ABSTRACT
OBJECTIVE: The purpose was to investigate the calcium required for calpain-mediated degradation of selected cardiac myofibril proteins modified by diabetes, sulfhydryl (SH) and hydrophobic reagents. METHODS: After 20 weeks of streptozotocin-induced (55 mg.kg-1) diabetes, calcium sensitive calpain (1.5 U.ml-1) degradation rates of purified cardiac myofibrillar proteins (1 mg.ml-1) were measured, in vitro, and compared to degradation rates for N-ethylmaleimide (NEM) and 2-p-toluidinylnapthalene-6-sulfonate (TNS) treated samples. RESULTS: Diabetes (blood glucose of 550 +/- 32 mg.dl-1) reduced the yield of purified myofibrillar protein with minimal change in fibril protein composition. Total SH group reactivities (nmol.mg-1.30min) were 220 +/- 21, 163 +/- 17 and 156 +/- 24 for control, diabetic and NEM-treated (0.5 mM) myofibrils (p < or = 0.05). Calpain degradation rates were faster for all diabetic and SH modified myofibrillar proteins (p < or = 0.05), with a 45 and 35% reduction in the pCa50 for a 37 kDa protein of diabetic and NEM-treated fibril complexes. For control myofibrils, both 100 and 200 uM TNS, reduced calpain degradation rates to a similar extent for all substrate proteins. In contrast, diabetic and NEM-treated samples showed a further reduction in calpain degradation rates with increasing TNS from 100 to 200 uM. CONCLUSION: Our results support the hypothesis that in diabetes the calcium requirements for calpain degradation rates are reduced and dependent upon sulfhydryl group status and Ca(2+)-induced hydrophobic interactions, implicating a 37 kDa myofbillar-complexed protein.
