Fig. 4: Heparin–spermine condensates create a metal-ion-rich, alkaline environment for enhanced activity of CPA3.
From: Mast cell extracellular granules are bioactive condensates assembled by heparin and polyamine
a, CPA3 protease activities of intact native granules and of dissociated granules (treated with heparinase I) were measured using chromogenic substrate and normalized to CPA3 densitometry unit from western blot analysis (n = 6 biological replicates). P values were determined using a two-tailed Mann–Whitney test. b, Protease activities of recombinant CPA3 in Tyrode’s buffer combined with heparin and spermine or in heparin–spermine condensates were measured (n = 6 biological replicates). P values were determined using a Kruskal–Wallis test with Dunn’s multiple-comparison test. c, Inductively coupled plasma mass spectrometry analysis of the ion concentrations in the dilute phase and the dense phase of heparin–spermine condensates (n = 3 biological replicates, each with three technical replicates). P values were determined using a two-tailed unpaired t-test. d, Protease activities of recombinant CPA3 in 50 mM HEPES pH 7.4 buffer containing varying concentrations of NaCl, CaCl2 or ZnCl2 as indicated (n = 3 biological replicates). e, pH difference between the dense phase and surrounding dilute phase of heparin–spermine condensates was determined with ratiometric C-SNARF-4 dye. Right, pH values were calculated from the ratio of fluorescence intensities at 540–590 nm and 610–660 nm using a calibration curve (n = 59 condensates). Scale bar, 10 μm. P values were determined using a two-tailed unpaired t-test. f, Protease activities of recombinant CPA3 at different pH (n = 6 biological replicates). P values were determined using an ordinary one-way ANOVA with Tukey’s multiple-comparison test. g, Protease activities of recombinant CPA3 in pH-adjusted and salt-adjusted buffers mimicking the dilute or dense conditions of heparin–spermine condensates (n = 3 biological replicates). P values were determined using a two-tailed unpaired t-test. Data are presented as the mean ± s.d. One representative of two (a,b,e) or three (d,f,g) independent experiments is shown.
