Calcium-supported calpain degradation rates for cardiac myofibrils in diabetes. Sulfhydryl and hydrophobic interactions.
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.


CONCLUSIONS
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.
Belcastro
A N
AN
Laboratory of Cell Physiology and Exercise, University of British Columbia, Vancouver, Canada.
Gilchrist
J S
JS
Scrubb
J A
JA
Arthur
G
G
D000818:Animals; D002154:Calpain / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D008297:Male; D009124:Muscle Proteins / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism*; D051381:Rats; D011920:Rats, Inbred WF; D012995:Solubility; D013439:Sulfhydryl Reagents; D014867:Water / Q000737:chemistry
D009124:Muscle Proteins; D013439:Sulfhydryl Reagents; D014867:Water; D002154:Calpain