scholarly journals Cytochemical model system for microsomal rat liver glucose-6-phosphate.

1976 ◽  
Vol 24 (5) ◽  
pp. 643-651 ◽  
Author(s):  
A S De Jong ◽  
P Van Duijn ◽  
W T Daems
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Rat Liver ◽  
Ammonium Persulfate ◽  
Model System ◽  
Significant Loss ◽  
Microscopical Investigation ◽  
Rapid Inactivation ◽  
Electron Microscopical ◽  
Liver Fraction

A method is described for the incorporation of a microsomal rat liver fraction into polyacrylamide films without significant loss of its glucose-6-phosphatase activity. The enzymatic activity was completely lost when the films were prepared with ammonium persulfate as initiator of the polymerization as previously described for alkaline phosphatase, but modification of this method showed that about 90% of the glucose-6-phosphatase activity could be retained. The enzyme in the films prepared with the new method was completely inhibited by alloxan, HgCl2, and preincubation in 0.05 M acetate buffer (pH 5.0) at 37 degrees C, as determined biochemically. Similar results were obtained for the enzyme in films determined histochemically according to the lead method of Wachstein and Meisel. In this respect the behavior of the incorporated enzyme is similar to that in suspension. Films fixed with 1.5% glutaraldehyde showed rapid inactivation of glucose-6-phosphatase. There was good correlation between the biochemical and histochemical activity determined after fixation. A method to embed polyacrylamide films in Epon for electron-microscopical investigation is also described. Dimethyl sulfoxide was used as the dehydrating agent instead of ethanol/acetone.

Macromolecular alkaline phosphatase and an immunoglobulin G that inhibited alkaline phosphatase in a patient's serum.

1983 ◽  
Vol 29 (2) ◽  
pp. 375-378 ◽  
Author(s):  
H Nakagawa ◽  
K Umeki ◽  
K Yamanaka ◽  
N Kida ◽  
S Ohtaki
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Immunoglobulin G ◽  
Significant Loss ◽  
Placental Alkaline Phosphatase ◽  
Type Ii ◽  
Intestinal Alkaline Phosphatase ◽  
Adult Type ◽  
Heat Stable

Abstract Macromolecular alkaline phosphatase (EC 3.1.3.1) was found in the serum of a patient suffering from myasthenia gravis (adult type II) complicated with thymoma, and was shown by immunoelectrophoresis to be bound to immunoglobulins A and G (IgG). Placental alkaline phosphatase, complexed with either the patient's serum or IgG purified from the patient's serum, remained at the origin on electrophoresis, with significant loss of activity. Intestinal alkaline phosphatase, complexed with either the patient's serum or the patient's IgG, migrated to a position similar to that of the macromolecular alkaline phosphatase in the patient's serum on electrophoresis. About 50% of the placental alkaline phosphatase activity was inhibited with 0.1-0.2 g of the patient's IgG per liter, but 6.93 g of the IgG per liter was required for about 20% inhibition of the intestinal alkaline phosphatase activity. The complex of intestinal alkaline phosphatase with the patient's IgG was fairly heat stable. From these results, we concluded that the macromolecular alkaline phosphatase in the patient's serum consisted of intestinal alkaline phosphatase and IgG that was specific for placental alkaline phosphatase.

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.

Alkaline Phosphatase Activity in Human and Rat Liver Tumors

Oncology ◽  
1991 ◽  
Vol 48 (2) ◽  
pp. 144-148 ◽  
Author(s):  
Ilona Kovalszky ◽  
Judit Kralovánszky ◽  
A. Jeney ◽  
K. Lapis ◽  
S. Karácsonyi ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Rat Liver ◽  
Alkaline Phosphatase Activity ◽  
Liver Tumors

Two new methods for separating and quantifying bone and liver alkaline phosphatase isoenzymes in plasma.

1984 ◽  
Vol 30 (7) ◽  
pp. 1182-1186 ◽  
Author(s):  
S B Rosalki ◽  
A Y Foo
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Wheat Germ ◽  
Placental Alkaline Phosphatase ◽  
Diagnostic Laboratory ◽  
New Methods ◽  
Cellulose Acetate Membranes ◽  
Wheat Germ Lectin ◽  
Liver Fraction

Abstract We describe two new methods for the separation and quantification of the bone and liver isoenzymes of alkaline phosphatase (EC 3.1.3.1) in plasma. In the first, we use wheat-germ lectin to precipitate the bone isoenzyme. About 80% of this, but minimal liver isoenzyme, is precipitated. The activity of the bone isoenzyme is calculated from measuring the alkaline phosphatase activity in the precipitate, that of liver alkaline phosphatase by subtracting the activity of the bone isoenzyme from total alkaline phosphatase activity. The liver fraction will also contain biliary, intestinal, and placental alkaline phosphatase if these are present in the original plasma, but correction for such activity is readily made. In the second method, samples are separated on cellulose acetate membranes that, before electrophoresis, have been soaked in buffer containing wheat-germ lectin. The bone isoenzyme is retarded and clearly separated from the liver fraction, allowing these isoenzymes to be quantified by densitometry. Both methods are rapid, reproducible, and suitable for use in the diagnostic laboratory.

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