A labile acid phosphatase isozyme associated with the surface of vegetative Dictyostelium discoideum cells

A minor acid phosphatase isozyme (acid phosphatase I) of vegetative Dictyostelium discoideum amebae has been shown to be associated exclusively with the external surface of the plasma membrane. The isozyme is not present in phagocytic vacuoles isolated with latex beads.The isozyme disappears from cells removed from nutrient medium and does not reappear during differentiation. When inhibitors of protein synthesis (e.g. cycloheximide, choral hydrate, concanavallin A) are added to cells growing in nutrient medium, acid phosphatase I is rapidly lost. It appears that the level of protein synthesis need only be moderately reduced (less than 25%) to induce loss of enzyme activity. Treatment with inhibitors of DNA and RNA synthesis for up to 2 h had no effect on isozyme activity. It is postulated that the cells are able to “sense” (through the reduction in levels of protein synthesis) when external conditions become unfavorable, and immediately respond by reducing the activity of enzymes involved in maintaining contact with the extracellular environment. The closed system thought necessary for differentiation would then be created.     

An examination of the age-related patterns of decay of acid phosphatase (ACP1) in human red cells from individuals of different phenotypes

A study has been made of the decay of acid phosphatase (ACP₁) in the human red cell using red cell fractions of different mean ages prepared by density gradient centrifugation. Red cells from acid phosphatase type A and type B individuals were used in the study. Acid phosphatase activity of the red cell fractions was determined by two different assay methods. The results obtained were comparable and have been combined. Acid phosphatase type A and type B showed a biphasic decay pattern with a rapid early loss of activity, followed by a more gradual rate of decline. Type A appeared to decay more rapidly than type B in both decay phases. It is proposed that differences in stability between type A and type B in vivo may explain the observed differences in activity between the enzyme types. There was no evidence for the generation of secondary isozymes by acid phosphatase type A or type B during red cell aging.     

Golgi associated acid phosphatase in muscle and nerve cells fromArion ater (L.)

Summary Golgi associated acid phosphatase has been demonstrated within muscle and nerve cells from the sub-epithelial layers of the crop, intestine, and digestive gland ofArion ater. Enzyme activity was detected in the saccules, vacuoles, and vesicles of the Golgi apparatus of both nerve ganglia and muscle cells. Other vacuolar sources of acid phosphatase could also be distinguished within the cytoplasm of these cells.     

High-level expression of endogenous acid phosphatase inhibits growth and vectorial secretion in Saccharomyces cerevisiae

The secretion pathway of Saccharomyces cerevisiae was challenged by constitutively overexpressing plasmid-encoded acid phosphatase, a secreted endogenous glycoprotein. A 2-μm-based multicopy plasmid carrying the coding sequence of acid phosphatase under the control of a truncated variant of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter was used for expression. Selection for the promoterless dLEU2 marker leads to a growth arrest. This is not per se due to leucine starvation, but due to intracellular accumulation of highly glycosylated enzymatically active acid phosphatase. Immunofluorescence and cytological analysis indicate that intracellular accumulation of acid phosphatase occurs in a subpopulation of cells. By Ludox-AM density centrifugation, these cells can be enriched on the basis of their higher density. The dense accumulating cells have a higher average plasmid copy number and produce more acid phosphatase than non-accumulating cells of low density. These cells are defective in directed secretion and bud formation, therefore can no longer grow and show dramatic changes in cell morphology. We suggest that the secretion pathway in these cells is overloaded with the high level of acid phosphatase leading to a shutdown in vectorial secretion, subsequently to a standstill in growth and to the intracellular accumulation of further expressed acid phosphatase. We have indications that accumulation of acid phosphatase occurs in the late Golgi, suggesting a limitation of the overall secretion at this stage.     

Acid hydrolases and their Release in Food Vacuole-Less Mutants of Tetrahymena thermophila*

SYNOPSIS. Mutants (NP1 and PSJ5) of Tetrahymena thermophila strains B and D 1968 exist that are unable to construct a functional oral apparatus and form food vacuoles at 37 C but which do so normally at 30 C. Food vacuole-less cells starved in dilute salt solution released similar amounts of acid phosphatase, β-N-acetyl-glucosaminidase and ±-glucosidase activity into the medium as wildtype cells during an 8-h period. Actively growing, food vacuole-less cells had ?50% less total protein, acid phosphatase, β-N-acetyl-glucosamin-idase, and ±-glucosidase per cell than wildtype cells after 72-h growth. During this time food vacuole-less cells released significant amounts of the 3 acid hydrolases into the growth medium. For each hydrolase, the total activity released from growing, food vacuole-less cells was less, on a per cell basis, than the amount released from food vacuole formers. The proportion of the total activity secreted by the mutant and the wildtype cells was the same for acid phosphatase and β-N-acetyl-glucosaminidase and somewhat lower for ±-glucosidase. It is concluded that the release of a significant amount of acid hydrolase activity from Tetrahymena is independent of food vacuole formation and may be analogous to the secretory activity of other nonphagocytic eukaryotic cells.     

Effect of endocytosis upon acid phosphatase activity ofTetrahymena pyriformis

Summary Increased endocytosis inTetrahymena pyriformis, produced by presenting starved cells with either peptone-yeast extract medium or killed yeast cell suspension, results in increased cellular acid phosphatase activity.Tetrahymena, grown in peptone-yeast extract medium, showed increased acid phosphatase activity after phagocytosis of yeast cells. This increase was not apparent until about one hour after presentation and was maximal at about 2.5 hours.Tetrahymena, grown on yeast suspension, showed little increase in acid phosphatase activity on presentation with peptone-yeast extract medium. These results may indicate that endocytosis, of either particles or solutes, produces an adaptive increase in acid phosphatase activity (presumably lysosomal in nature) which is related to feeding.Histochemical examination failed to localise the increase in acid phosphatase activity cellularly, but small particles, of about 1 diameter, which showed acid phosphatase activity and were presumably lysosomes were noted. Closely orientated yeast cells showed varying intensities of lead deposition, from absence to intense staining. This suggests that newly ingested yeast cells may be ingested initially in a single phagosome and that thereafter one or more lysosomes may fuse with them.     

A quantitative cytochemical study of acid phosphatases in rat liver parenchymal cells of different ploidy values

Synopsis The Vickers M86 integrating microdensitometer has been used to quantify the cytochemical reaction for acid naphthol AS-BI phosphatase activity in isolated, rat liver parenchymal cells. Data are presented which valudate the method. The use of this method together with that of the Feulgen reaction to estimate nuclear ploidy value in the same cell, has shown that there is an increase in acid phosphatase activity of up to 100% when the euploidy value of the liver cell doubles. It has been further shown that 70–80% of this enzyme activity is ouabain-sensitive, regardless of the euploidy value. The data may be interpreted to indicate that the extra gene copies of the polyploid cells are operative.     

Role of an acid phosphatase isoenzyme in callus tissue during cytodifferentiation

Summary Both histological and isoenzyme patterns of acid phosphatase were observed in callus tissue of Vigna unquiculate (L.) Walp. up to the tenth passage from its initiation. It was observed that when un-differentiated cells begin to transform into a differentiated condition in the form of tracheids, and xylem vessels, a new acid phosphatase band appears at the anionic end of the polyacrylamide gel. The transformation of living cells into a dead, empty tracheid during cellular differentiation and the biosynthesis of the acid phosphatase enzyme are functionally related to the autolysis of the cell contents and lignin synthesis.     

Histochemical aspects of the differentiation of adventitious cartilage on the membrane bones of the embryo chick

Summary The histochemistry of the adventitious cartilage of the chick has been studied and compared with both primary cartilage and the bone on which the adventitious cartilage develops. The distribution of DNA, RNA, collagen, acid mucopolysaccharide, mucoprotein, glycogen, lipid, alkaline phosphatase and inorganic phosphate has been studied. Adventitious cartilage was found to have the histochemistry of primary hypertrophic cartilage and to calcify. The appearance of lipid and alkaline phosphatase activity coincided with the onset of calcification.The proliferating osteogenic and chondrogenic cells of the chick embryo have been classified and compared histochemically. Collagen synthesis was found to be high in the osteogenic cells and acid mucopolysaccharide and mucoprotein synthesis high in the chondrogenic cells.It has been postulated that the morphogenetic switch from osteogenesis to adventitious chondrogenesis most probably involves a change in the rate of collagen and acid mucopolysaccharide synthesis by the germinal cells of the membrane bones.     

