scholarly journals CYTOPHOTOMETRIC DETERMINATION OF ALKALINE PHOSPHATASE ACTIVITY OF INDIVIDUAL NEUTROPHILIC LEUKOCYTES WITH A BIOCHEMICALLY CALIBRATED MODEL SYSTEM

1968 ◽  
Vol 16 (11) ◽  
pp. 693-706 ◽  
Author(s):  
M. VAN DER PLOEG ◽  
P. VAN DUIJN
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Azo Dye ◽  
Enzyme Concentration ◽  
Model System ◽  
Coupling Method ◽  
Dye Coupling ◽  
Neutrophilic Leukocytes

A model system consisting of polyacrylamide films into which cell sonicate is incorporated was applied to investigate quantitative aspects of the cytochemical azo dye coupling method for alkaline phosphatase activity in neutrophilic leukocytes. The films were assayed in media containing naphthol AS-MX phosphate and 4-aminodiphenylamine diazonium sulfate. Optimal reaction conditions under which proportionality existed between the amount of reaction product and incubation time or enzyme concentration were determined. Since the enzyme activity in the films could also be measured chemically, using disodium phenyl phosphate as a substrate, a direct relation between the cytochemical and biochemical activity could be established. The azo dye coupling method was applied for the quantification of the enzyme activity. Air-dried microscopic preparations of exudate neutrophils were stained and the amount of dye formed in the cytoplasm of individual leukocytes was measured with a cytospectrophotometer based on the two-wavelength principle. By reference to the relation between biochemical and cytochemical activities in the model films, the cytochemically determined enzyme activity could be expressed in biochemical units. Independently, the average alkaline phosphatase activity per cell was determined by direct biochemical assay of the leukocyte suspension. The results showed that about 60% of the biochemically assayed activity in sonicate was demonstrated in the cell preparations by the cytochemical staining method. The discrepancy between the results of the two methods is discussed. The applicability of the model system for elucidation of the relationship between the results of cytochemical and biochemical enzyme determination in cells is stressed.

The onset of phosphatase activity in early mammalian embryos

Development ◽  
1977 ◽  
Vol 42 (1) ◽  
pp. 305-308
Author(s):  
V. Ishiyama ◽  
L. Izquierdo
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Azo Dye ◽  
Mouse Embryos ◽  
Dye Coupling ◽  
Cell Stage ◽  
Acid And Alkaline Phosphatase ◽  
Mammalian Embryos

The onset of acid and alkaline phosphatase activity is determined by means of Burstone's azo dye coupling methods in oocytes and embryos of the mouse, rat and hamster, and in mouse embryos cultured in vitro. Acid phosphatase activity is detected in all cases while alkaline phosphatase activity begins during the 4-cell stage and is always present thereafter. The method is sensitive regarding detection of activity but does not permit the quantification nor a precise localization of the enzymes.

scholarly journals Increase in alkaline phosphatase activity in calvaria cells cultured with diphosphonates

1979 ◽  
Vol 183 (1) ◽  
pp. 73-81 ◽  
Author(s):  
R Felix ◽  
H Fleisch
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Enzyme Activity ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Heat Inactivation ◽  
High Concentration ◽  
Control Value ◽  
Km Value ◽  
Inhibitor Of Protein Synthesis

1. Dichloromethanediphosphonate and to a lesser degree 1-hydroxyethane-1,1-diphosphonate, two compounds characterized by a P-C-P bond, increased the alkaline phosphatase activity of cultured rat calvaria cells up to 30 times in a dose-dependent fashion. 2. Both diphosphonates also slightly inhibited the protein synthesis in these cells. 3. Thymidine, an inhibitor of cell division, did not inhibit the induction of the enzyme, indicating that the increase in enzyme activity was not due to the formation of a specific population of cells with high alkaline phosphatase activity. 4. The effect on alkaline phosphatase was suppressed by the addition of cycloheximide, an inhibitor of protein synthesis. 5. After subculturing the stimulated cells in medium without diphosphonates, the enzyme activity fell almost to the control value. 6. Bovine parathyrin diminished the enzyme activity of the control cells and the cells treated with dichloromethanediphosphonate; however, at high concentration the effect of parathyrin was greater on the diphosphonate-treated cells than on the control cells. 7. The electrophoretic behaviour, heat inactivation, inhibition by bromotetramisole or by phenylalanine, and the Km value of the induced enzyme were identical with that of the control enzyme.

scholarly journals Induction of germ-cell alkaline phosphatase by butyrate and cyclic AMP in BeWo choriocarcinoma cells

1993 ◽  
Vol 296 (1) ◽  
pp. 59-65 ◽  
Author(s):  
J F Telfer ◽  
C D Green
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Cyclic Amp ◽  
Germ Cell ◽  
Cholera Toxin ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Sodium Butyrate ◽  
Dibutyryl Camp ◽  
Choriocarcinoma Cells

BeWo choriocarcinoma cells synthesize two alkaline phosphatase isoenzymes: germ-cell alkaline phosphatase and tissue-unspecific alkaline phosphatase. We have made use of the differential heat-stabilities of these two isoenzymes to study the induction of germ-cell alkaline phosphatase by sodium butyrate and cyclic AMP (cAMP). Sodium butyrate causes a large induction of germ-cell alkaline phosphatase activity (approx. 35-fold after 96 h) after an initial lag period of 12-24 h. We showed that butyrate increases germ-cell alkaline phosphatase mRNA. Dibutyryl cAMP also induces germ cell alkaline phosphatase (approx. 2.5-fold after 96 h). When optimal concentrations of butyrate and dibutyryl cAMP were added simultaneously to cells, they caused a synergistic induction of activity. This suggested that these compounds use separate mechanisms to induce germ-cell alkaline phosphatase activity and that it is the cAMP moiety of dibutyryl cAMP that induces enzyme activity. This was confirmed by the use of two additional cAMP analogues, 8-(4-chlorophenylthio) cAMP and 8-bromo cAMP, and of two compounds, 3-methyl-1-isobutylxanthine and cholera toxin, which raise the endogenous concentration of cAMP. All four compounds caused a 2-fold increase in enzyme activity. Treatment of cells with 8-(4-chlorophenylthio) cAMP, 8-bromo cAMP and cholera toxin increased germ-cell alkaline phosphatase mRNA between 2- and 7-fold. These data suggest that this alkaline phosphatase isoenzyme is regulated at the level of its mRNA by cAMP, in a manner distinct from that of butyrate.

