Alkaline Phosphatase: Properties in Crude Homogenates of Human Diploid Fibroblasts

Vanneuville, F. J.; Elsen, August F. Van; Leroy, Jules

doi:10.1042/bst0051117

Cited by 4 publications

(2 citation statements)

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“…These three main classes of human alkaline phosphatase can be sharply discriminated one from another by inhibition (1,3), thermostability (4)(5)(6)(7), electrophoretic (8), and immunologic studies (9)(10)(11)(12). In many cultured human cell lines the alkaline phosphatase has a low, though rather variable, activity and generally appears to be of the liver/bone/kidney type (13)(14)(15). Certain human cell lines, however, show relatively high alkaline phosphatase activity.…”

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confidence: 99%

Human cell lines expressing intestinal alkaline phosphatase.

Benham¹,

Harris²

1979

Proc. Natl. Acad. Sci. U.S.A.

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At least three loci determine human alkaline phosphatases [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3. (16)(17)(18)(19)(20). This has generally been interpreted as due to derepression or activation of the placental alkaline phosphatase locus. HeLa was originally derived from a cervical carcinoma in the early 1950s. It has since been propagated in many different laboratories. Consequently, the various sublines have different cultural histories. In addition, studies of isozyme (21-23) and chromosomal markers (24-25) and also, most recently, endonuclease restriction enzyme analysis of ribosomal genes* have led to the conclusion that a number of long-term human cell lines, which were originally thought to have different origins, are in all probability HeLa lines that arose by contamination of the original line at some point in its history.In a recent study of the alkaline phosphatase in a number of different HeLa and presumptive HeLa sublines (20) we demonstrated, as expected, the presence of a placental-like alkaline phosphatase in most of the lines. However, in one cell line, D98/AH-2, although there was considerable alkaline phosphatase activity, its properties were quite different from placental alkaline phosphatase and no evidence for even small amounts of placental alkaline phosphatase was obtained. We have now investigated the characteristics of this alkaline phosphatase in some detail and conclude that it corresponds to intestinal alkaline phosphatase. Furthermore, it appears to resemble the fetal form of intestinal alkaline phosphatase more closely than the adult form. MATERIAL AND METHODCell lines D98/AH-2, D98/S, D98/AH-R, and Detroit-98 were obtained from the American Type Culture Collection. On arrival and thereafter at routine intervals, the cells were tested and found free of mycoplasmas. The cells were cultured as monolayers in plastic flasks with RPMI 1640 medium supplemented with 10% fetal calf serum. Cells at harvest were washed with EDTA and detached by a brief exposure to trypsin. The cell pellets were then thoroughly washed and stored at -209C.Alkaline phosphatase activity was assayed in sonicated cell homogenates as described (1), with 5.0 mM p-nitrophenylphosphate (pH 9.8). Total protein was measured by the method of Lowry et al. (26). Inhibition, thermostability, and electrophoretic studies were carried out on extracts prepared from cell homogenates by butanol extraction as described (20). Control tissue samples (liver, adult intestine, fetal intestine, and placenta) were obtained as reported (1,2), and alkaline phosphatase solutions for analysis were prepared by butanol extraction. The intestinal samples were heated at 560C for 90 min as described (1, 2), to destroy any liver-type alkaline phosphatase in the crude tissue extract. Inhibition studies were carried out as described (1), with p-nitrophenylphosphate (5.0 mM) as substrate and L-phenylalanine (2.5 mM), L-homoarginine (10 mM), L-leucine (5 mM), L-leucylglycylglycine (5 mM), and Lphenylalanylglycylgl...

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confidence: 99%

Human cell lines expressing intestinal alkaline phosphatase.

Benham¹,

Harris²

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Induction of AP activity may occur by catalytic activation of preexisting AP polypeptides [25] or by de novo synthesis of more AP. A constitutive AP and a different inducible AP have also been suggested [71]. It is likely that both mechanisms exist.…”

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confidence: 96%

Alteration of human breast tumor cell membrane functions by chromosome‐mediated gene transfer

Muneer

Gray

1979

J Supramol Struct

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BOT-2 cells (human breast tumor origin) have an impaired ability to utilize exogenous thymidine. Previous studies revealed this deficiency to be the permeation event rather than phosphorylation, since the cells have active thymidine kinase. Chromosome-mediated gene transfer was used to transfer genetic information in the form of metaphase chromosomes, from HeLa-65 cells to the BOT-2 cells, correcting the permease deficiency. Poly-L-ornithine or lipochromes were used for facilitation of chromosome uptake. After selection on HAT medium, transferant clones were isolated at a frequency of 4 x 10(-5) and 1 x 10(-5), respectively. Transferants MGP-1 and MGL-1 are stable after 18 months and have been characterized on the bases of purine and pyrimidine nucleoside uptake, relative thymidine kinase activities, alkaline phosphatase activities, and hydrocortisone-induced alkaline phosphatase activity. MGP-1 demonstrates positive thymidine uptake and incorporates radiolabeled thymidine into DNA. MGL-1 remains thymidine transport-deficient and surveys on HAT by increasing endogenous dihydrofolate reductase activity. Alkaline phosphatase activity in MGL-1 is similar to HeLa-65, 2% of that in BOT-2, and in addition, is inducible 25-30-fold by 3 micro M hydrocortisone. We have separated, genetically, a thymidine permease function from phosphorylation in cells of human origin and have transferred genetic information for the regulation of alkaline phosphatase.

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