INTRACELLULAR REGULATION OF TRANSGLUTAMINASE IN INTACT AND H202 INJURED CLONED BOVINE ENDOTHELIAL CELLS

Vascular endothelial cells are normally in a quiescent, nonproliferating state. After intimal injury, they are likely to undergo proliferation. We found that cloned bovine aortic endothelial cells (EC) in culture contain high activity of tissue-type transglutaminase (TG). The enzyme was Ca⁺⁺ dependent, the Km and vmax for putrescine were 0.203 and 18.5 nmol/min/mg protein. Primary amines were capable of inhibiting its activity but not methylated dansylcadaverine. EC and TG molecular weight estimated by gel filtration or by SDS-PAGE, was 88±5,000 Kd. Immunologically it was cross-reactive with purified rat liver TG. TG activity and antigen were found to increase significantly, when the cultures reached confluence and even more when cultures arrested at G₀/G₁ state by serum depletion. The half-life of EC-TG in G₀/G₁ arrested cultures was 128 minutes, determined in the presence cycloheximide; no decrease was observed in confluent cultures, indicating active accumulation of TG at the non-pro-liferative state. Most of cellular TG activity was found in the 15,000 g soluble fraction, with the crude membrane fraction containing 4-22% of the total TG activity, depending on the state of cell proliferation. However, immunoblots revealed that the membrane fraction contained similar amounts of TG antigen as did the soluble fraction. When the membranes were treated with detergents (0.1% SDS, 0.5 M NaCl, 75 mM KSCN, 75 mM dithio-threitol), the activity increased significantly, resulting in as much as 4- to 7-fold increases above control. Similar treatment of the soluble fraction did not increase TG activity. This data strongly suggests that the cell membranes contain a latent or cryptic form of TG that can undergo activation. Indeed, when cultures were injured by exposing them to H₂O₂ (10-10 mM) , a rapid 75-200% increase in TG activity was observed. One hour later, the activity was at control level. If protein synthesis inhibitors were present, no decrease was noted up to 8 hours. It is proposed that significant amounts of TG is stored in an inactive form in endothelial cell membranes, and that under different conditions, e.g., cell injury, activation and translocation of the enzyme can occur, out of and back into the cells membrane.