COOH-TERMINAL PROCESSING OF NASCENT POLYPEPTIDES BY THE GLYCOSYLPHOSPHATIDYLINOSITOL TRANSAMIDASE IN THE PRESENCE OF HYDRAZINE IS GOVERNED BY THE SAME PARAMETERS AS GLYCOSYLPHOSPHATIDYLINOSITOL ADDITION

Proteins anchored to the cell membrane via a glycosylphosphatidylinosi tol (GPI) moiety are found in all eukaryotes. After NH2-terminal pepti de cleavage of the nascent protein by the signal peptidase, a second C OOH-terminal signal peptide is cleaved with the concomitant addition o f the GPI unit. The proposed mechanism of the GPI transfer is a transa midation reaction that involves the formation of an activated carbonyl intermediate (enzyme-substrate complex) with the ethanolamine moiety of the preassembled GPI unit serving as a nucleophile, Other nucleophi lic accepters like hydrazine (HDZ) and hydroxylamine have been shown t o be possible alternate substrates for GPI. Since GPI has vet to be pu rified, the use of readily available nucleophilic substitutes such as HDZ and hydroxylamine is a viable alternative to study COOH-terminal p rocessing by the putative transamidase. As a first step in del eloping a soluble system to study this process, we have examined the amino ac id requirements at the COOH terminus for the transamidation reaction u sing HDZ as the nucleophilic acceptor instead of GPI. The hydrazide-fo rming reaction shows identical amino acid requirement profiles to that of GPI anchor addition, Additionally, we have studied other parameter s relating to the kinetics of the transamidation reaction in the conte xt of rough microsomal membranes, The findings with HDZ provide furthe r evidence for the transamidase nature of the enzyme and also provide a starting point for development of a soluble assay.