The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N(2) and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [(125)I]iodide. 5'-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH-cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH-cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.     

Plasma membrane specialization and intracellular polarity of freshly isolated rat hepatocytes

It was investigated whether rat hepatocytes maintain their plasma membrane specialization (sinusoidal, lateral and bile canalicular sites) and their intracellular polarity (peribiliary region, rich in lysosomes and poor in mitochondria) after isolation. The morphology of the hepatocytes and the cytochemical localization of marker enzymes for the bile canalicular membrane (alkaline phosphatase, adenosine triphosphatase and 5' nucleotidase), for the lysosomes (acid phosphatase) and for the mitochondria (beta-hydroxybutyrate dehydrogenase and succinate dehydrogenase) were studied in situ and directly after isolation using both light and electron microscopy. The morphology of the cells and the cytochemical activity of acid phosphatase, succinate dehydrogenase and beta-hydroxybutyrate dehydrogenase showed that in isolated cells, as in situ, the lysosomes were concentrated in bands, devoid of mitochondria. Unlike in situ the reaction product of alkaline phosphatase, adenosine triphosphatase and 5'nucleotidase was evenly distributed along the entire plasma membrane of the isolated cells. Morphologically, no tight or gap junctions or desmosomes could be detected in the isolated cells, while the plasma membrane appeared to be homogeneously covered with uniform microvilli. In conclusion it can be stated that during isolation the hepatocytes loose their distinct plasma membrane specialization, but maintain their peribiliary region rich in lysosomes and poor in mitochondria.     

Induction of plasma membrane alkaline phosphatase in rat liver

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.     

Plasma membranes of human neutrophils: a one-step isolation procedure by cell disruption in paraffin oil

Plasma membranes of high purity and good yield have been prepared from human polymorphonuclear neutrophils by a one-step procedure involving disruption of cells suspended in paraffin oil and forced by pressure through an annular slit. This results in a band floating above the oil which is composed of large sheets of plasma membranes. Enrichment values for the plasma membrane marker alkaline phosphatase and 125I-labeled protein after surface labeling performed at the whole cell level were 23-fold and 22-fold, respectively. Contamination of the plasma membrane by other organelles was negligible and approximately 2 mg of membrane protein was obtained from 10(9) neutrophils. The procedure is very fast and the use of paraffin oil avoids lengthy high-speed centrifugation. The technique also allows isolation of granules devoid of plasma membrane and can probably be applied to other cell types.     

Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases

Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase.     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane.

Mouse leukemia L-1210 cells were iodinated with 125I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of 125I over that of the cell homogenate and a 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide (Mr 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel system for the analysis of membrane proteins is emphasized.     

Isolation of brush-border membranes from rat and rabbit colonocytes: Is alkaline phosphatase a marker enzyme?

Summary A method for the isolation of brush-border membranes of large intestinal epithelial cells was developed, which is based on the purification of intact brush-border caps by Percoll^® density-gradient centrifugation followed by separation of the vesiculated brush-border membranes on sucrose gradients. The procedure has two major advantages in comparison to known methods: 1) its first step does not depend on the determination of marker enzymes and 2) the method is applicable to rats as well as rabbits without major modifications. Due to the lack of an accepted marker for the colonic brush-border membrane the validity of the isolation procedure was tested by its application to the small intestine. Rat small intestinal brush-border membranes were enriched 21-fold when compared to the homogenate. The method was used to evaluate alkaline phosphatase as a marker enzyme for the colonic brush-border membrane. The results suggest that alkaline phosphatase is not exclusively localized in the brush-border membrane since this enzyme was also associated with membranes having different physical properties.     

Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum

Summary Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.     

Intracellular alkaline phosphatase activity in cultured human cancer cells

Summary The effect of saponin treatment in demonstrating intracellular portion of alkaline phosphatase activity in human cancer cell lines was evaluated. Previous reports using standard lead-salt techniques visualized enzyme almost exclusively on the plasma membrane and sometimes in the lysosomes. However, by treating cells with saponin before or during the cytochemical incubation, intracellular alkaline phosphatase became demonstrable at the endoplasmic reticulum, Golgi apparatus, Golgi-derived vesicles and mitochondria as well as lysosomes and plasma membrane. These intracellular catalytic activities were significantly inhibited by the specific amino acid inhibitors characteristic for each cell line, and this suggested that intracellular alkaline phosphatase is the same isoenzyme as that present in the plasma membrane. The results of our current and previous studies therefore indicate that saponin reveals latent intracellular alkaline phosphatase activity by changing the membrane's physical state; thereby increasing the availability of both catalytic and antigenic sites of the enzyme to substrate and to antibody respectively.This work was supported by National Institutes of Health Grant No. CA 21967     

Differential expression of alkaline phosphatase and ATPase activities in human colon carcinoma cell line HT-29.18 during differentiation

The expression and cytochemical localization of alkaline phosphatase and Na+-pump sites were investigated in the human adenocarcinoma cell line HT-29.18 during differentiation. In the undifferentiated state, HT-29.18 cells expressed ATPase activity on plasma membrane whereas they displayed no alkaline phosphatase activity. In differentiated HT-29.18 cells, strong alkaline phosphatase activity was present on the apical membrane, whereas ATPase activity was restricted to the basolateral membrane. Intra- and intercellular lumina (cysts) observed in undifferentiated cells were devoid of both enzyme activities. In differentiated cells, cysts bearing well developed microvilli were strongly positive for alkaline phosphatase activity, while this activity seemed to be lacking in cysts without microvilli. ATPase activity was not found in either type of structure. Finally, HT-29.18 differentiated cells expressed, at pH 9.0, a p-nitrophenylphosphatase activity six-fold greater than that of undifferentiated cells.     

Expression of plasma membrane alkaline phosphatase in normal and regenerating choriocapillaris in the rabbit.

Ultrastructural histochemistry for plasma membrane nonspecific alkaline phosphatase was performed on the normal and regenerating choriocapillaris (CC) of rabbits. In normal animals the CC endothelium expressed little or no staining, whereas in regenerating CC the endothelium exhibited staining. The staining was most intense at the unfenestrated plasma membrane. As the capillaries matured and the fenestrated plasma membrane became more extensive, the staining was reduced and eventually eliminated. Pericytes did not stain in normal or regenerating CC.     

Localization of phosphatases in Trypanosoma rhodesiense

Phosphatase activity in Trypanosoma rhodesiense has been examined histochemically by light and electron microscopy and by enzymatic assay in homogenate fractions. Using a method with lead as capture ion, acid phosphatase was found in lysosome-like vesicles and in the flagellar pocket. No alkaline adenosine triphosphatase (ATPase) was detectable by this method. Direct assay of p-nitrophenylphosphatase activity in homogenate fractions showed that acid phosphatase activity was strongly membrane-bound, but that activity at pH 9 was minimal in both soluble and particulate fractions. "Endogenous" ATPase activity was localized specifically and reproducibly in the mitochondrial membranes and under the plasma membrane of he flagellum. This nonenzymic reaction product could not be eradicated by glycerol extraction or glucose depletion. Unlike the membrane staining, which was manifest only after lead treatment, heat-resistant electron-dense material was found in the matrix of lysosomal vesicles in trypanosomes fixed in glutaraldehyde only and not subjected to further treatment with heavy metal reagents. X-ray emission analysis showed the presence of calcium and phosphorus, indicating that the matrix might have a phosphate storage function.     

Placental alkaline phosphatase expression at the apical and basal plasma membrane in term villous trophoblasts.

Human placental alkaline phosphatase (PLAP) was localized at the apical and basal plasma membrane of syncytiotrophoblasts and at the surface of cytotrophoblasts in term chorionic villi using immunoelectron microscopy. Similarly, apical and basolateral PLAP expression was found in polarized trophoblast-derived BeWo cells. Trophoblasts isolated from term placentas exhibited mainly vesicular PLAP immunofluorescence staining immediately after isolation. After in vitro differentiation into syncytia, PLAP plasma membrane expression was upregulated and exceeded that observed in mononuclear trophoblasts. These data call for caution in using PLAP as a morphological marker to differentiate syncytiotrophoblasts from cytotrophoblasts or as a marker enzyme for placental brush-border membranes. (J Histochem Cytochem 49:1155-1164, 2001)     

Plasma membrane localization of alkaline phosphatase in HeLa cells.

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.     

Regulated internalization of caveolae

Caveolae are specialized invaginations of the plasma membrane which have been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to determine the fraction of internal versus plasma membrane caveolae. The GPI-anchored protein, alkaline phosphatase, was clustered in caveolae after antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phosphatase activity which was dependent on antibody-induced clustering. The decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a protein kinase C activator. Internalization of clustered alkaline phosphatase in the presence or absence of okadaic acid was blocked by cytochalasin D and by the kinase inhibitor staurosporine. Electron microscopy confirmed that okadaic acid induced removal of caveolae from the cell surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell close to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clusters was shown to be dependent on an intact microtubule network. Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and require an intact actin network.     

