TY - JOUR
T1 - Incorporation of (3H) ethanolamine into a single cytosolic protein in a cell free system
T2 - Ethanolaminylation of EF-1α in Vitro
AU - Whiteheart, Sidney W.
AU - Hart, Gerald W.
PY - 1994/3
Y1 - 1994/3
N2 - Ethanolamine containing modifications of cytosolic proteins have only been described for elongation factor 1α (EF-1α) which contains two ethanolamine-phosphoglycerol moieties at Glu 301 and 374 (Whiteheart et al. (1989) J. Biol. Chem. 264, 14334-14341 and Dever et al. (1989) J. Biol. Chem. 264, 20518-20525). In this report, we describe a cell-free, cytosolic extract which specifically incorporates [3H]ethanolamine into a single cytosolic protein with properties identical to EF-1α. The incorporation reaction is dependent on time and extract and is independent of any membrane-bound components. The single in vitro-radiolabeled protein is modified on two sites and chromatographic analysis of pronase-digested peptides is consistent with the addition of an unmodified ethanolamine. Ethanolaminylation does not require divalent cations or ATP but is inhibited by N-ethylmaleimide and stimulated by reducing agents (β-mercaptoethanol and dithiothreitol), indicating the requirement for free sulfhydryls. The nucleophile, hydroxylamine, at low concentrations, greatly inhibits the incorporation reaction, indicating the importance of an electrophilic center. This cytosolic extract appears to be able to carry out only the initial step in the addition of the ethanolamine-phosphoglycerol moieties to EF-1α, and subsequent addition of the phosphoglycerol moiety appears to require membrane components.
AB - Ethanolamine containing modifications of cytosolic proteins have only been described for elongation factor 1α (EF-1α) which contains two ethanolamine-phosphoglycerol moieties at Glu 301 and 374 (Whiteheart et al. (1989) J. Biol. Chem. 264, 14334-14341 and Dever et al. (1989) J. Biol. Chem. 264, 20518-20525). In this report, we describe a cell-free, cytosolic extract which specifically incorporates [3H]ethanolamine into a single cytosolic protein with properties identical to EF-1α. The incorporation reaction is dependent on time and extract and is independent of any membrane-bound components. The single in vitro-radiolabeled protein is modified on two sites and chromatographic analysis of pronase-digested peptides is consistent with the addition of an unmodified ethanolamine. Ethanolaminylation does not require divalent cations or ATP but is inhibited by N-ethylmaleimide and stimulated by reducing agents (β-mercaptoethanol and dithiothreitol), indicating the requirement for free sulfhydryls. The nucleophile, hydroxylamine, at low concentrations, greatly inhibits the incorporation reaction, indicating the importance of an electrophilic center. This cytosolic extract appears to be able to carry out only the initial step in the addition of the ethanolamine-phosphoglycerol moieties to EF-1α, and subsequent addition of the phosphoglycerol moiety appears to require membrane components.
UR - http://www.scopus.com/inward/record.url?scp=0028180069&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028180069&partnerID=8YFLogxK
U2 - 10.1006/abbi.1994.1128
DO - 10.1006/abbi.1994.1128
M3 - Article
C2 - 8135552
AN - SCOPUS:0028180069
SN - 0003-9861
VL - 309
SP - 387
EP - 391
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -