Inhibition of XnGroEL binding to BBMV by different chitin
derivatives. A, detection of XnGroEL binding to BBMV after
chitinase treatment. The BBMV were incubated with chitinase followed by
incubation with XnGroEL, and the membrane proteins were processed and resolved
by SDS-PAGE as above. The gel was stained with Coomassie Blue; lane
1, untreated BBMV proteins; lane 2, treated with chitinase.
Samples in lanes 1 and 2 were blotted with anti-GroEL
antibodies after Western transfer; M, prestained markers; lane
3, untreated BBMV; lane 4, BBMV with chitinase and protease
inhibitor; lane 5, with chitinase, no protease inhibitor. B,
the binding assay mixture contained 20 μg of BBMV, 10 μg of XnGroEL, and
the competing compounds. 1, no inhibitory compound; 2, 3,
and 4, 0.05, 0.1, and 0.5 mg of solubilized chitin, respectively;
5, 1.0 mg of cellulose; 6, 1.0 mg of chitosan. C,
XnGroEL binding in the presence of lanes 1–5, 0, 50, 100, 150,
and 200 mm GalNAc, respectively (A); lanes
1–5, 0, 50, 100, 150, and 200 mm GlcNAc, respectively
(B), lanes 1–5, 0, 50, 100, 150, and 200 mm
diacetyl chitobiose, respectively (C), lanes 1–5, 0,
50, 75, 100, and 125 mm triacetyl chitotriose, respectively
(D); lanes 1–5, 0, 20, 40, 60 and 80 mm
hexaacetyl chitohexaose, respectively (E); loading control, BBM
blotted with antibody against N-aminopeptidase (F). The table shows
ID50 values calculated by plotting the integrated density values of
the bands shown in panel C. The experiments were repeated three
times, and data from one of the representative experiments are presented.