Probing the transglutaminase-mediated, posttranslational modification of proteins during development.
Sphaerechinus granularis eggs were fertilized in seawater in the presence of 0.2 mM dansylcadaverine, and development was allowed to take place with this compound in the medium. gamma-Glutamyldansylcadaverine, indicative of the utilization of the amine tracer by intrinsic transglutaminase, was isolated from the embryonic proteins, and identity of the product with the chemically synthesized gamma-glutamyl derivative of dansylcadaverine was confirmed. Covalent labeling of proteins occurring during development was examined by means of electrophoresis in NaDodSO4, followed by immunoblotting with an antibody that specifically recognized the dansyl hapten. There was an increase in the total uptake of the tracer at an essentially constant rate with each cell division, from 2- to 8- and 64-cell stages. Moreover, multiple protein labeling was evident in all specimens. The described concept of studying posttranslational modifications in vivo by transglutaminase through detection of the haptenic or specific ligand recognizable group of an incorporated small amine substrate will undoubtedly be of general utility for probing the functions of this family of enzymes in other cell types as well.
Cariello
L
L
Stazione Zoologica, Naples, Italy.
Velasco
P T
PT
Wilson
J
J
Parameswaran
K N
KN
Karush
F
F
Lorand
L
L
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002103:Cadaverine / Q000031:analogs & derivatives / Q000378:metabolism; D002478:Cells, Cultured; D005307:Fertilization in Vitro; D005456:Fluorescent Dyes / Q000378:metabolism; D006241:Haptens / Q000276:immunology; D008297:Male; D010063:Ovum / Q000201:enzymology; D011073:Polyamines / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012617:Sea Urchins / Q000196:embryology; D013379:Substrate Specificity; D011503:Transglutaminases / Q000276:immunology / Q000502:physiology*
D000906:Antibodies; D005456:Fluorescent Dyes; D006241:Haptens; D011073:Polyamines; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine