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Chapter 44,587

The cyclic AMP-mediated induction of alkaline phosphatase in mouse L-cells

Firestone, G.L.; Heath, E.C.

Journal of Biological Chemistry 256(3): 1396-1403

1981


ISSN/ISBN: 0021-9258
PMID: 6256395
Accession: 044586717

A cell surface-localized glycoprotein that exhibits alkaline phosphatase activity was induced by treatment of mouse L-cell cultures with dibutyryl cyclic AMP. Treatment of cells with 1.5 mM dibutyryl cyclic AMP for a period of 7 days resulted in a approximately 2000-fold increase in the specific activity of the enzyme. Enzyme induction was dependent upon de novo RNA and protein biosynthesis since this induction was completely suppressed when actinomycin D (0.5 microgram/ml) or cycloheximide (5 microgram/ml) was administered with dibutyryl cyclic AMP. Further, the overall rates of incorporation of either [3H]glucosamine or [3H]leucine into macromolecules were identical in the presence or absence of dibutyryl cyclic AMP. Alkaline phosphatase was immunotitrated in 0.5% Triton X-100-solubilized cell extracts with antisera prepared against purified native enzyme and the results indicated that dibutyryl cyclic AMP stimulated that de novo synthesis of the enzyme. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis of specifically immunoprecipitated protein from cells incubated with either [35S]methionine or [6-3H]glucosamine demonstrated that dibutyryl cyclic AMP induced a 76,000-dalton glycoprotein that was characterized as alkaline phosphatase by its identity with native alkaline phosphatase that had been labeled with 32P in its active site. Electrophoretic analysis of specifically immunoprecipitated translation products from an in vitro protein-synthesizing system supplemented with L-cell RNA isolated from uninduced and cAMP-induced cells indicated that dibutyryl cyclic AMP induced the production of alkaline phosphatase-specific mRNA. These results suggest that dibutyryl cyclic AMP directly or indirectly influences the regulation of transcription of the alkaline phosphatase gene in L-cells.

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