The effect of heat hardening on the heat resistance of some enzymes from plant leaves

Summary Heat hardening of leaves which leads to an increase in the heat resistance of their cells, also increases the heat resistance of their enzymes (urease, acid phosphatase, ATPase). As judged by the temperature reducing enzyme activity by 50%, the heat resistance increased by about 6° and 4°, respectively, for urease and acid phosphatase of cucumber, about 7° for acid phosphatase of wheat, and 1,5° for ATPase of Caragana. Increased heat resistance of acid phosphatase and ATPase caused by heat hardening was accompanied by a decrease in the activity ofthese enzymes. The activity of urease was not affected by heat hardening. It is assumed that the cause of this increase in thermal resistance of enzymes is a stabilization of protein macromolecules during heat hardening of leaves.     

Localization of Adenyl Cyclase in Meristems of Young Pea Hypocotyls

Electron micrographs of Pisum sativum L. hypocotyl tips treated to localize adenyl cyclase revealed discrete deposits on the internal membranes of cytoplasmic vacuoles which correspond to previously localized enzymes described as acid phosphatases. It remains to be determined whether the specificity of the substrate, adenylyl-imidodiphosphate, used in the present study is such as to exclude all phosphatase activity other than adenyl cyclase. The acid phosphatase localized in earlier studies by other investigators may be an adenyl cyclase. In the differentiated cells of the root cap, lead precipitate was localized in distinct areas bound to the smooth endoplasmic reticulum.     

Localization of acid phosphatase activity in the liver of pregnant rats

Synopsis In the liver of pregnant rats, fedad libitum, there was an increase in acid phosphatase specific activity which occurred in two peaks, one at the 15th day and the other at the end of gestation. By light and electron microscopic histochemistry, the activity was found to be localized in parenchymal cell peribiliary dense bodies and also in phagosomes present in macrophages and parenchymal cells. There was an increase in liver weight which reached a peak at the 17th day of gestation. Total DNA also rose to the 17th day; there was a high rate of cell division in the hepatic parenchyma at the 17th and 18th days of gestation. During this period single cell deletion by apoptosis was relatively frequent and in late pregnancy there was evidence of cell deletion by lysis.During pregnancy there was a slight increase in sinusoidal macrophages as a proportion of the total cell population but there did not appear to be significant changes in macrophage enzymic activity. It is suggested that the acid phosphatase activity present in macrophages makes a minor contribution to total liver activity, most of which is present in parenchymal cells. Acid phosphatase activity associated with single cell deletion appears to be quantitatively negligible.There was a direct relationship between total hepatic acid phosphatase activity and the numbers of peribiliary dense bodies, which were most numerous at the 15th day and at the end of gestation. It is suggested that these residual bodies contain products of detoxification processes and also cell structural elements resulting from enhanced liver metabolism and intracellular turnover during pregnancy.     

Studies of vegetative compatibility-incompatibility in higher plants

Summary In order to elucidate the events that lead to cellular autolysis, and thus better understand the mechanism of cellular incompatibility betweenSedum telephoides andSolanum pennellii stems, we have followed the appearance and fate of the hydrolytic enzyme acid phosphatase in both the compatibleSedum autograft and the incompatibleSedum/Solanum heterograft. Acid phosphatase was localized by a modified Gomori-type reaction. Following an initial association with the endoplasmic reticulum and dictyosomes by 6–10 hours after grafting, acid phosphatase activity in the compatibleSedum autograft was associated primarily with the plasmalemma, tonoplast, and vacuole. This strict compartmentation in membranes or organelles and absence of enzyme from the cytosol was maintained throughout the development of the compatible autograft inSedum. Although acid phosphatase activity in the incompatible heterograft betweenSedum andSolanum was initially similar to the compatible autograft inSedum, a marked difference in enzyme localization occurred in the two graft partners over time.Solanum cells accumulated increased amounts of acid phosphatase, but the enzyme remained sequestered in the plasmalemma, tonoplast, and vacuole. In comparableSedum cells, however, there was a dramatic increase in acid phosphatase activity in the cytosol, often without any prior compartmentation within the vacuole. This high activity of acid phosphatase in theSedum cytosol was correlated with cellular autolysis, death, and eventual cell collapse to form the characteristic necrotic layer that insulates the stock from the scion. These results suggest that the lethal cellular senescence associated withSedum cells of the incompatible heterograft is correlated with a cytoplasmic release of acid phosphatase. A similar release of the enzyme does not occur in theSolanum stock or in the compatibleSedum autograft. Thus, while acid phosphatase synthesis and/or activation is induced in both the compatible and incompatible grafts, incompatibility betweenSedum andSolanum involves a failure ofSedum cells to isolate hydrolytic enzymes from the cytosol, which subsequently leads to cellular necrosis.Supported in part by grants from the Academic Senate of UCLA, Sigma Xi, the American Philosophical Society, and the URC of Baylor University.     

