|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 2. Mapping the binding sites within Vn and PrPC. (a) 125I-Vn was incubated over PrPC coated wells in the presence of PrPC peptides. Binding to PrPC was set as 100% and binding in the presence of peptides was expressed as a percentage thereof. PrPC peptides 103-122 and 113-132 interfered with binding to Vn (*P<0.01, Student's t-test). (b) 125I-Vn binding to deletion mutant proteins 
105-112, 113-119, or 105-128 His6-PrPC was markedly reduced relative to binding to wild-type PrPC, which was set as 100% (*P<0.01, Student's t-test). 51-90 and 120-125 His6-PrPC proteins exhibited 125I-Vn binding that did not differ significantly from the wild-type protein. (c,d) The hydropathy plots compare the mouse PrPC amino acid sequence from a.a. 104 to 127 and human Vn peptides with complementary hydropathy pattern: (c) Vn262-275 and (d) Vn309-322. (e) 125I-Vn was incubated in PrPC-coated wells in the presence or absence of the indicated concentrations of Vn peptides and radioactivity levels were determined. 125I-Vn binding was disrupted in the presence of Vn309-322Hu and Vn307-320Mo (*P<0.01 vs binding to PrPC alone, Student's t-test) at the two higher concentrations tested. (f) Binding of peptide Vn307-320Mo to immobilized PrPC. Wells were coated with wild-type, 105-119, 113-119 or 105-128 His6-PrPC proteins and incubated with 1 µM 125I-Vn307-320Mo. 125I-Vn307-320Mo binding to immobilized PrPC was reduced in 105-128, 105-119 or 113-119 His6-PrPC proteins relative to wild-type PrPC controls (*P<0.001, Tukey's test). Inset shows an overlay assay, where His6-PrPC or PrPc peptides 43-62 and 103-122 spotted into a membrane were incubated with biotin labeled Vn307-320Mo followed by streptavidin-HRP.
|
