New method to discriminate between cathepsin B and cathepsin L in crude extracts from fish muscle based on a simple acidification procedure

Abstract

A new and simple method to distinguish between cathepsin B and cathepsin L in crude extracts of herring (Clupea harengus) muscle has been established. An acid treatment of crude extracts (exposed to pH 3 for 5 min) activated a latent form of cathepsin L and inactivated cathepsin B. Furthermore, in neutral crude extract, the hydrolysis of benzyloxycarbonyl-l-phenylalanyl-l-arginyl-4-methylcoumarine (Z-Phe-Arg-MCA) (cathepsin B and cathepsin L substrates) was between 0% and 15% of the hydrolysis of benzyloxycarbonyl-l-arginyl-l-arginyl-7-amino-4-methylcoumarine (Z-Arg-Arg-MCA; cathepsin B substrate). Cathepsin B activity is measured in neutral extract using the specific cathepsin B substrate Z-ArgArg-MCA and cathepsin L activity is measured in acid-treated extract with Z-Phe-Arg-MCA as substrate. The specific cathepsin B inhibitor, CA-074, did not inhibit the Z-Arg-Arg-MCA significantly without affecting the Z-Phe-Arg-MCA activity. An acid treatment of the crude extract is therefore a more advantageous approach to discriminate between cathepsin B and cathepsin L activities.

Recovery of cathepsins from marinating brine waste

“…The activity originated mainly from aspartyl proteases (Fig. At this pH, the highest activity was expressed by cysteine cathepsins, especially B and L ones (Godiksen & Nielsen, 2007). In turn, the optimum activity of cysteine proteases was noted at pH 5.0 and reached ca.…”

“…1, Table 1). The recovery of cathepsins D + E at pH 5.0 or cathepsins B + L at pH 4.0 was lower (Table 1) owing to a close isoelectric point and/or reduced stability in the acidic/alkaline medium of these proteases (Godiksen & Nielsen, 2007;Miranowska et al, 2009;Szymczak & Lepczy nski, 2016). Therefore, aspartyl cathepsin recovery yield is higher at alkaline than at acidic pH.…”

“…Aspartyl and cysteine proteases predominate in the brine; therefore, the activity was also measured with and without the addition of (i) pepstatin-Aa specific inhibitor of aspartyl proteases and (ii) E64an inhibitor of cysteine proteases that inhibits most of the activity of cathepsins B and L (Godiksen & Nielsen, 2007). Proteolytic preparations from brine contain many cathepsins that express cooperative action against substrate.…”

“…Hence, general proteolytic activity (GPA) was determined against haemoglobina natural substrate specific to all acidic proteases. Aspartyl and cysteine proteases predominate in the brine; therefore, the activity was also measured with and without the addition of (i) pepstatin-Aa specific inhibitor of aspartyl proteases and (ii) E64an inhibitor of cysteine proteases that inhibits most of the activity of cathepsins B and L (Godiksen & Nielsen, 2007).…”

Cathepsin L Acutely Alters Microvessel Integrity within the Neurovascular Unit during Focal Cerebral Ischemia

“…Cell extracts incubated with Z-phe-arg-MNA at pH3.0 for 5 minutes resulted in 98% inactivation of cathepsin B. 23 For cathepsin B-like activity, Z-arg-arg-MNA was pre-incubated with 100 mM 2-(N-morpholino)-ethanesulfonic acid pH6.0, 1 mM DTT, and 1 mM EDTA, while for cathepsin L-like activity 0.5 mM Z-phe-arg-MNA was preincubated with 100 mM 2-(N-morpholino)-ethanesulfonic acid pH3.5, 1 mM DTT, and 1 mM EDTA for 15 minutes at 37°C. Cell homogenates (100 μL each) were then added to the assay buffer/substrate mixture and incubated at 37°C for 10 minutes.…”