Localization of cellular transglutaminase on the extracellular matrix after wounding: characteristics of the matrix bound enzyme.
Extending our previous observation that tissue transglutaminase (TGase) binds to extracellular matrix (ECM) fibronectin, we report here that endogenous tissue TGase is localized on the adjacent ECM after puncture wounding embryonic human lung fibroblasts (WI-38). The bound TGase persisted at the wound site for many hours, demonstrated by immunofluorescence and by catalytic activity using an overlay assay. The binding characteristics of TGase with ECM were studied further by the addition of exogenous TGase to cell monolayers and monitoring by immunofluorescence or overlay catalytic activity assays. Binding occurred equally well at 4 degrees C or 37 degrees C. Prior incubation of exogenous TGase with guanosine 5'-triphosphate (GTP), guanosine 5'-diphosphate (GDP), or adenosine triphosphate (ATP) had little effect on the amount bound to matrix, but prior treatment with calcium, magnesium, strontium, or manganese ions enhanced binding 2- to 3-fold. The Ca(++)-dependent change was a concentration-dependent effect on soluble exogenous TGase, rather than an effect on ECM. Immunofluorescent techniques showed that binding of exogenous TGase to ECM was prevented by prior mixing with fibronectin or collagen, but not with several other ECM components, including laminin, elastin, chondroitin sulfate, heparan sulfate, and hyaluronic acid. ECM-bound TGase was released by 2 M potassium thiocyanate (KSCN) treatment but was not released by treatment with a variety of amino acids, salts, reducing agents, glycerol, or other chaotropic agents.
Samuel Roberts Noble Foundation, Inc., Ardmore, Oklahoma 73402.
H F
HF
Upchurch
E
E
Conway
M K
MK
Patterson
M D
MD
Maxwell
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D005109:Extracellular Matrix / Q000201:enzymology / Q000502:physiology* / Q000648:ultrastructure; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011503:Transglutaminases / Q000378:metabolism*; D014947:Wounds and Injuries
D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011503:Transglutaminases; D002118:Calcium