Dibutyryl cAMP-inducible alkaline phosphatase in animal cell plasma membranes: fluorescence detection of mutant clones on polyester cloth.

AUTOR(ES)

Gum, J R

RESUMO

We have developed a rapid screening assay that allows us to estimate the alkaline phosphatase content of mouse L-M cell colonies immobilized on polyester cloth. This permitted the identification and isolation of two mutant clones with increased constitutive alkaline phosphatase activity and six clones that fail to express this activity when treated with dibutyryl cyclic AMP. Both of the strains with increased constitutive activity have basal enzymatic activities that are 6- to 7-fold higher than the activity of the parental strain. The extents to which the cyclic nucleotide further induces alkaline phosphatase in these two strains are different, however, indicating that they represent two classes of mutants. Studies using amino acids and synthetic peptides as alkaline phosphatase inhibitors suggest that only one alkaline phosphatase isoenzyme predominates, in both the parental and the mutant cell lines, with or without induction by cyclic nucleotide. Comparison to mouse tissues indicates that our cell lines express an isozyme resembling that found in kidney and bone. The six clones that fail to express alkaline phosphatase activity when treated with dibutyryl cyclic AMP also have extremely low basal levels of the enzyme. All of these mutant strains continue to synthesize protein when treated with dibutyryl cyclic AMP and undergo growth cessation and morphological changes in the presence of this agent. Thus, the mutations all appear to affect factors specific to the expression of alkaline phosphatase activity rather than factors that affect general cellular responsiveness or permeability to dibutyryl cyclic AMP. The characterization of these strains may help elucidate mechanisms of eukaryotic membrane protein biogenesis, enzyme induction, and regulation of gene expression by cyclic nucleotides.

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