The activity of transglutaminase (TGase) was measured in cultured C6 glioma cells after their stimulation by either isoproterenol and isobutyl-methylxanthine or by a serum-containing medium. The activity fluctuated in a biphasic manner, with the peaks at 2-3 hr and 7-8 h r poststirnulation. The first peak of TGase activity was affected neither by cycloheximide nor by actinomycin D, which inhibited protein synthesis. The second peak, on t h e other hand, was completely eliminated by cycloheximide and was reduced by actinomycin D. Immunological procedures were employed to find out whether or not the activity of TGase corresponded with the presence of the TCase antigen in the cultured cells. Indirect immunofluorescent staining and radioimmunoblot techniques suggested that unstimulated cells contained an inactive enzyme. This inactive, or cryptic, enzyme had t h e same molecular weight as its active counterpart. Activation of t h e enzyme was mediated by cell stimulation, probably by its release from the membrane. This step did not require protein synthesis, unlike the second step, which was dependent on d e novo protein synthesis.
Transglutaminase (R-glutaminyl-peptide: amine y- glutamyl transferase, EC 2.3.2.13, TGase) catalyzes a Ca2+-dependent acyl transfer reaction between the y- carboxamide group of peptide bound glutamine and various primary amines, to yield new y-amide bonds of glutamic acid and ammonia (Clarke et al., 1959;Folk and Chung, 1973). Recently, several investigators demonstrated the presence of y-glutamyl-putrescine and y- glutamyl-spermidine residues in cells and extracellular fluids (Folk et al., 1980;Williams-Ashman and Canellakis, 1980). These findings suggest that polyamines and diamines are physiological substrates for TGase. Although stabilization of fibrin clot via -E(y-glutamyl) lysine formation is the physiological function of plasma TGase (Lorand et al., 1968; Pisano et al., 19681, the role of cellular TGase is still unknown.
Cell cultures
CG-BU-1 glioma cells were grown in DMEM medium supplemented with 5% fetal bovine serum, 200 units of penicillin, and 200 units of streptomycin per liter of medium. Approximately 9 x lo5 cells were seeded on