Endogenous phosphorylation and dephosphorylation of rat liver plasma membrane proteins, suggesting a 18 kDa phosphoprotein as a potential substrate for alkaline phosphatase.

Purified rat liver plasma membranes were incubated for 0-60 min with [gamma-32P]ATP and analysis of 32P-labeled proteins by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the presence of two shifted kinetic phenomena. The use of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinases, allowed the identification of one as the endogenous protein phosphorylation. The other was shown to be the labeling of two phospho-intermediate forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1.], which have apparent molecular masses of 151 and 135 kDa. Bromolevamisole, a potent inhibitor of the enzyme, stabilized these phospho-intermediates, and consequent on this inhibition the labelling of a 18 kDa phosphoprotein was augmented. So, when alkaline phosphatase was studied in its native plasma membrane environment, a specificity of this enzyme over the endogenous phosphoproteins was established.     

Quantitative histochemical study of acid phosphatase activity in rat liver using a semipermeable membrane technique

Acid phosphatase activity has been demonstrated in rat liver with the semipermeable membrane technique using naphthol AS-BI phosphate as substrate and hexazotized pararosaniline (HPRA) as simultaneous coupling agent. With this method the final reaction product (FRP) appeared in rat liver as intensely colored red granules in liver parenchymal cells and in Küpffer cells. The absorbance spectrum of the FRP peaks between 510 and 550 nm. A nonspecific reaction product, as has been found in skeletal muscle, did not occur in rat liver. A substrate concentration of 5 mM and a HPRA concentration of 10 mM result in optimum localization and activity. We concluded from the results with different enzyme inhibitors that lysosomal acid phosphatase was demonstrated. The mean absorbance of the FRP increased linearly with incubation time (15-60 min). Furthermore, we found a linear increase of the FRP with increasing section thickness (4-10 micron). When the simultaneous coupling method was replaced by a post-coupling technique, the colored reaction product was diffusely located throughout the cytoplasm. In conclusion, the simultaneous coupling technique in combination with the semipermeable membrane method is a valuable tool for detecting and quantifying lysosomal acid phosphatase activity in rat liver. We demonstrated that acid phosphatase activity is 1.2 times higher periportally than pericentrally in rat liver, and that 24 hr fasting before the experiments did not change the acid phosphatase activity.     

Evidence for the non-identity of proteins having synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in rat liver.

Synthase phosphatase, phosphorylase phosphatase and histone phosphatase in rat liver were measured using as substrates purified liver synthase D, phosphorylase alpha and 32P-labelled phosphorylated f1 histone, respectively. The three phosphatase enzymes had different sedimentation characteristics. Both synthase phosphatase and phosphorylase phosphatase were found to sediment with the microsomal fraction under our experimental conditions. Only 10% of histone phosphatase was in this fraction; the majority was in the cytosol. No change in histone phosphatase was observed in the adrenalectomized fasted rat whereas synthase phosphatase and phosphorylase phosphatase activities were decreased 5-10 fold. Fractionation of liver extract with ethanol produced a dissociation of the three phosphatase activities. When a partially purified fraction was put on a DEAE-cellulose column, synthase phosphatase and phosphorylase phosphatase both exhibited broad elution profiles but their activity peaks did not coincide. Histone phosphatase eluted as a single discrete peak. When the supernatant of CaCl2-treated microsomal fraction was put on a Sepharose 4B column, the majority of synthase phosphatase was found to elute with the larger molecular weight proteins whereas the majority of phosphorylase phosphatase eluted with the smaller species. Histone phosphatase migrated as a single peak and was of intermediate size. Synthase phosphorylase phosphatase by synthase D (Ki approximately 2 units/ml). The inhibition of synthase phosphatase by phosphorylase alpha was kinetically non-competitive with substrate. Histone phosphatase activity was not inhibited by synthase D or by phosphorylase alpha. The above results suggest that different proteins are involved in the dephosphorylation of synthase D, phosphorylase alpha and histone in the cell.     

Dopaminergic inhibition of DNA synthesis in pituitary tumor cells is associated with phosphotyrosine phosphatase activity.

Dopaminergic D2 receptor agonists, such as bromocriptine, are potent anti-proliferative agents in the treatment of human pituitary adenomas. We have reproduced the anti-proliferative effect of dopamine in an established pituitary cell line stably transfected with the rat D2 dopamine receptor cDNA. We found that dopaminergic inhibition of DNA synthesis parallels the stimulation of a phosphotyrosine phosphatase activity. Both actions are blocked by pertussis toxin and by the phosphotyrosine phosphatase inhibitor, vanadate. We suggest that the anti-proliferative action of dopamine is mediated, at least in part, by the dopaminergic stimulation of a phosphotyrosine phosphatase.     

