IDENTIFICATION OF THE 30-KDA POLYPEPTIDE IN POST-MORTEM SKELETAL-MUSCLE AS A DEGRADATION PRODUCT OF TROPONIN-T

Although a 30 kDa polypeptide frequently is seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of post mortem (pm) skeletal muscle and in turn is used as an indicator of proteolysis, its origin has not been conclusively identified. We used antibodies to selected myofibrillar proteins, including some known to be degraded pm, to identify this polypeptide. The left side of eight beef carcasses was electrically stimulated (ES) within 1 h after slaughter, and the right side served as the non-stimulated (NS) control. The longissimus lumborum (LL) muscle was removed from the carcass at 24 h pm and was stored at 2 degrees C. Myofibrils were prepared from the LL muscle immediately after stimulation (0 day) and from the stored muscle sample at 1, 3, 7, 14 and 28 days pm for analysis of SDS-PAGE and Western blots. By SDS-PAGE, troponin-T (TN-T) decreased in amount more rapidly pm in ES samples than in NS samples. By SDS-PAGE, a 30 kDa band increased and became a prominent band by 7 days pm in both NS and ES samples. A monoclonal antibody (mAb) to TN-T labeled purified TN-T, as well as the TN-T in myofibrils, a promi nent 30 kDa polypeptide and a family of lower molecular mass polypeptides in pm muscle. This mAb also labeled a 30 kDa band that had been electrophoretically purified from pm muscle. The 30 kDa band in blots of myofibrils was prominent from day 3 through 28 in bath ES and NS samples, but was exaggerated in the ES samples. Antibodies to other myofibrillar proteins (titin, nebulin, alpha-actinin and desmin) did not label the 30 kDa band. We conclude, on the basis of labeling with a mAb to TN-T, that the prominent 30 kDa polypeptide often observed in pm bovine skeletal muscle is a degradation product of TN-T and, furthermore, that an entire family of lower molecular mass polypeptides (one of which is the 30 kDa polypeptide) originates from TN-T.