Cytochemical localization of alkaline phosphatase in the cell membrane ofDictyostelium discoideum amebae

Histochemistry and Cell Biology - We demonstrated that alkaline phosphatase was localized on the cell membrane ofDictyostelium discoideum amebae and on isolated plasma membranes. The enzyme...     

Cytochemical localization of alkaline phosphatase in the cell membrane ofDictyostelium discoideum amebae

Summary We demonstrated that alkaline phosphatase was localized on the cell membrane ofDictyostelium discoideum amebae and on isolated plasma membranes. The enzyme activity was specifically inhibited by 0.01 M KCN or cysteine. The same method could also be applied to baker's yeast and MDCK cells (dog kidney cells in vitro).     

Studies on the alkaline phosphatase and 5'-nucleotidase of Dictyostelium discoideum.

The alkaline phosphatase and 5′-nucleotidase activities of Dictyostelium discoideum are due to two distinct enzymes. Both enzymes are membrane bound, but over 90% of the 5′-nucleotidase activity is solubilized when the crude membrane fraction of the cell is treated with phospholipase C under conditions that release only 10% of the alkaline phosphatase.Part of the alkaline phosphatase activity can be detected in whole cells, suggesting that some of the enzyme molecules are located on the exterior surface of the plasma membrane. In contrast very low 5′-nucleotidase activity can be detected in whole cells. When membrane preparations, isolated from cells that had been surface labeled with ¹²⁵I, were subjected to sedimentation equilibrium on sucrose density gradients, the majority of the ¹²⁵I-radioactivity cosedimented with the alkaline phosphatase and 5′-nucleotidase activites, suggesting that both enzymes are plasma membrane components.The two enzymes have distinctly different pH optima, but otherwise their properties are remarkably similar. Both enzymes are inhibited by cyanide, sulfhydryl inhibitors and sulfhydryl reagents, although in each case the 5′-nucleotidase is slightly more susceptible. Both enzymes are inhibited by the levamisole analogue, R 8231, but the alkaline phosphatase is inhibited to a somewhat greater extent. Both enzymes are activated by incubation at 50 °C but inactivated by higher temperatures.The two enzymes increase in activity at identical times during differentiation, suggesting that they are under coordinate developmental control.     

Studies on the mechanism of action of the alkaline phosphatase from Dictyostelium discoideum

The Dictyostelium discoideum alkaline phosphatase was investigated kinetically in an attempt to elucidate its mechanism of action. Analysis of the hydrolysis of p-nitrophenyl phosphate by stopped-flow spectrophotometry revealed biphasic kinetics, suggesting a double displacement enzyme mechanism. Furthermore, Tris stimulated activity in an uncompetitive manner, a result that was consistent with this interpretation. The enzyme was inhibited reversibly by phosphate at low ionic strength, but the inhibition was irreversible at high ionic strength and the latter effect was enhanced at alkaline pH values. These results indicate that high ionic strength and alkaline pH conditions bring about a conformational change that renders the enzyme susceptible to irreversible inhibition by phosphate.     

Cytochemical localization of alkaline phosphatase in intestinal metaplasia of the human stomach

Summary Alkaline phosphatase in the brush border of areas of intestinal metaplasia of human stomach was studied cytochemically. All absorptive cells in the upper part of the villi of the duodenum had strong alkaline phosphatase activity but, in areas of intestinal metaplasia, the metaplastic glands consisted of alkaline phosphatase-positive and negative absorptive cells. Alkaline phosphatase activity was found in tall dense microvilli of absorptive cells in areas of intestinal metaplasia and in the duodenum. However, in some areas of metaplastic epithelium, the activity was very weak in some tall dense microvilli of absorptive cells but strong in those of neighbouring absorptive cells. No alkaline phosphatase activity was found in short sparse microvilli of absorptive cells in areas of intestinal metaplasia. The difference in alkaline phosphatase activity in microvilli of different cells in areas of intestinal metaplasia, which is not seen in the duodenum, indicates abnormal morphological and enzymatic differentiation in intestinal metaplasia.     

Cytochemical localization of alkaline phosphatase in the ependyma of the rat medulla oblongata

Summary The ependyma of the IVth ventricle and the central canal of the rat medulla oblongata was investigated using the cytochemical technique for alkaline phosphatase (AlPase) which revealed two types of ependymal cells in the medulla. The central canal type of the ependymal cell occupying the dorsal part of the central canal in the lower medulla exhibited intense AlPase activity with light microscopy. These cells had reaction products in all plasma membranes, including the microvilli and the cilia at the luminal cell surface. Some cells appeared to be tanycytes, since the process reached the basement membrane of the parenchymal blood vessel. The ventricular type of ependymal cells, which form the floor of the IVth ventricle and the central canal, contained no reaction products in any structure of the luminal cell surface.The possible relationship between the cerebrospinal fluid and the nervous tissues through the ependymal linings is discussed.     

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.     

The membrane-bound alkaline phosphatase and 5'-nucleotidase activities of vegetative cells of Dictyostelium discoideum

Earlier reports suggested that the adenosine monophosphate (AMP)- and the p-nitrophenyl phosphate (pNPP)-hydrolyzing activities of Dictyostelium discoideum membrane preparations are due to different proteins. These results have been apparently contradicted by the recent purification to homogeneity of the two activities from culmination phase cells as a single protein [D. R. Armant and C. L. Rutherford (1981) J. Biol. Chem. 256, 12710-12718]. Results presented here from studies on the activities of vegetative cells support the concept of a single protein. Nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Triton X-100 extracts of cell membrane preparations of D. discoideum showed identical migration of pNPPase and AMPase activities. Furthermore, the previously reported different pH optima of the two activities was due to the fact that pH optima are dependent upon the substrate concentration, and the selective solubilization of AMPase from membrane preparations by phospholipase C can probably be accounted for by the finding that phospholipase C preparations from the same commercial source contain 5'-nucleotidase activity. Moreover, there are alterations in the Km and the stability of both AMPase and pNPPase in a strain with a mutationally altered alkaline phosphatase, further supporting the concept that the two activities are due to a single protein. Both substrates serve as transphosphorylation donors demonstrating that the enzyme activity is mechanistically an alkaline phosphatase.     

Biochemical and genetic analysis of a mutant with altered alkaline phosphatase activity in Dictyostelium discoideum

Alkaline phosphatase is one of several enzymes that accumulate in a temporally regulated sequence during the development of Dictyostelium discoideum. These enzymes can be used to monitor specific gene expression; moreover, isolation and analysis of mutations in the structural gene(s) can serve to indicate some of the essential steps in programmed synthesis and morphogenesis. A mutation (alpA) which affects the activity and substrate affinity of alkaline phosphatase was isolated in D discoideum using a procedure for screening large numbers of clones. Alkaline phosphatase activity at all stages of vegetative growth and development was altered by the mutation. Several physical properties of the enzyme from growing cells and developed cells were compared and found to be indistinguishable. It is likely that a single enzyme is responsible for the majority of alkaline phosphatase activity in growth and development. The mutation is coexpressed in diploids heterozygous for alpA and maps to linkage group III. One of the haploid segregants isolated from these diploids carries convenient markers on each of the six defined linkage groups and can be used for linkage analysis of other genetic loci.     

Analysis of microtubule arms and bridges in mitotic and interphase amebae of Dictyostelium discoideum

We have analyzed transparencies of electron micrographs from ultrathin longitudinal sections through mitotic spindles of undifferentiated amebae of Dictyostelium discoideum for the presence of arms on microtubules (MTs) and bridges between them. We used the technique of microdensitometer scanning and computer-based model matching by cross-correlation and autocorrelation. We also determined that spindle MTs are composed of 13 protofilaments. Although regularly arranged lateral appendages are not a universal feature of MTs in these cells, both cross-correlation and autocorrelation analysis revealed that bridges between a kinetochore MT and its neighbor, and between MTs in the zone of overlap of the central spindle were significantly arranged on a 12-dimer superlattice. In addition, the autocorrelation analysis indicated a slight match with the 12-dimer model for neighboring non-kinetochore MTs. Although electron micrographs revealed putative arms on cytoplasmic and astral MTs, as well as bridges between central spindle MTs outside the zone of overlap, their arrangement did not match any of the models tested. Bridges between non-kinetochore MTs in the half-spindles possibly serve to reinforce the spindle scaffold. Bridges between kinetochore MTs and their neighbors may contribute to the mechanical stability of kinetochore fibers or they may be involved in poleward movements of the chromosomes. In the zone of overlap of the central spindle, the occurrence of frequent and regularly spaced bridges is consistent with models predicting that a sliding mechanism operates between MTs of opposite polarity in this region of the spindle to produce its elongation.     