A method for determining alkaline phosphatase activity in marine phytoplankton using spectrofluorometry

A method for determining relative percent intensity alkaline phosphatase activity (APA) using enzyme labeled fluorescence coupled with spectrofluorometry is presented. Compared to traditional microscopy and flow cytometry, we increase statistical power and reduce sample-handling issues. Combined with a biological standard, our method can quantify APA of natural plankton assemblages.     

A comparative study of fluorescence-labelled enzyme activity methods for assaying phosphatase activity in phytoplankton. A possible bias in the enzymatic pathway estimations

We compared different fluorescence-labelled enzyme activity (FLEA) methods for assaying phosphatase activity in phytoplankton. Unfixed and liquid incubations are devised. We demonstrated that the presence of intracellular labelling was persistent, which could point out a source of bias in ectoenzymatic activities measurements based either on the FLEA or classical methods.     

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.     

Constant expression and activity of protein phosphatase 2A in synchronized cells.

The levels of the A, B, and C subunits of protein phosphatase 2A in extracts from synchronized embryonic bovine tracheal cells were determined by immunoblotting with subunit-specific antibodies. A constant amount of each subunit was found in resting cells as well as in growing cells from all stages of the cell cycle. The phosphatase activity of protein phosphatase 2A was also constant. A quantitative comparison showed that the A and C subunits were present in similar amounts, whereas the B subunit was present at a significantly lower level. Together, the A, B, and C subunits represented approximately 0.2% of the total cellular protein.     

Endogenous ethylene production is a potential problem in the measurement of nitrogenase activity associated with excised corn and sorghum roots

Endogenous ethylene production was evaluated as a source of ethylene during acetylene reduction assays with freshly collected roots of field-grown corn, Zea mays L. cv Funks G-4646, and sorghum, Sorghum bicolor (L.) Moench. cv CK-60A. Ethylene production was not detected when roots were incubated in air without acetylene. The presence of endogenous ethylene production was confirmed when roots were incubated anaerobically and in the presence of 40 millimolar sodium hydrosulfite. Ethylene oxidase activity was also associated with excised roots. The rate of ethylene oxidation was higher than the rates of ethylene accumulation during either acetylene reduction assays or anaerobic incubations. These results indicate that the procedure of incubating roots of grasses in air to monitor endogenous ethylene production is not a valid control in acetylene reduction studies with grasses. The presence of endogenous ethylene production during acetylene reduction assays was demonstrated by using either CO to inhibit nitrogenase activity or chloramphenicol to inhibit nitrogenase synthesis in freshly excised roots.     

X-ray microanalysis of colloidal-gold-labelled lysosomes in rat liver sinusoidal cells after incubation for acid phosphatase activity

Summary The lysosomal apparatus of the Kupffer and endothelial cells of the sinusoidal lining of the rat liver was found to take up colloidal-gold particles with a mean diameter of 5 nm, prepared according to a modified method. After incubation of the glutaraldehyde-perfusion-fixed tissue in a lead-containing medium for the demonstration of acid phosphatase activity, a reaction product was observed in the gold-loaded lysosomes. By X-ray microanalysis of such lysosomes, the presence of osmium, gold and lead was detected qualitatively in the unstained sections from the tissue, which after the incubation had been post-fixed with an OsO₄-solution to which K₄Fe(CN)₆ had been added to enhance the contrast. The quantitative computer-assisted processing of the X-ray microanalytical data from such lysosomes enabled to determine the gold-to-lead ratio and the individual gold and lead peak intensities derived from both the M and L values in the spectra. On the basis of these results and those obtained similarly in control lysosomes containing either only gold or only lead phosphate precipitate, it was found that only the L values were reliable, whereas the M values from the same lysosomal spectra were unrealistic, due to deconvolution problems in the computer programs applied. Based upon the L values it was found that among the population of lysosomes in single Kupffer cells, studied after a 60-min interval between the injection of the gold colloid and fixation, three types of lysosomal contents could be quantitated by X-ray microanalysis, viz. one type with only gold, one with only lead, one with gold and lead, in various ratios. This quantitative approach might make it possible to detect variations in lysosomal composition associated with ageing.The unit for analytical electron microscopy was established by collaboration between: the Erasmus University of Rotterdam (W.C. de Bruijn), the University of Leiden, and the Organization for Health Research TNO. The analytical electron microscope was purchased with funds from the Netherlands Organization for Pure Scientific Research (ZWO)     

X-ray microanalysis of colloidal-gold-labelled lysosomes in rat liver sinusoidal cells after incubation for acid phosphatase activity

