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Fig. 6. Functional analysis of gephyrin constructs. (A) Moco activity of E. coli MoeA in cells expressing gephyrin mutant constructs (L1, L2B, L2C), a gephyrin variant unable to bind glycine receptors (P713E), gephyrin-GCEE, the isolated CE-domain, gephyrin (GCE) and as negative controls, the isolated G-domain (G) or empty vector (pQE30). Activities were determined with the nit-1 reconstitution assay in the absence of external molybdate (black bars). Different dilutions of crude protein extracts were used according to the linear range of the assay and units are defined as reconstituted enzyme activity (A₅₄₀ over 20-minute reaction time per mg crude protein extract). Error bars (s.e.) are derived from at least three independent experiments. Enzymatic activity was normalized to the expression levels of gephyrin variants (white bars) measured by densitometry in western blots (B). (B) Western blot analysis of crude protein extracts that were used for Moco synthesis analysis (A) using monoclonal (mAB, upper blot) and polyclonal gephyrin antibodies (pAB, Puszta serum; lower blot); arrows indicate the relevant bands. The amount of protein loaded is stated for each lane. For comparison, different dilutions of gephyrin (GCE) are shown that were used for densitometric quantification of band intensities. The upper blot shows dotted lines for the molecular weight standards at 116 and 67 kDa.

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