Benzamidine hydrochloride hydrate

N-Ethylmaleimide, 6-aminocaproic acid, benzamidine hydrochloride hydrate, dithiothreitol (DTT), iodoacetamide, ammonium bicarbonate (AMBIC), formic acid, HPLC grade acetonitrile and A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) solutions for LC-MS were purchased from Sigma-Aldrich (St. Louis, USA). Guanidine hydrochloride (GdnHCl) and anhydrous sodium acetate (NaAc) were purchased from Merck (Darmstadt, Germany). Trypsin gold MS grade was purchased from Promega (Madison, WI). The water used in this study was purified using a MilliQ apparatus (Millipore, Billerica, MA).

Selected protease inhibitors were added to digestion reactions in order to define the types of proteases present in midgut fluids. Bestatin and chymostatin (Roche Diagnostics, Indianapolis, IN, USA) were resuspended in methanol and DMSO respectively according to the manufacturer’s recommendations and added to reactions at final concentrations of 130 µM and 100 µM respectively. benzamidine hydrochloride hydrate (Sigma-Aldrich) was resuspended in 50 mM Tris-HCl (pH 8.0) and added to reactions at a final concentration of 5 mM. For insect bioassays, benzamidine was added directly to the prepared proteins in 50 mM Tris-HCl (pH 8.0) at final concentrations of 9 mM, 3 mM, 1 mM and 0.3 mM relative to the diet volume of 1.5 mL per well.

All steps of the SR vesicle preparation were performed on ice and/or at 4°C. For lipid bilayer experiments, five to seven hearts were homogenised in cardiac homogenising buffer (CHB, containing, in mM: sucrose 290, imidazole 10 and NaN₃ 3, pH 6.9). The homogenate was then centrifuged at 12,000 g for 20 min, then the pellet discarded and the supernatant centrifuged at 43,000 g for 2 h. The pellet was resuspended in Buffer A (CHB plus 649 mM KCl) and centrifuged at 46,000 g for 1.5 h. This pellet was re-suspended in 125 µl per g of mouse heart (∼5 hearts) of buffer A plus protease inhibitor mixture and stored in 8 μl aliquots at −80°C for use in lipid bilayer experiments. All individual protease inhibitors were obtained from Sigma-Aldrich and were added to the final suspension at the following final concentrations: benzamidine hydrochloride hydrate (catalogue #B6506), 1.0 mM; pepstatin A (catalogue #P4265), 2.1 µM; leupeptin (catalogue #L2884), 1 µM; AEBSF/Pefabloc SC (catalogue #76307), 0.5 mM; calpain inhibitor I –(catalogue #A6185), 3 µM; calpain inhibitor II (catalogue no. A6060), 3 µM.

The dual contrast agent was composed of CA4 + (5,5′‐(malonylbis(azanediyl))bis(N¹,N³‐bis(2‐aminoethyl)‐2,4,6‐triiodoisophthalamide, q =  +4, M = 1,499.88 g/mol) and gadoteridol (Prohance®; Bracco International B. V., Amsterdam, Netherlands, q = 0, M = 558.69 g/mol) diluted with phosphate‐buffered saline (PBS). Two dual contrast agent immersion baths (for 2 and 72 h immersions) containing 5 mg I/ml and 10 mg Gd/ml were prepared. Inhibitors of proteolytic enzymes (5 mM ethyleneadiaminetetra‐acetic acid [EDTA; VWR International, Fontenay‐sous‐Bois, France] and 5 mM benzamidine hydrochloride hydrate [Sigma‐Aldrich Inc., St. Louis, MO]) were added to both immersion baths. Furthermore, penicillin–streptomycin–amphotericin B (antibiotic antimycotic solution: 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B, Sigma‐Aldrich Inc., St. Louis, MO) was added to the 72‐h immersion bath to minimize the sample degeneration. The osmolalities of the contrast agent mixtures were ~308 and ~297 mOsm/kg for the 2‐ and 72‐h immersion baths, respectively. The osmolalities of the contrast agents and PBS were measured with a freeze‐point osmometer (Halbmikro‐osmometer; GWB, Knauer & CO GmbH, Berlin, West‐Germany).