Specific activity of skeletal alkaline phosphatase in human osteoblast-line cells regulated by phosphate, phosphate esters, and phosphate analogs and release of alkaline phosphatase activity inversely regulated by calcium

Abstract Earlier we described a kinetic assay for quantifying skeletal alkaline phosphatase (ALP) isoenzyme activity in serum. The precision of the assay depends on including ALP standards for the skeletal, hepatic, intestinal, and placental isoenzymes. We wondered whether human osteosarcoma cells could provide an efficient alternative to human bone or Pagetic serum as a source of the skeletal ALP standard. ALP activities prepared from five human osteosarcoma cell lines were compared with a bone-derived ALP standard with respect to heat stability and sensitivity to chemical effectors. Two of the cell lines (SaOS-2 and TE-85) contained ALP activities that resembled the bone-derived standard. We selected SaOS-2 cells for additional evaluation (as a potential source of isoenzyme standard), because they contained 40-50 times more ALP activity than did the TE-85 cells. To include the SaOS-2 cell-derived ALP activity in the quantitative isoenzyme assay, we diluted the enzyme in a solution containing heat-inactivated (i.e., ALP-negative) human serum. Surprisingly, this dilution caused a 60-125% increase in maximum enzyme activity. In the quantitative assay of ALP isoenzyme in serum, the SaOS-2 derived ALP was indistinguishable from the serum skeletal ALP standard, with respect to the above criteria and assay variations. Evidently ALP from SaOS-2 cells is suited as a standard for measuring skeletal ALP activity in this assay.

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Skeletal alkaline phosphatase activity in serum

Clinical Chemistry ◽

10.1093/clinchem/41.11.1551 ◽

1995 ◽

Vol 41 (11) ◽

pp. 1551-1553 ◽

Cited By ~ 4

Author(s):

J Farley ◽

D J Baylink

Keyword(s):

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Skeletal Alkaline Phosphatase

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A Comparison of Human Leukocyte Phosphatase Activity toward Sodium β-Glycerophosphate, Adenosine 5'-Phosphate and Glucose 1-Phosphate

Blood ◽

10.1182/blood.v14.4.415.415 ◽

1959 ◽

Vol 14 (4) ◽

pp. 415-422 ◽

Cited By ~ 6

Author(s):

JAMES H. FOLLETTE ◽

WILLIAM N. VALENTINE ◽

JOHN REYNOLDS

Keyword(s):

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Cell Population ◽

Alkaline Phosphatase Activity ◽

Human Leukocyte ◽

Maximal Activity ◽

Phosphate Esters ◽

Chronic Myelocytic Leukemia ◽

Myelocytic Leukemia ◽

Hydrolysis Of

Abstract The ability of human leukocyte enzymes to hydrolyze phosphorus is compared in terms of the conventional substrate sodium β-glycerophosphate and the metabolically important phosphate esters, adenosine 5'-phosphate and glucose 1-phosphate. At pH 9.9, there is marked and comparable variation in phosphatase activity toward all three substrates, this being low in chronic myelocytic leukemia and high in the presence of infection and certain "stressful" states. Moreover, substrate mixture experiments show no increased hydrolysis of phosphorus when two substrates are present in the incubation mixture. Increased phosphatase activity toward both glucose 1-phosphate and sodium β-glycerophosphate resulted when corticosteroids were administered in large doses for 72 hours. The data, while not providing absolute proof, are compatible with the hydrolysis of phosphorus at pH 9.9, being due in the case of all three substrates to the activity of the same phosphomonoesterase or group of phosphomonesterases. At pH 5.5, phosphatase activity toward both sodium β-glycerophosphate and adenosine 5'-phosphate was likewise demonstrated, but, in leukocytes, the pH of maximal activity varies from subject to subject and is dependent to a large extent on the amount of the highly variable "alkaline phosphatase" activity present in any given cell population at the time of analysis.

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Decreased Abundance of Alkaline Phosphatase and Calbindin-D9K mRNAs in the Intestine of the Spontaneously Hypertensive Rat

Clinical Chemistry ◽

10.1093/clinchem/38.12.2526 ◽

1992 ◽

Vol 38 (12) ◽

pp. 2526-2531 ◽

Cited By ~ 1

Author(s):

D T Leetun ◽

M E Bruns ◽

D E Bruns

Keyword(s):

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Specific Activity ◽

Spontaneously Hypertensive Rat ◽

Similar Extent ◽

Spontaneously Hypertensive ◽

Wky Rats ◽

Calbindin D9k ◽

The Mean

Abstract Alkaline phosphatase activity and calbindin-D9K immunoreactivity are decreased in the intestines of spontaneously hypertensive rats (SHRs). To investigate the potential role of altered gene expression in these decreases, we measured, by Northern blot analyses, the abundances of alkaline phosphatase and calbindin-D9K mRNAs in the proximal regions of the small intestines of 14-week-old SHR and control Wistar-Kyoto (WKY) rats. Alternate 4-cm segments of intestine were used for measurements of the proteins (0-4 cm, 8-12 cm, and 16-20 cm from pylorus, segments A1, B1, and C1, respectively) and mRNAs (4-8 cm, 12-16 cm, and 20-24 cm, segments A2, B2, and C2). Calbindin-D9K (immunoassay) was decreased in SHR vs WKY rats by 27%, 64%, and 67% in segments A1, B1, and C1, respectively (P < 0.01); its mRNA was decreased to a similar extent (69%, 82%, and 80%, respectively; P < 0.002 by analysis of variance). Alkaline phosphatase activity was decreased in SHRs by 58%, 54%, and 51% in segments A1, B1, and C1, respectively (P < 0.01); the abundance of its 3.0-kb mRNA was decreased to a similar extent: 57%, 80%, and 69% in segments A2, B2, and C2, respectively (P < 0.02). The mean decreases of the 2.7-kb mRNA of alkaline phosphatase were statistically significant (P < 0.02) but smaller (38%, 40%, and 35%). The mean abundance of vitamin D receptor mRNA in the same animals was decreased slightly in SHR vs WKY rats (3%, 36%, and 20% in segments A2, B2, and C2, respectively), but the difference in the values was not statistically significant. Decreases in alkaline phosphatase activity and calbindin-D9K immunoreactivity may reflect decreased mRNA abundance and not decreased enzyme-specific activity or increased protein degradation.

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