Synchronous Cultures of Chlamydomonas reinhardti: Properties and Regulation of Repressible Phosphatases

De novo synthesis of phosphatase (derepression) in orthophosphate deprived synchronously growing Chlamydomonas reinhardti has been demonstrated by using a double labelling isotope technique coupled with cellulose acetate electrophoresis. Repressed and derepressed phosphatase exhibited different enzymatic properties as pH optimum, electrophoretic pattern, K_m and K_i values. Especially the acid phosphatase was located near the cell surface. Inorganic, cold TCA-extractable ³²P, decreased during the first 1–2 h after phosphate deprivation when there was little or no net synthesis of phosphatase. Results of experiments with additions of orthophosphate and cycloheximide to derepressed cells, indicated that the derepressible enzyme was relatively unstable, while its m-RNA was relatively stable.     

Docosahexaenoic acid induces apoptosis in lung cancer cells by increasing MKP-1 and down-regulating p-ERK1/2 and p-p38 expression

Different agents able to modulate apoptosis have been shown to modify the expression of the MAP-kinase-phosphatase-1 (MKP-1). The expression of this phosphatase has been considered a potential positive prognostic factor in lung cancer, and smoke was shown to reduce the levels of MKP-1 in ferret lung. Our aim was to assess whether the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), known to inhibit the growth of several cancer cells mainly inducing apoptosis, may exert pro-apoptotic effect in lung cancer cells by modifying MKP-1 expression. We observed that DHA increased MKP-1 protein and mRNA expression and induced apoptosis in different lung cancer cell lines (mink Mv1Lu adenocarcinoma cells, human A549 adenocarcinoma and human BEN squamous carcinoma cells). We inhibited the pro-apoptotic effect of DHA by treating the cells with the phosphatase inhibitor Na₃VO₄ or by silencing the MKP-1 gene with the specific siRNA. This finding demonstrated that the induction of apoptosis by DHA involved a phosphatase activity, specifically that of MKP-1. DHA reduced also the levels of the phosphorylated MAP-kinases, especially ERK1/2 and p38. Such an effect was not observed when the MKP-1 gene was silenced. Altogether, the data provide evidence that the DHA-induced overexpression of MKP-1 and the resulting decrease of MAP-kinase phosphorylation by DHA may underlie the pro-apoptotic effect of this fatty acid in lung cancer cells. Moreover, they support the hypothesis that DHA may exert chemopreventive action in lung cancer.     

Position effect variegation of an acid phosphatase gene in Drosophila melanogaster

Summary X-ray mutagenesis has produced a series of deficiencies in a duplication of part of the third chromosome containing the acid phosphatase gene (Acph-1) in Drosophila melanogaster. In one of these deficiencies, Acph-1 is shown to be undergoing position effect variegation. Naturally occurring electrophoretic variants of the enzyme were used to visualize and determine quantitatively the extent of variegation of the allele which is cis to the heterochromatic breakpoint. Alteration of genotypic background and temperature provided further evidence for position effect. Rocket immunoelectrophoresis was used to correlate the levels of acid phosphatase activity and protein in flies containing the deficiency. A novel result indicates that the variegation is not the consequence of an averaging of active and inactive cells, but rather due to a quantitative alteration of gene activity within at least some individual cells.     

