Sj. Berger et al., ANALYSIS OF RECOMBINANT HUMAN ADP-RIBOSYLATION FACTORS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND ELECTROSPRAY MASS-SPECTROMETRY, Analytical biochemistry (Print), 264(1), 1998, pp. 53-65
Citations number
60
Categorie Soggetti
Biology,"Biochemical Research Methods","Chemistry Analytical
Two complementary approaches utilizing reverse-phase high-performance
liquid chromatography and liquid chromatography/mass spectrometry were
developed to analyze recombinantly produced Group I and Group II huma
n ADP-ribosylation factors (ARFs), We observe that the NH2 termini of
Group II ARFs (ARF4 and ARF5) are efficiently processed by removal of
the initiating methionine. In contrast, the NH2 termini of Group I ARF
s (ARF1 and ARF3), although fully deformylated, undergo only partial m
ethionine cleavage. This result is unexpected as ARFs are canonical su
bstrates for methionine processing in both bacterial and eukaryotic sy
stems, but it may explain the difficulties encountered by many researc
hers attempting to produce myristoylated ARFs in Escherichia coli. Add
itionally, we observe that a significant fraction of purified ARF4 con
tains a modification which we demonstrate to be consistent with mono-g
lutathionation. Both methionine retention and glutathione modification
may impact ARF function and the methods presented here should be empl
oyed to determine the duality of recombinant ARFs. (C) 1998 Academic P
ress.