Studies on Leukocyte Phosphatases

1971 ◽  
Vol 17 (3) ◽  
pp. 210-213 ◽  
Author(s):  
Lawrence R DeChatelet ◽  
James V Volk ◽  
Charles E McCall ◽  
M Robert Cooper
Keyword(s):  
Amino Acids ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Leukocyte Alkaline Phosphatase ◽  
Complete Reversal

Abstract The activity of leukocyte alkaline phosphatase is inhibited by a number of amino acids, most notably cysteine and histidine. The mechanism of this inhibition involves chelation of Zn2+ by the amino acids, as indicated by the complete reversal of the inhibition by added Zn2+. The concentrations of amino acids and Zn2+ required to affect the enzyme activity are such that their interaction might represent an in vivo mechanism for the control of leukocyte alkaline phosphatase activity.

THE EFFECT OF THYROPROTEIN AND PROPYLTHIOURACIL FEED SUPPLEMENTATION ON CALCIUM LEVELS AND ALKALINE PHOSPHATASE ACTIVITY IN THE PLASMA AND UTERUS OF LAYING HENS

1970 ◽  
Vol 64 (3) ◽  
pp. 398-409 ◽  
Author(s):  
N. Snapir ◽  
M. Perek
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Blood Plasma ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Calcium Level ◽  
Laying Hens ◽  
Marked Decrease ◽  
Feed Supplementation ◽  
Plasma Alkaline Phosphatase

ABSTRACT In both young and old laying hens the protein-bound calcium fraction in blood plasma decreased following feed supplementation of 0.05% thyroprotein, whereas the plasma alkaline phosphatase activity significantly increased. The enzyme response was found to be delayed in old as compared to young hens, significantly higher absolute levels being found in the latter. Propylthiouracil treatment caused a marked decrease in protein-bound calcium levels and alkaline phosphatase activity in both young and old hens. The uterine calcium levels and alkaline phosphatase activity in the thyroprotein treated birds did not show any significant differences as compared to the controls, except in the group of old hens receiving the highest dose, in which the calcium level was significantly decreased and the enzyme activity significantly increased. Both propylthiouracil treated groups showed a significant decrease in uterine calcium and alkaline phosphatase activity.

scholarly journals Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2+-binding glycoprotein.

1986 ◽  
Vol 103 (4) ◽  
pp. 1615-1623 ◽  
Author(s):  
B de Bernard ◽  
P Bianco ◽  
E Bonucci ◽  
M Costantini ◽  
G C Lunazzi ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Protein A ◽  
Matrix Vesicles ◽  
Immune Reactivity ◽  
The Matrix ◽  
Immunohistochemical Evidence

A glycoprotein that exhibits alkaline phosphatase activity and binds Ca2+ with high affinity has been extracted and purified from cartilage matrix vesicles by fast protein liquid chromatography. Antibodies against this glycoprotein were used to analyze its distribution in chondrocytes and in the matrix of calcifying cartilage. Under the light microscope, using immunoperoxidase or immunofluorescence techniques, the glycoprotein is localized in chondrocytes of the resting zone. At this level, the extracellular matrix does not show any reaction. In the cartilage plate, between the proliferating and the hypertrophic region, a weak immune reactivity is seen in the cytoplasm, whereas in the intercolumnar matrix the collagen fibers appear clearly stained. Stained granular structures, distributed with a pattern similar to that of matrix vesicles, are also visible. Calcified matrix is the most stained area. These results were confirmed under the electron microscope using both immunoperoxidase and protein A-gold techniques. In parallel studies, enzyme activity was also analyzed by histochemical methods. Whereas resting cartilage, the intercellular matrix of the resting zone, and calcified matrix do not exhibit any enzyme activity, the zones of maturing and hypertrophic chondrocytes are highly reactive. Some weak reactivity is also shown by chondrocytes of the resting zone. The observation that this glycoprotein (which binds Ca2+ and has alkaline phosphatase activity) is synthesized in chondrocytes and is exported to the extracellular matrix at the time when calcification begins, suggests that it plays a specific role in the process of calcification.

scholarly journals Morphofunctional properties of stromal stem cells incubating in vitro under dynamic conditions

2009 ◽  
Vol 8 (4) ◽  
pp. 28-32 ◽  
Author(s):  
I. V. Zapuskalov ◽  
O. I. Krivosheina ◽  
I. A. Khlusov ◽  
Ya. A. Martusevich ◽  
N. M. Shevtsova ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Steady State ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Intracellular Enzyme ◽  
Dynamic Conditions ◽  
Stromal Stem Cells

The culture of stromal stem cells was incubated under constant and dynamic conditions. The incubation period was lasting 48 h. Intracellular enzyme activity of stromal stem cells were carried out with cytochemical methods. Alkaline phosphatase activity was increased under dynamic conditions, differentiation of stromal stem cells being accelerated comparatively steady-state conditions.

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