Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase

Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.

Download Full-text

Problems inherent in obtaining the alkaline phosphatase reaction.

Journal of Histochemistry & Cytochemistry ◽

10.1177/28.1.6153197 ◽

1980 ◽

Vol 28 (1) ◽

pp. 66-68 ◽

Cited By ~ 11

Author(s):

S B Doty

Keyword(s):

Electron Microscopy ◽

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Enzyme Inhibitors ◽

Light And Electron Microscopy ◽

Histochemical Localization ◽

Inorganic Pyrophosphatase ◽

Pyrophosphatase Activity ◽

Alkaline Phosphatase Reaction

Problems encountered in the histochemical localization of alkaline phosphatase activity are discussed and solutions presented. The purpose is to achieve a reaction that can be studied by light and electron microscopy and to distinguish alkaline glycerophosphatase from inorganic pyrophosphatase activity. Details are presented concerning fixatives, fixation times, incubation media, enzyme inhibitors, activators, and associated techniques that can be used to obtain optimal histochemical results.

Download Full-text

Association of inorganic pyrophosphatase activity with normal calcification of rat costal cartilage in vivo

Biochemical Journal ◽

10.1042/bj1120505 ◽

1969 ◽

Vol 112 (4) ◽

pp. 505-510 ◽

Cited By ~ 36

Author(s):

Nancy W. Alcock ◽

Maurice E. Shils

Keyword(s):

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Inorganic Phosphate ◽

Costal Cartilage ◽

Hydrolytic Activity ◽

Inorganic Pyrophosphatase ◽

Inorganic Pyrophosphate ◽

Pyrophosphatase Activity

1. Dialysed extracts of rat costal cartilage were shown to possess an enzyme that hydrolyses inorganic pyrophosphate. 2. Inorganic pyrophosphatase activity assayed in the presence of 2mm substrate was maximal at pH6·8. 3. Mg2+ was essential for activity, which was greatest with 10mm or higher concentrations of Mg2+. 4. Extracts prepared from cartilage taken from suckling rats (<20g.) showed little or no hydrolytic activity, but as rat weight increased inorganic pyrophosphatase activity was detected, increased to a maximum in tissue from animals weighing about 40g., and then rapidly declined. 5. The increase in inorganic pyrophosphatase activity was associated with an increase in the uptake of 45Ca by the cartilage in vivo. 6. Accumulation of calcium, inorganic phosphate and magnesium occurred when inorganic pyrophosphatase activity was at its maximum. 7. Alkaline phosphatase activity, measured in the same extracts used to determine pyrophosphatase activity, was highest in the tissues of the animals weighing <20g., and decreased as inorganic pyrophosphatase activity increased to its maximum. 8. There was no direct relationship between alkaline phosphatase activity and the onset of calcification.

Download Full-text

Alkaline Phosphatase Activity of Human Cell Cultures.

Experimental Biology and Medicine ◽

10.3181/00379727-107-26678 ◽

1961 ◽

Vol 107 (3) ◽

pp. 532-534 ◽

Cited By ~ 11

Author(s):

H. M. Nitowsky ◽

F. Herz

Keyword(s):

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Cell Cultures ◽

Alkaline Phosphatase Activity ◽

Human Cell ◽

Human Cell Cultures

Download Full-text

Studies on the Mechanism of Substrate Induction and L-Cyst(e)ine Repression of Alkaline Phosphatase in Mammalian Cell Cultures

Journal of Cell Science ◽

10.1242/jcs.2.4.545 ◽

1967 ◽

Vol 2 (4) ◽

pp. 545-555

Author(s):

M. J. GRIFFIN ◽

R. P. COX

Keyword(s):

Tissue Culture ◽

Alkaline Phosphatase ◽

Cell Cultures ◽

Phosphate Esters ◽

Kidney Cell Line ◽

Monkey Kidney Cell ◽

African Green Monkey Kidney ◽

Mammalian Cell Cultures ◽

Green Monkey ◽

Substrate Induction

The mechanisms of substrate induction and L-cyst(e)ine repression of alkaline phosphatase were studied in tissue culture using an established African green monkey kidney cell line (BS-C-I). L-Cyst(e)ine repression and substrate induction are mutually antagonistic. Evidence is presented which suggests that the increase in alkaline phosphatase levels induced by mono-phosphate esters may in part be due to protection of the enzyme from cellular degradation, while L-cyst(e)ine is believed to act either by repressing the synthesis of the enzyme or by selectively increasing its catabolism.

Download Full-text