Histochemical analysis of molluscan stomach and intestinal alkaline phosphatase: A sialoglycoprotein

Summary Though sialoprotein nature of alkaline phosphatase of certain mammalian organs has been suggested by biochemical investigations, no histochemical techniques have yet been applied to elucidate this concept. With this view, the alkaline phosphatase of stomach and intestine of a mollusc—Semperula maculata—was analysed histochemically to elucidate its sialoglycoprotein nature. The localisation of alkaline phosphatase and sialic acid was investigated by employing well known and standard histochemical techniques.Alkaline phosphatase was localised selectively in the brush border of the mucosa of stomach and intestine, it was Mg⁺⁺ nonsensitive but showed a structure-linked sensitivity to phenylalanine. The sialomucins were selectively localised in the brush border, whereas the goblet cells contained both the sialomucins and sulfomucins, and the connective tissue of lamina propria contained sulfomucins. The localisation of alkaline phosphatase and sialomucins in the brush border uniquely coincided with each other. The alkaline phosphatase activity in the brush border was completely lost after neuraminidase treatment at 37.5° C for 16 h. Such effect of neuraminidase on alkaline phosphatase activity was pH dependent and controlled by velocity of reaction. Heat-inactivated neuraminidase showed no effect on alkaline phosphatase activity.These histochemical results have been interpreted as suggesting a sialoglycoprotein nature of alkaline phosphatase in the brush border, and sialic acid somehow seems to be essential for enzyme activity. These results, thus, indicate necessity of visualising some of the sialo-glycoproteins as macromolecules with catalytic activity.     

Is acid phosphatase activity present in bone matrix at sites of endochondral ossification in rabbit fracture callus?

It has been suggested that acid phosphatase activity is present in newly formed bone matrix at sites of endochondral ossification in rabbit fracture calluses. Because acid phosphatases are usually found intracellularly, it was decided to test this possibility more rigorously. Tissue from 10- and 14-day healing rabbit fractures was subjected to a series of critical tests for acid phosphatases with a pH optimum of 5.0. Fluoride, tartrate and molybdate were used as potential inhibitors of acid phosphatase activity. The effects of several counterstaining protocols were also investigated. A fluoride- and tartrate-resistant acid phosphatase is located in osteoclasts and mononuclear phagocytes. Diffuse staining of the bone matrix is seen, but it is dependent upon the length of incubation in the substrate medium and the distance from the acid phosphatase-reacting cells. It is concluded that the coloration of the bone matrix is probably caused by diffusion of the dye and reaction product and is, therefore, artifactual. © 1998 Chapman & Hall     

Acid phosphatase activity and cell death in mouse thymus

Summary The distribution of acid phosphatase activity in the thymus of young (8 week) and old (42 week) mice is presented. In 8 week old mice acid phosphatase positive cells represent 1.27±0.13% of the total population whereas in 42 week old mice, showing involution of the thymus, acid phosphatase positive cells represent 2.40±0.17% of the total population. Loci of free acid phosphatase activity have been interpreted as sites of cell lysis and death. This has been confirmed at electron microscope level where free acid phosphatase has been demonstrated in the cytoplasm of lysing thymic lymphocytes. Vacuolar sites of acid phosphatase activity have been demonstrated in macrophages which appear to dispose of the lymphocytes. Extensive autophagic activity occurs in the epithelial reticular cells. The role of acid phosphatase in thymic lymphocyte deletion and in the tissue dynamics of the thymus is discussed.     

Histochemical study of a number of hydrolases,including a new acid phosphatase,in various tissues of men and mice

Summary Two acid phosphatases have been demonstrated histochemically in mouse ventral prostate, seminal vesicles, coagulating glands, and liver and in human prostate. The first is the lysosomal acid phosphatase demonstrable by the Gomori technique. The second differs from thisβ-glycerophosphatase in that it splits naphthol AS phosphates but notβ-glycerophosphate; it has a different histochemical pH optimum and it is not inhibited by MoO₄ or NaF. The enzyme does not represent the “tail” of alkaline phosphatase activity as it is not inhibited by inhibitors of alkaline phosphatase and it has a different localization in liver and in human prostate. The enzyme may be membrane-bound but a lysosomal localization has still to be confirmed.