Fig. 6. hNMD3 binds to 60S subunits in vitro. (A) Saturable binding of recombinant hNMD3 to 60S ribosomal subunits. Twenty-five pmol of purified HeLa cell 60S subunits (lanes 3 to 12) were incubated with increasing amounts of 6His-hNMD3 (10, 20, 40, 60 or 80 pmol). Ribosomal subunits and associated hNMD3 were pelleted by sedimentation. The proteins in the pellet (P) and supernatant (S) were separated by 10% SDS-PAGE followed by Coomassie blue staining. Load of proteins in the unbound fractions (supernatants) equals the load of the ribosomal pellet fractions. Note that there is no sedimentation of 6His-hNMD3 in the absence of 60S subunits (lanes 1 and 2). (B) hNMD3 cosediments with 60S subunits. A mixture of each 25 pmol purified 40S and 60S subunits was incubated with 60 pmol of 6His-hNMD3 and then fractionated on a 10-40% sucrose density gradient. Fractions from the gradient were analyzed by Western blotting using antibodies directed to rpL10 and rpS6 as markers for the migration of 60S and 40S subunits, respectively. Detection of hNMD3 was by an antibody that recognizes the N-terminal 6His-tag of recombinant hNMD3. Note that hNMD3 comigrates with 60S subunits and that, in addition, it is found on the top of the gradient as free protein but not in association with 40S subunits.

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