The effects of cell population density on the plasma membrane of Acanthamoeba castellanii

Plasma membranes were isolated from both exponential and stationary phase cells and their properties compared, to determine whether alterations are sustained coincident with the transition to plateau phase growth. Polyacrylamide gel electrophoresis revealed no significant differences in macromolecular composition between the two types of membrane. However, the specific activity of alkaline phosphatase (EC 3.1.3.1), an enzyme which shows enrichments in purified plasma membrane fractions relative to homogenates, was markedly reduced in preparations from stationary as compared with exponentially growing cells. The total activity per cell did not change, but in cell fractionation experiments the stationary phase cells yielded a higher proportion of the enzyme in microsomal fractions than did exponentially growing cells. This indicates that once plateau phase is attained, a greater proportion of the membrane bearing alkaline phosphatase activity is internalized as opposed to being associated with the plasmalemma.Alkaline phosphatase is known to be present on the contractile vacuole membrane. During discharge this vacuole becomes associated with the plasmalemma, an event which presumably accounts for at least part of the alkaline phosphatase in plasma membrane preparations. Thus one interpretation of the decreased levels of alkaline phosphatase in plasma membrane fractions from stationary phase cells is that they reflect a decline in the rate of water expulsion. This in turn suggests that the plasmalemma of stationary phase cells may have undergone changes leading to a decreased rate of water influx.     

Cytochrome b, flavins, and ubiquinone-50 in enucleated human neutrophils (polymorphonuclear leukocyte cytoplasts)

Neutrophilic granulocytes contain an oxidase system in their plasma membrane that can be activated to generate superoxide radicals and hydrogen peroxide. Cytochrome b, flavoprotein, and ubiquinone-50 have been proposed as components of this oxidase system. These components have been quantitated, but the results are obscured by different isolation procedures for plasma membranes from resting and activated neutrophils. This problem has now been avoided by the use of enucleated neutrophils (polymorphonuclear leukocyte cytoplasts), which are almost completely devoid of intracellular structures but contain an intact, activatable oxidase system (Roos, D., Voetman, A.A., and Meerhof, L.J. (1983) J. Cell Biol. 97, 368-377). Membranes of resting and phorbol myristate acetate-stimulated cytoplasts contain equal amounts of cytochrome b (4 pmol/milliunit of alkaline phosphatase) and also equal amounts of noncovalently bound FAD (2 pmol/milliunit of alkaline phosphatase). These findings refute the hypothesis that incorporation of cytochrome b and/or a flavoprotein into the plasma membrane constitutes the mechanism of activation of the oxidase system. Ubiquinone-50 is present neither in intact neutrophils nor in cytoplasts, excluding a role for this compound in the generation of bactericidal oxygen species by neutrophils.     

The respiratory burst of bovine neutrophils. Role of a b type cytochrome and coenzyme specificity

A new method of preparation of bovine polymorphonuclear leukocytes (PMN) is described. The subcellular distribution of cytochrome b in resting and activated bovine PMN was compared to that of the O2-.-generating oxidase (assessed as NADPH cytochrome c reductase inhibited by superoxide dismutase). In resting PMN and in PMN activated by phorbol myristate acetate (PMA), cytochrome b was located into two membrane fractions, one of which was enriched in plasma membrane and cosedimented with alkaline phosphatase, while the other consisted of a denser material cosedimenting with markers of the specific and azurophil granules, i.e. the vitamin-B12-binding protein and myeloperoxidase respectively. During activation of PMN by PMA, 15-20% cytochrome b migrated from dense granules to the plasma membrane. The distribution of the O2-. generating oxidase and cytochrome b in subcellular particles was studied during the course of phagocytosis of PMA-coated latex beads by bovine PMN. At the onset of the respiratory burst, the phagocytic vacuoles arising from internalization of the plasma membrane were enriched in oxidase and alkaline phosphatase, but their specific content of cytochrome b was limited; in contrast, cytochrome b was predominant in denser membrane fractions cosedimenting with myeloperoxidase and the vitamin-B12-binding protein. After a few minutes of phagocytosis, a fraction of light vacuoles, slightly denser than the phagocytic vacuoles, became enriched in O2-.-generating oxidase, cytochrome b, the vitamin-B12-binding protein and myeloperoxidase. These vacuoles probably arose from the fusion of the phagocytic vacuoles with dense granules. In bovine PMN supplemented with glucose and maintained in anaerobiosis, activation by PMA induced slow reduction of cytochrome b (60-70% in 15 min at 37 degrees C). Similar results were obtained with cytoplasts after activation by PMA (30% reduction in 3 min at 37 degrees C). Cytochrome b in a particulate fraction obtained by centrifugation at 100 000 X g of an homogenate of PMA-activated PMN, was slowly reduced upon addition of NADPH under anaerobiosis (less 20% in 20 min at 37 degrees C). No reduction occurred in the 100 000 X g fraction prepared from non-activated PMN. The Soret band of cytochrome b reduced by dithionite was displaced by CO only by 1-2 nm. At subsaturating concentrations, CO had no effect on the rate of O2 uptake by activated bovine PMN.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.