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J. Biol. Chem., Vol. 267, Issue 16, 10946-10949, 06, 1992
H Shirataki, K Kaibuchi, T Yamaguchi, K Wada, H Horiuchi and Y Takai
The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein
implicated in intracellular vesicle traffic, particularly in
neurotransmitter release from the presynapse. In the present study, we
attempted to identify a target protein in bovine brain crude membranes that
might be interacted with the GTP-bound form of smg p25A. When the
guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of
radioiodinated smg p25A and the crude membrane fraction of bovine brain
were incubated with a cross-linker, disuccinimidyl suberate, and the sample
was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of
about 110,000 was detected. This radioactive band appeared to be composed
of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This
molecule, tentatively termed here smg p25A target, was extracted from the
membranes by a detergent and highly purified by column chromatographies and
sucrose density gradient ultracentrifugation. The purified smg p25A target
was sensitive to heat boiling and tryptic digestion, indicating that smg
p25A target is a protein molecule. The M(r) of the purified smg p25A target
was estimated to be about 85,000-86,000 from SDS-PAGE and to be about
100,000 from the S value. The cross-linking of radioiodinated smg p25A with
the purified smg p25A target was inhibited by the GTP gamma S- bound form
of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form
of smg p25A was much less effective. Other small GTP- binding proteins,
such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective.
These results indicate that a protein with a M(r) of about 85,000-100,000
is a target for smg p25A.
A possible target protein for smg-25A/rab3A small GTP-binding protein
Department of Biochemistry, Kobe University School of Medicine, Japan.
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