Expression of alkaline phosphatase in murine B lymphocytes. Correlation with B cell differentiation into Ig secretion

Alkaline phosphatases (ALPase) (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) are implicated in many biologic phenomena including ossification and differentiation of human neutrophils and choriocarcinoma cells. Another trait, demonstrated by microinjection into Xenopus oocytes, is their ability to block the first mitotic division. Previous work in our laboratory has established that ALPase is also present on murine B lymphocytes activated by either polyclonal mitogens or Th cells. We have now characterized the ALPase present on murine B cells as belonging to the liver-bone-kidney isoenzyme and found it to be implicated in B cell differentiation into antibody secretion. Thus, B cell proliferative responses, elicited either by high concentrations of rabbit anti-IgM antibodies or by LPS in the presence of PMA, are characterized by the lack of both antibody secretion and expression of ALPase activity. In contrast, B cells stimulated to differentiate into Ig-secreting cells by B cell differentiation factors, nearly in the absence of a proliferative response, express high levels of ALPase activity, as did those that were LPS-stimulated. These data showing the association of the ALPase expression with the process of B cell differentiation into antibody-secreting cells are discussed in the context of the possible role that phosphorylation-dephosphorylation mechanism may play in controlling the growth/differentiation rate in the B cell lineage.     

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.     

Characterization of KB cell alkaline phosphatase. Evidence of similarity to placental alkaline phosphatase.

The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.     

Expression of intestinal alkaline phosphatase in human organs.

Human intestinal alkaline phosphatase was immunohistochemically identified and localized in the pancreas, liver and kidney by use of a monoclonal antibody specific for intestinal alkaline phosphatase isozyme and by amplified biotin-streptavidin staining. In all the examined organs, the intestinal isozyme was found to be localized in the epithelial cells of ducts: bile ducts in the liver, distal convoluted tubules and collecting tubules in the kidney and ducts in the secretory epithelium in the pancreas. In the liver the antibody also stained some sinus-lining cells. In all the examined organs the endothelial cells of the capillaries and some vessels were stained. By use of immunoelectron microscopy, intestinal alkaline phosphatase was, as expected, found to be localized to the microvillar region of the small intestine. The isozyme was abundantly expressed in the apical area of the microvilli and in membrane remnants in the fuzzy coat. Capillaries and vessels in the submucosa were also stained, as well as small vesicles in the endothelial cells. The present investigation demonstrates the expression and localization of the intestinal alkaline phosphatase in several organs, though previously believed to be expressed only in the intestine.     

The activation of chick alkaline phosphatase by calmodulin.

Calmodulin, an activator protein in most calcium-dependent processes, was isolated to apparent homogeneity from the femurs of 1-day old chicks using phenyl-Sepharose and high performance liquid chromatography. The purified calmodulin was found to produce a 6-fold increase in the activity of alkaline phosphatase isolated from the same source. A Ca2+ concentration of 10(-5) M was required for the activation. Purification of alkaline phosphatase involved acetone precipitation, DEAE-Sephacel and Sephadex G-200 column chromatography. The enzyme was purified to 540-fold and had a specific activity of 10.75 U/mg protein.     

Alkaline phosphatase on activated B cells characterization of the expression of alkaline phosphatase on activated B cells. Kinetics and membrane anchor.

Recently we reported that the expression of the enzyme alkaline phosphatase (APase) is a marker for B cell activation. Enzymatic activity was found only in activated B cells and not T cells. Using flow cytometry we showed that some of the APase was found on the cell membranes (mAPase) and by functional assays, some was spontaneously released into the tissue culture medium. In the present report the expression of mAPase on activated B lymphocytes is more fully characterized. Two mAb specific for rat APase were used to measure the kinetics of the membrane expression of mAPase. Within 48 h of activation, mAPase is detected by flow cytometry and increases coordinately with both the transferrin receptor and IL-2R. Maximal membrane expression of mAPase in terms of number of positive cells and mean fluorescent intensity, is detected by day 4 to 5 of culture. Using hydroxyurea and demecolcine to block cells at G1/S and G2/M, respectively, it appeared that the initial expression of mAPase occurred as cells progressed into S phase of the cell cycle. This was confirmed using two-color flow cytometric analysis with the Hoechst DNA stain 33342 and the FITC-labeled APase-specific mAb. Finally, using phosphatidylinositol-specific phospholipase C we were able to show that 60 to 80% of the mAPase is linked to the membrane via a glycosyl-phosphatidylinositol linkage. From this we have concluded that mAPase can be added to a growing list of glycoproteins that are anchored to the membrane by the glycosyl-phosphatidylinositol linkage and are expressed on differentiating B cells. This list now includes Thy-1, BLAST-1, Jlld, and mAPase.     

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...     

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.     

Tissue alkaline phosphatase activity in selected freshwater teleosts.

1. Kinetic studies of tissue alkaline phosphatase activity were carried out on eight species of teleost fishes taken from the Mississippi River. 2. Liver alkaline phosphatase activity expressed in mumoles of p-nitrophenol released per minute per gram of protein were as follows: Micropterus salmoides, 20.31; Stizostedion vitreum, 19.22; Pomoxis nigromaculatus, 18.76; Lepisosteus platostomus, 16.26; Esox lucius, 16.14; Hiodon tergisus, 13.83; Ictalurus punctatus, 10.87; Aplodinotus grunniens, 7.94. 3. Specific activity of the kidney alkaline phosphatase was about ten times the level of liver alkaline phosphatase for the species examined.     

Neutrophil alkaline phosphatase activity in the early puerperium.

Neutrophil alkaline phosphatase studied cytochemically according to Kaplow's method in 20 healthy women aged 20 to 38 years, shows statistically significant, although transient, increase on the second day after physiological delivery and returns to initial values after several days. The above increase is unrelated to the increased enzyme activity observed in the terminal months of pregnancy in a majority of healthy women. It is associated with an increase in neutrophilic granulocytosis in the puerperial period.     

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

We demonstrated that alkaline phosphatase was localized on the cell membrane of Dictyostelium 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).     

Presence and activity of alkaline phosphatase in two human osteosarcoma cell lines

The presence and activity of alkaline phosphatase in SAOS-2 and TE-85 human osteosarcoma cells grown in culture were examined at the ultrastructural level. A monoclonal antibody raised against purified human bone osteosarcoma alkaline phosphatase was used to localize the enzyme in cultures of the osteosarcoma cells. Similar cultures were analyzed for alkaline phosphatase activity using an enzyme cytochemical method with cerium as the capture agent. Alkaline phosphatase was immunolocalized at the light microscopic level in an osteogenic sarcoma and ultrastructurally on the SAOS-2 cell membrane and the enclosing membrane of extracellular vesicular structures close to the cells. In contrast, the TE-85 cells were characterized by the absence of all but a few traces of immunolabeling at the cell surface. Enzyme cytochemical studies revealed strong alkaline phosphatase activity on the outer surface of the SAOS-2 cell membrane. Much lower enzyme activity was observed in the TE-85 cells. The results support biochemical data from previous studies and confirm that SAOS-2 cells have a significantly greater concentration of alkaline phosphatase at the plasma membrane.