Identification by direct photoaffinity labeling of an altered phosphodiesterase in a mutant S49 lymphoma cell.
Extracts of a mutant S49 lymphoma cell line, termed K30a, hydrolyze cAMP and cGMP at rates much faster than do wild type S49 extracts. This elevated phosphodiesterase activity, called K-PDE, elutes as a single peak of activity on DEAE-cellulose columns (Brothers, V. M., Walker, N., and Bourne, H. R. (1982) J. Biol. Chem. 257, 9349-9355). Direct photoaffinity labeling of K30a extracts with [32P]cGMP results in radiolabeling of a unique polypeptide, not observed in wild type extracts, which migrates in sodium dodecyl sulfate polyacrylamide gels with an Mr = 106,000. The 106-kDa band was identified as the catalytic K-PDE polypeptide based on the following observations: competitive inhibitors and substrates of K-PDE inhibit photolabeling of the 106-kDa band, indicating that [32P] cGMP photolabels the enzyme at its catalytic site; on DEAE-cellulose chromatography the polypeptide that is susceptible to photolabeling co-elutes with K-PDE activity; the 106-kDa band is detectable in extracts of WT X K30a hybrids (where WT denotes wild type) in amounts proportional to the K-PDE activity in the hybrids, but is undetectable in wild type. The hybrid phenotype strongly suggests that the K30a phenotype is not due to mutations that affect either a diffusible regulator of transcription or an enzyme that modifies K-PDE. Although wild type cells contain a minor cGMP phosphodiesterase activity distinct from the major cAMP phosphodiesterase, the wild type cGMP phosphodiesterase is not susceptible to radiolabeling with [32P]cGMP; this rules out the possibility that the K30a phenotype is caused by overexpression of a wild type phosphodiesterase. We conclude that the K30a mutation produced expression of a new species of phosphodiesterase molecule that is not detectably expressed in the parental S49 wild type cell line.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D008223:Lymphoma / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D009374:Neoplasms, Experimental / Q000201:enzymology; D010777:Photochemistry
D000345:Affinity Labels; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP