Visualization of purified fibronectin-transglutaminase complexes.
It has been reported previously (Turner, P.M., and Lorand, L. (1989) Biochemistry 28, 628-635) that human erythrocyte transglutaminase forms a noncovalent complex with human plasma fibronectin near its collagen-binding domain. In the present study, we show by nondenaturing electrophoresis that guinea pig liver transglutaminase, similarly to the erythrocyte enzyme, forms a complex with human fibronectin. Studies of anisotropic shifts of fluorescein-labeled liver and erythrocyte transglutaminases, upon addition of fibronectin, indicated that both transglutaminases bind to fibronectin with a stoichiometry of about 2:1. Polymerization of fibrinogen by human erythrocyte transglutaminase was inhibited after complex formation with fibronectin. Complexes of fibronectin with either erythrocyte or liver transglutaminase were isolated by glycerol gradient zone sedimentation and examined by rotary shadowing electron microscopy. The globular transglutaminase could be readily identified binding to the thin fibronectin strand. The binding site for transglutaminase was within 5-10 nm of the N terminus of fibronectin, consistent with its proximity to the collagen-binding domain. Under some experimental conditions, the complex of fibronectin with erythrocyte transglutaminase appeared as a ring-shaped structure in which two transglutaminase molecules had probably dimerized. The molecular weight of the erythrocyte transglutaminase was determined by sedimentation equilibrium to be 71,440 +/- 830.
LeMosy
E K
EK
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
Erickson
H P
HP
Beyer
W F
WF
Radek
J T
JT
Jeong
J M
JM
Murthy
S N
SN
Lorand
L
L
D000818:Animals; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology; D005353:Fibronectins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D005454:Fluorescence Polarization; D006168:Guinea Pigs; D006801:Humans; D008099:Liver / Q000201:enzymology; D008854:Microscopy, Electron; D016622:Silver Staining; D013045:Species Specificity; D011503:Transglutaminases / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D005353:Fibronectins; D011503:Transglutaminases