Prevalence of nucleoside diphosphate kinase autophosphorylation in human colon carcinoma versus normal colon homogenates

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Abstract
The G‐regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product require the production of GTP from ATP in order to exert their effects within the cell. This implies that the activity of nucleoside diphosphate kinase (NDPK) plays a major role in the regulation of cellular events requiring GTP and that the level of activity of this enzyme is critical. This report presents a simple method for trapping a specific isozyme of NDPK in its high‐energy phosphorylated form (NDPK∼P) using EDTA and demonstrates that this NDPK∼P is tenfold higher in malignant colon tumor tissue than in normal colon tissue. This autophosphorylation of the 21,000 and 24,000 Mr subunits of NDPK occurs rapidly at Oß, will use either [γ32P]ATP, [γ32P]GTP, or the corresponding 8‐azidopurine photoprobes, is intramolecular, displays saturation effects, and is prevented from forming if GTPγS is added. Dephosphorylation in the homogenate occurs rapidly upon addition of Mg2+ or any nucleoside‐5′‐diphosphate. The subunits autophosphorylated in the homogenates are mostly in the soluble phase, and they comigrate with the subunits of pure NDPK from human erythrocytes. Cross‐addition of normal and malignant homogenates does not decrease the level of autophosphorylation of NDPK, which indicates that the level of NDPK∼P may be a quantitative measure of the level of this specific NDPK isozyme form. Assays for NDPK activity show correspondingly elevated levels in the malignant homogenates. Using western blot and photoaffinity labeling techniques, we distinguished the NDPK∼P subunits from two closely migrating GTP‐binding proteins. These were identified as the ras gene protein product and a 20,000 Mr protein, which comigrates identically with ADP‐ribosylating factor (ARF). The ARF also comigrates in a tight band that is phosphorylated by [γ32P]ATP or [γ32P]GTP when Mg2+ is present.