Figure 5.

The Azurosome Promotes Translocation across Granule Membranes

(A and B) Calcein release from synthetic PC/PS liposomes. Calcein fluorescence was measured after 15 min of incubation and normalized to liposomes alone and liposomes permeabilized with NP-40. Error bars indicate SD in duplicate samples.

(A) Calcein release from synthetic liposomes incubated with control or ΔMPO azurosome, monitored by fluorescence dequenching of released calcein. The azurosome was quantified based on NE content as measured by ELISA and expressed in moles (x axis). Black and white squares: flowthrough buffer from the purification of control (black) and ΔMPO (white) azurosome. Black and white rhombuses: boiled samples (black) and ΔMPO azurosome (white) at the highest concentration. Fitting was used to calculate the concentration of azurosome required for 50% release (R₅₀) and the apparent cooperativity coefficient (n_H).

(B) Calcein release from synthetic liposomes incubated with control azurosome or purified MPO, monitored by fluorescence. The azurosome was quantified based on MPO content as measured by ELISA and expressed in moles (x axis). Fitting was used to calculate the concentration of azurosome required for 50% release (R₅₀).

(C) CASY impedance cell counter analysis of calcein-loaded synthetic PC/PS liposomes, either untreated or incubated with azurosome from a control donor or NP-40.

(D) Release of LYZ from specific and gelatinase granules incubated with control azurosome, ΔMPO azurosome, or NP-40. Samples were separated into soluble (S) and total (T) fractions by ultracentrifugation and immunoblotted against LYZ. Complexes without granules were used as controls for the background levels of LYZ from azurosomes alone.

(E) NE release by azurophilic granules as it was captured and detected by NE ELISA. Duplicate reactions of intact azurophilic granules, untreated or treated with NEi and activated with 100 μM H₂O₂ for 30 min. Additional reactions in the same conditions but treated with NP-40 were used for total to calculate the fraction of NE released. Error bars indicate SD in duplicate samples.

(F) AZU and MPO release from isolated native azurophilic granules alone or after incubation with H₂O₂ in the absence or presence of NEi. Samples were incubated for 30 min and insoluble granules were removed by centrifugation to yield soluble (S) protein. Total protein (T) prior to centrifugation.

See also Figures S4 and S5.