Fig. 3.

Released μ1N is sufficient for promoting activity. (A) (i) ISVPs of T1L (L+L) or T3D (D+D), or a mixture of equal parts of both (L+D), were incubated at 37°C for 60 min. ISVP* conversion was assayed by TRY sensitivity of the particle-associated μ1 species (μ1C, μ1δ, and δ). (ii) T1L ISVPs were incubated at 37°C for 65 min (lane 1); at 53°C for 5 min (lane 2); or preconverted at 53°C for 5 min, chilled and supplemented with an equal part of fresh ISVPs, and incubated at 37°C for 60 additional min (lane 3). ISVP* conversion was assayed by TRY sensitivity. (B) ISVPs were preconverted at 52°C and centrifuged to pellet particles. (i) Equivalent amounts of input particles and supernatant, demonstrating efficient particle clearance (μ1N and φ are too small to be resolved on this gel). (ii) 10 μl of buffer or supernatant were added to ISVPs, and reactions were incubated at 37°C for 30 min followed by the TRY-sensitivity assay. (C) (i) WT-pRCs, N42A-pRCs, ISVPs, and dpISVPs were preconverted at 52°C. Preconversion of a duplicate sample was confirmed by the TRY-sensitivity assay (“pre-conv.”). Preconverted particles were chilled and supplemented with fresh WT ISVPs, incubated at 37°C for 30 min, and assayed by TRY sensitivity (“promoting”). Samples containing ISVPs incubated at 0°C, instead of 52°C, were analyzed in parallel. (ii) Quantitation of three or more experiments such as shown in i. Two different preparations of each particle type were used. The amount of δ remaining after the TRY-sensitivity assay is expressed relative to the amount in samples containing only unconverted ISVPs (0° lanes in i). The 50% protease-resistant δ in the N42A-pRC lane reflects preconversion of the N42A-pRCs but no promotion of the target ISVPs, which are present in equal numbers. Error bars indicate SD. (D) ISVPs or dpISVPs were preconverted at 52°C and centrifuged to pellet particles. The indicated amounts of supernatant were added to fresh WT ISVPs, and reactions were incubated at 37°C for 30 min followed by TRY-sensitivity assay. (E) ISVPs were incubated with 50 μg/ml synthetic μ1N peptide or vehicle control (1% DMSO) at 37°C for 60 min followed by TRY-sensitivity assay. All reactions contained a total final ISVP concentration of 5 × 10¹² particles per milliliter.