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Fig. 2.
Peptide maps of HMWCaMBP, calpastain I, and calpastatin II. Bands from SDS–PAGE (10%) stained with Coomassie Blue were digested by placing gel slices containing these polypeptides as described under Materials and methods. Lane 1; HMWCaMBP, lane 2; calpastatin I, lane 3; calpastatin II.
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Inhibition of m-calpain by HMWCaMBP, calpastatin I, and calpastatin II
Furthermore, we examined the inhibitory properties of HMWCaMBP, calpastatin I, and calpastatin II. Dose-dependent inhibition assays were carried out with HMWCaMBP, calpastatin I and calpastatin II in presence of m-calpain as described in Materials and methods. All three Angiotensin 1/2 (1-7) showed a concentration-dependent inhibition of m-calpain (data not shown). The IC50 values for the inhibition are summarized in Table 1. These results suggest that HMWCaMBP, calpastatin I, and calpastatin II have similar affinities for m-calpain.
Discussion
In this study, we used five different antibodies in order to find out the difference between the isolated homologs (HMWCaMBP, calpastatin I, and calpastatin II) at their N-termini and at other domains. Of the five antibodies, four antibodies (Type IXL, Type IIXL, Type XL, and Type Ex3T) were based on the peptide sequence of porcine calpastatin isoforms: Type I, Type II, and Type III, the sequences of which are nearly identical to the sequences of bovine calpastatin isoforms [14]. The antigen sequence for Type IXL, Type IIXL, and Type Ex3T antibodies are identical to bovine sequences except that Type IXL and Type Ex3T each have only one amino acid difference compared to bovine sequence (Fig. 1A). However, although Type XL has eight residues different than bovine, it cross-reacted with all three bovine homologs (Fig. 1B).