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.     

Expression pattern of alkaline phosphatase in Dictyostelium

We used two different methods to study the expression pattern of alkaline phosphatase (alp) in Dictyostelium. In situ staining of the endogenous enzyme activity at different stages of development showed that the enzyme was active early in the aggregation stage and localized to the area where the tip of the first finger was initiated. The activity was localized to the anterior region of developing slugs, then became restricted to the region between the prestalk and prespore cells at the culmination stage. In the complete fruiting body, the activity was confined to the lower and upper cup. A second method to study alp expression utilized a beta-galactosidase reporter gene under the control of the alp promoter. A low level of beta-galactosidase activity was observed in vegetative cells, then increased during development. Reporter gene activity was restricted to PstO cells at the slug stage. At the culmination stage, the expression was restricted to prestalk cells at the interface between the prestalk and prespore cells. In the completed fruiting body, the expression was observed in the upper and lower cup.     

Cytoplasmic pH of Dictyostelium discoideum amebae during early development: identification of two cell subpopulations before the aggregation stage

Development of the cellular slime mold Dictyostelium discoideum is initiated by the removal of nutrients, and results in formation of a mature fruiting body composed of two cell types, the stalk and spore cells. A considerable body of evidence supports the hypothesis that cytoplasmic pH may be an essential regulator of the choice to differentiate in either the prestalk or prespore pathway. We have devised methods for measurement and analysis of intracellular pH in developing Dictyostelium amebae in order to assess directly the potential role of cytoplasmic pH in regulating the pathway of differentiation. The intracellular pH of single D. discoideum amebae during development and in intact slugs has been measured using the pH-sensitive indicator pyranine in a low light level microspectrofluorometer. We have used the ATP-mediated loading method to introduce pyranine into these cells. Cells loaded by the ATP method appear healthy, have no detectable defects in development, and exhibit a similar population distribution of intracellular pH to those loaded by sonication. The intracellular pH of populations comprised of single amebae was found to undergo a transient acidification during development resulting in a bimodal distribution of intracellular pH. The subpopulations were characterized by fitting two gaussian distributions to the data. The number of cells in the acidic intracellular pH subpopulation reached a maximum 4 h after initiation of development, and had returned to a low level by 7 h of development. In addition, a random sample of single amebae within a slug had a median intracellular pH of 7.2, nearly identical to the median pH (7.19) of similarly treated vegetative cells. No gradient of intracellular pH along the anterior to posterior axis of the slug was detected. Our data demonstrate the existence of two distinct subpopulations of cells before the aggregation stage of development in Dictyostelium, and offers support for the hypothesis that changes in intracellular pH contribute to development in D. discoideum.     

Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum

Membrane interaction in aggregating cells of Dictyostelium discoideum can be blocked by univalent antibodies directed against specific membrane sites. Using a quantitative technique for measuring cell association, two classes of target sites for blocking antibodies were distinguished and their developmental dynamics studied. One class of these sites is specific for aggregation-competent cells, their quantity rising from virtually 0-level during growth, with a steep increase shortly before cell aggregation. The serological activity of these structures is species specific; they are not detectable in a nonaggregating mutant, but present in a revertant undergoing normal morphogenesis. Patterns of cell assembly in the presence of antibodies show that selective blockage of these membrane sites abolishes the preference for end-to-end association which is typical for aggregating cells. A second class of target sites is present in comparable quantities in particle fractions from both growth-phase and aggregation-competent cells. Blockage of these sites leads to aggregation patterns in which the side-by-side contacts of aggregating cells are abolished. The target sites of aggregation-inhibiting antibodies are suggested to be identical or associated with the molecular units of the cell membrane that mediate cell-to-cell contacts during aggregation. The results indicate that in one cell, two independent classes of contact sites can be simultaneously active.     

Cytochemical localization of alkaline phosphatase and Na+-pump sites in adult rat colon.

The cellular and subcellular locialization of alkaline and K+-dependent phosphatase activities in the colonic mucosa of adult rats and rabbits was studied with the electron microscope. The 1-cysteine-sensitive alkaline phosphatase activity was observed in the brush border membrane of the chief cells. The contraluminal plasma membrane of chief cells was devoid of this enzyme activity. In contrast, the cardiac glycoside-sensitive K+-dependent phosphatase was predominantly localized in this region of the cheif cells.     

The plasma membrane ATPase of Dictyostelium discoideum

During centrifugation of Dictyostelium membranes on sucrose and metrizamide gradients, an ATPase activity resistant to azide and molybdate but sensitive to diethylstilbestrol was found to copurify with the plasma membrane markers alkaline phosphatase and ¹²⁵I in cells surface-labelled by lactoperoxidase catalyzed iodination. This ATPase was enriched 50-fold in purified plasma membranes and could be separated from the mitochondrial ATPase on metrizamide gradients. The plasma membrane ATPase is very specific for ATP as substrate and Mg²⁺ as essential cofactor. Its pH optimum is 6.5 and it is inhibited by dicyclohexylcarbodiimide, diethylstilbestrol, vanadate, mercurials and Cu²⁺, but not by ouabain, molybdate, azide or oligomycin. It was not specifically affected by either monovalent cations or anions. These results suggest that the plasma membranes of Dictyostelium contain an ATPase similar to the proton-pumping ATPases recently identified in fungal and plant plasma membranes (Serrano, R. (1984) Curr. Top. Cell. Regul. 23, 87–126).     

Cytochemical studies on the localization of alkaline phosphatase and HCO-3-activated adenosine triphosphatase in the brush border membrane of rat duodenal enterocytes

Cytochemical techniques were used to demonstrate, with appropriate controls, alkaline phosphatase and HCO-3-activated adenosine triphosphatase (ATPase) in rat duodenal brush border microvillus membranes. Intense activity of ecto-alkaline phosphatase activity was demonstrated with 2-glycerophosphate as substrate. Although biochemical assays suggested that L-phenylalanine inhibited both alkaline phosphatase and HCO-3-activated ATPase, cytochemical studies indicated that there was marked inhibition of alkaline phosphatase revealing a specific HCO-3-activated ATPase on the inner aspect of the microvillus membrane. While it is tempting to suggest that this HCO-3-activated ATPase is implicated in active bicarbonate secretion by the duodenum, decisive identification is not yet possible.     

Plasma membrane proton pump inhibition and stalk cell differentiation in Dictyostelium discoideum

The choice of the stalk cell differentiation pathway in Dictyostelium is promoted by an endogenous substance, DIF-1, which is 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone. It is also favoured by weak acids and two inhibitors of the plasma membrane proton pumps of fungi and plants, diethylstilbestrol (DES) and zearalenone, and antagonised by ammonia and other weak bases, which promote spore differentiation. These observations led to the proposal that the choice of differentiation pathway is regulated by intracellular pH. They also prompted the conjecture that DIF-1 itself is a plasma membrane proton pump inhibitor. We report here experiments showing that DIF-1 is not a plasma membrane proton pump inhibitor. We demonstrate that diethylstilbestrol and zearalenone do inhibit the plasma membrane proton pump of Dictyostelium and we show that there is an excellent qualitative and quantitative correlation between the inhibitory activity of these agents, and of a number of other substances, and their ability to divert differentiation from the spore to the stalk pathway. We conclude that inhibition of the plasma membrane proton pump does shift the choice of differentiation pathway in Dictyostelium towards the stalk pathway, but that DIF does not act by this route, and we propose a model for the actions of DIF and plasma membrane proton pump inhibitors in which the differentiation pathway is controlled by the pH of intracellular vesicles rather than by intracellular pH itself. The model invokes a DIF- and proton-activated vesicular chloride channel whose opening permits acidification of the vesicles and lowers cytosolic Ca++ concentration.