The lysosomal apparatus of the Kupffer and endothelial cells of the sinusoidal lining of the rat liver was found to take up colloidal-gold particles with a mean diameter of 5 nm, prepared according to a modified method. After incubation of the glutaraldehyde-perfusion-fixed tissue in a lead-containing medium for the demonstration of acid phosphatase activity, a reaction product was observed in the gold-loaded lysosomes. By X-ray microanalysis of such lysosomes, the presence of osmium, gold and lead was detected qualitatively in the unstained sections from the tissue, which after the incubation had been post-fixed with an OsO4-solution to which K4Fe(CN)6 had been added to enhance the contrast. The quantitative computer-assisted processing of the X-ray microanalytical data from such lysosomes enabled to determine the gold-to-lead ratio and the individual gold and lead peak intensities derived from both the M chi and L chi values in the spectra. On the basis of these results and those obtained similarly in control lysosomes containing either only gold or only lead phosphate precipitate, it was found that only the L chi values were reliable, whereas the M chi values from the same lysosomal spectra were unrealistic, due to deconvolution problems in the computer programs applied. Based upon the L chi values it was found that among the population of lysosomes in single Kupffer cells, studied after a 60-min interval between the injection of the gold colloid and fixation, three types of lysosomal contents could be quantitated by X-ray microanalysis, viz. one type with only gold, one with only lead, one with gold and lead, in various ratios. This quantitative approach might make it possible to detect variations in lysosomal composition associated with ageing.     

A radioligand binding assay for antitubulin activity in tumor cells

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.     

Alkaline phosphatase activity as a membrane marker for activated B cells

Alkaline phosphatase activity was assayed by a microtiter assay (with p-nitrophenylphosphate used as substrate) in the plasma membrane of mouse spleen cells activated in vitro by the B cell mitogen LPS and the T cell-dependent B cell mitogen, PWM. No activity was detected in spleen cells cultured in the presence of the T cell mitogens PHA and Con A. This alkaline phosphatase activity was detected in the blast-enriched 30 to 40% fraction of discontinuous Percoll gradients in LPS-treated cultures, and the enzymatic activity assayed was susceptible to inhibition by the alkaline phosphatase inhibitors EDTA and L-phenylalanine. These data support the idea that the membrane alkaline phosphatase activity could be used as a marker for activated B cells.     

Telomerase activity as a measure for monitoring radiocurability of tumor cells.

Radiotherapy plays a key role in the treatment of many tumors. It is difficult to determine what fraction of tumor cells survives after treatment with ionizing radiation. A convenient and sensitive biochemical assay could be efficacious in determining the potential success of radiotherapy. Since telomerase activity is frequently associated with the malignant phenotype, we sought to determine whether a correlation existed between ionizing radiation-induced cell killing and telomerase activity. We evaluated telomerase activity in two telomerase-positive and one telomerase-negative human cell line exposed to ionizing radiation. Telomerase activity was determined using a PCR-based telomeric repeat amplification protocol coupled with ELISA. We found ionizing radiation treatment to decrease the telomerase activity (in plateau phase cells of RKO, HeLa; and growing cells of RKO) in a dose-dependent manner, which correlated with cell death in in vitro tests as well as during tumor regression in nude mice. In contrast, growing HeLa cells after 24 h postradiation treatment showed an increase in telomerase activity, but there was no increase in the levels of mRNA of hTERT. To assess the sensitivity of the telomerase activity assay, we performed mixing experiments of HeLa and AG1522 cell extracts. These studies showed that telomerase activity could be detected in lysate equal to a single HeLa cell when mixed with 10,000 AG1522 cells. Our results indicate that even a few surviving neoplastic cells can be detected by telomerase activity assay. Therefore, detection of telomerase activity may be a useful monitor of radiotherapeutic efficacy and an early predictor of outcome.     

Alkaline phosphatase activity in cultured urinary bladder cancer cells.

Established cell lines derived from human urinary bladder carcinomas produce heat-stable alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] which resembles the oncofetal enzyme of HeLa S3. Rat bladder cancer cell lines derived from chemically induced tumors produce heat-labile alkaline phosphatase. Corticosteroids and/or hyperosmolality do not influence the enzyme of rodent cells, but induce increased levels of activity in human cells. The increase is most pronounced when human cells multiply in hyperosmolar medium containing prednisolone. Under these conditions a rise of over 100-fold in specific activity is noted. This synergistic effect, not seen with other cultured heteroploid cells, may represent a specific characteristic of cells derived from human bladder tumors.     

Phosphatidyl glycerol phosphate phosphatase in BHK-21 cells. A comparison with rat liver mitochondria.

Unlike mitochondria from rat liver, BHK-21 mitochondria are not autonomous for polyglycerolphosphatide biosynthesis. The phosphatase which converts phosphatidyl glycerol phosphate to phosphatidyl glycerol is located in the post-microsomal supernatant. It is somewhat heat stable, non-dialyzable, sulfhydryl-containing and its activity is similar to the phosphatase that can be found in the supernatant from sonicated rat liver mitochondria.