Affinity of human erythrocyte transglutaminase for a 42-kDa gelatin-binding fragment of human plasma fibronectin.
Complex formation between the human erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) and fibronectin or its fragments was examined by immunoanalytical procedures and by fluorescence polarization. A 42-kDa gelatin-binding structure, obtained from human plasma fibronectin by thermolytic digestion, showed as high an affinity for the cytosolic enzyme as the parent fibronectin chains themselves. A 21-kDa fragment comprising type I modules 8 and 9, the last two modules in the 42-kDa fragment, bound with an affinity 100-fold less than the 42-kDa fragment. Binding was remarkably specific and could be exploited for the affinity purification of transglutaminase directly from the hemoglobin-depleted erythrocyte lysate. In spite of the high affinity, it was possible to elute active enzyme from the 42-kDa fragment column with 0.25% monochloroacetic acid. This solvent might have general applicability in other systems involving separation of tightly bound ligands.
Radek
J T
JT
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208.
Jeong
J M
JM
Murthy
S N
SN
Ingham
K C
KC
Lorand
L
L
D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000201:enzymology*; D005353:Fibronectins / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D011503:Transglutaminases / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D010446:Peptide Fragments; D005780:Gelatin; D011503:Transglutaminases