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 approximately P) using
EDTA and demonstrates that this NDPK approximately 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 0 degrees C, will use either [gamma-32P]
ATP, [gamma-32P]
GTP, or the corresponding 8-azidopurine photoprobes, is intramolecular, displays saturation effects, and is prevented from forming if
GTP gamma S 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 approximately 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 approximately 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 [gamma 32P]
ATP or [gamma-32P]
GTP when Mg2+ is present.