys 6 in the cytoplasmic tail of Phm5p disrupts its sorting, but sorting is restored, even in doa4 cells, by the biosynthetic addition of a single ubiquitin chain. In contrast, Sna3p enters internal vesicles in a ubiquitin-independent manner. Thus, ubiquitin acts as a signal for the partitioning of some, but not all, membrane proteins into invaginating endosomal vesicles.     

Phosphohydrolases of the phytopathogenic fungus Phytophthora infestans (Mont) de Bary]

ATPase, pyrophosphatase and tripolyhosphatase activities were found in a cell-free Phytophtora infestans micelium extract. No polyphosphatase activity, hydrolyzing high molecular weigh polyphosphates to orthophosphate, was observed in the fungi. It was demonstrated that, unlike ATPase, the activity of pyrophosphatase was inhibited by Ca2+ at concentrations from 0.1 to 20 mM, and it was considerably decreased in the presence of a Ca2+ transport inhibitor, ruthenium red (0.01--0.1 mM). Possible relation of Ph. infestans pyrophosphatase activity with the process of active calcium transport is suggested.     

Inorganic Polyphosphate and Cancer

This review presents data on the relationship between inorganic polyphosphate metabolism and carcinogenesis including participation of polyphosphates in the regulation of activity of mTOR and other proteins involved in carcinogenesis, the role of h-prune protein (human polyphosphatase) in cell migration and metastasis formation, the prospects for using polyphosphates and inhibitors of polyphosphate metabolism enzymes as agents for controlling cell proliferation and migration.     

Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.     

Protein import into cyanelles and complex chloroplasts

Higher-plant, green and red algal chloroplasts are surrounded by a double membrane envelope. The glaucocystophyte plastid (cyanelle) has retained a prokaryotic cell wall between the two envelope membranes. The complex chloroplasts of Euglena and dinoflagellates are surrounded by three membranes while the complex chloroplasts of chlorarachniophytes, cryptomonads, brown algae, diatoms and other chromophytes, are surrounded by 4 membranes. The peptidoglycan layer of the cyanelle envelope and the additional membranes of complex chloroplasts provide barriers to chloroplast protein import not present in the simpler double membrane chloroplast envelope. Analysis of presequence structure and in vitro import experiments indicate that proteins are imported directly from the cytoplasm across the two envelope membranes and peptidoglycan layer into cyanelles. Protein import into complex chloroplasts is however fundamentally different. Analysis of presequence structure and in vitro import into microsomal membranes has shown that translocation into the ER is the first step for protein import into complex chloroplasts enclosed by three or four membranes. In vivo pulse chase experiments and immunoelectronmicroscopy have shown that in Euglena, proteins are transported from the ER to the Golgi apparatus prior to import across the three chloroplast membranes. Ultrastructural studies and the presence of ribosomes on the outermost of the four envelope membranes suggests protein import into 4 membrane-bounded complex chloroplasts is directly from the ER like outermost membrane into the chloroplast. The fundamental difference in import mechanisms, post-translational direct chloroplast import or co-translational translocation into the ER prior to chloroplast import, appears to reflect the evolutionary origin of the different chloroplast types. Chloroplasts with a two-membrane envelope are thought to have evolved through the primary endosymbiotic association between a eukaryotic host and a photosynthetic prokaryote while complex chloroplasts are believed to have evolved through a secondary endosymbiotic association between a heterotrophic or possibly phototrophic eukaryotic host and a photosynthetic eukaryote.     

Heptameric association of light-harvesting complex II trimers in partially solubilized photosystem II membranes.

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of seven trimeric light-harvesting complex II proteins. The complex was readily observed in partially-solubilized Tris-washed photosystem II membranes from spinach but was also found to occur, with a low frequency, in oxygen-evolving photosystem II membranes. The structure reveals six peripheral trimers with the same rotational orientation and a central trimer with the opposite orientation. We conclude that the heptamer represents a naturally occurring aggregation state of part of the light-harvesting complex II trimers in the thylakoid membranes.     

Polyphosphate-Mediated Inhibition of Tartrate-Resistant Acid Phosphatase and Suppression of Bone Resorption of Osteoclasts

Inorganic polyphosphate (poly(P)) has recently been found to play an important role in bone formation. In this study, we found that tartrate-resistant acid phosphatase (TRAP), which is abundantly expressed in osteoclasts, has polyphosphatase activity that degrades poly(P) and yields Pi as well as shorter poly(P) chains. Since the TRAP protein that coprecipitated with anti-TRAP monoclonal antibodies exhibited both polyphosphatase and the original phosphatase activity, poly(P) degradation activity is dependent on TRAP and not on other contaminating enzymes. The ferrous chelator α, α’-bipyridyl, which inhibits the TRAP-mediated production of reactive oxygen species (ROS), had no effect on such poly(P) degradation, suggesting that the degradation is not dependent on ROS. In addition, shorter chain length poly(P) molecules were better substrates than longer chains for TRAP, and poly(P) inhibited the phosphatase activity of TRAP depending on its chain length. The IC50 of poly(P) against the original phosphatase activity of TRAP was 9.8 µM with an average chain length more than 300 phosphate residues, whereas the IC50 of poly(P) with a shorter average chain length of 15 phosphate residues was 8.3 mM. Finally, the pit formation activity of cultured rat osteoclasts differentiated by RANKL and M-CSF were markedly inhibited by poly(P), while no obvious decrease in cell number or differentiation efficiency was observed for poly(P). In particular, the inhibition of pit formation by long chain poly(P) with 300 phosphate residues was stronger than that of shorter chain poly(P). Thus, poly(P) may play an important regulatory role in osteoclastic bone resorption by inhibiting TRAP activity, which is dependent on its chain length.