CALCIUM-SUPPORTED CALPAIN DEGRADATION RATES FOR CARDIAC MYOFIBRILS INDIABETES - SULFHYDRYL AND HYDROPHOBIC INTERACTIONS

Objective: The purpose was to investigate the calcium required for cal pain-mediated degradation of selected cardiac myofibril proteins modif ied by diabetes, sulfhydryl (SH) and hydrophobic reagents. Methods: Af ter 20 weeks of streptozotocin-induced (55mg.kg(-1)) diabetes, calcium sensitive calpain (1.5 U.ml(-1)) degradation rates of purified cardia c myofibrillar proteins (1 mg.ml(-1)) were measured, in vitro, and com pared to degradation rates for N-ethylmaleimide (NEM) and 2-p-toluidin ylnapthalene-6-sulfonate (TNS) treated samples. Results: Diabetes (blo od glucose of 550 +/- 32 mg.dl(-1)) reduced the yield of purified myof ibrillar protein with minimal change in fibril protein composition. To tal SH group reactivities (nmol.mg(-1).30min) were 220 +/- 21, 163 +/- 17 and 156 +/- 24 for control, diabetic and NEM-treated (0.5mM) myofi brils (p less than or equal to 0.05). Calpain degradation rates were f aster for all diabetic and SH modified myofibrillar proteins (p less t han or equal to 0.05), with a 45 and 35% reduction in the pCa(50) for a 37 kDa protein of diabetic and NEM-treated fibril complexes. For con trol myofibrils, both 100 and 200 uM TNS, reduced calpain degradation rates to a similar extent for all substrate proteins. In contrast, dia betic and NEM-treated samples showed a further reduction in calpain de gradation rates with increasing TNS from 100 to 200 uM. Conclusion: Ou r results support the hypothesis that in diabetes the calcium requirem ents for calpain degradation rates are reduced and dependent upon sulf hydryl group status and Ca2+-induced hydrophobic interactions, implica ting a 37 kDa myofbillar-complexed protein.