A sensitive assay for the enzyme activity in hair follicles and epidermis that catalyses the peptidyl-arginine-citrulline post-translational modification.
In 1977, Rogers, Harding, and Llewellyn-Smith described an enzymic activity in hair follicles which has the property of converting arginine to citrulline in situ in proteins. The protein precursor substrate in the hair follicle was identified as trichohyalin in the inner root sheath and medulla. Epidermal activity of the same kind has been described, although the natural protein substrates have not been identified. Attempts to enrich the enzyme are made difficult by the colorimetric enzyme assay which was originally devised by Rogers and Taylor but which lacks sensitivity and reproducibility. With protein substrates the formation of a citrulline product was not reliably detected below 25 nmoles. Using radioactively-labelled synthetic substrates such as N-benzoyl-L-[guanido-14C] arginine (Bz-14C-arg), the formation of only 1 nmole N-benzoyl-L-[ureido14C] citrulline (Bz-14C-cit) could be detected. In this assay, the enzyme is incubated with 1.8 mumoles Bz-14C-arg (approximately 50 microCi/mmole) for 3 hr at 37 degrees C. The protein is removed and the reaction mixture paper electrophoresed at pH 4.7. The Bz-14C-cit product separates optimally from unreacted substrate and the conversion is quantitated by radioactive counting of the appropriate regions of paper. The possibility of further improvement of the procedure is being investigated, including using other, related substrates. Using the assay, the enrichment and properties of the enzyme are under study in the richly medullated follicles of the guinea pig. The enzyme has an apparent Km of 18 mM for Bz-14C-arg and an absolute requirement for Ca2+ with a broad maximum around 10 mM. It is stable at +4 degrees C and -80 degrees C for at least four weeks and can be freeze-dried with some activity loss.
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001345:Autoradiography; D001711:Biotransformation; D002118:Calcium / Q000494:pharmacology; D002956:Citrulline / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism*; D004590:Electrophoresis, Paper / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D004817:Epidermis / Q000201:enzymology*; D006168:Guinea Pigs; D006197:Hair / Q000201:enzymology*; D066298:In Vitro Techniques; D007700:Kinetics; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D004151:Dipeptides; D002956:Citrulline; D001120:Arginine; D002118:Calcium