76D-16 |
Identification of transglutaminase-induced myofibrillar polypeptides with electrophoretic peptide mapping |
J. C. RAMIREZ-SUAREZ, Dept. of Animal Sciences, Univ. of Kentucky, 200 Garrigus Bldg., Lexington, KY 40546 and Y. L. Xiong. Transglutaminase (TGase) catalyzes an acyl transfer reaction between g-carboxiamide groups of glutaminyl residues and primary amines in proteins. These interactions promote intra- as well as intermolecular cross-linking. Earlier studies showed that water suspensions of myofibrillar protein isolate (MPI) from chicken breast muscle treated with TGase at 5°C formed three distinctive electrophoretic bands during the first hour of reaction, one immediately below myosin heavy chain, another below actin, and the third at 31 kDa. An extended TGase incubation seemed to completely reverse the protein reactions. We hypothesize that these bands were derived from myosin and actin. Our objective was to determine the origin of the TGase-induced new myofibrillar protein bands by using peptide mapping with limited proteolysis and SDS-PAGE. SDS-PAGE of 2 mg/mL MPI treated with 0.1% TGase at 5°C for 0, 15 min, and 2 h was conducted to separate proteins. Bands of interest were excised, and the proteins were subjected to limited proteolytic digestion using trypsin and papain. The TGase-treated protein digests were electrophoresed to map the peptide fragments, and the results were compared with the digests of control proteins. Peptide maps of the TGase-induced sub-myosin, sub-actin, and the 31 kDa component generated by either protease essentially matched those without the TGase treatment. Peptide profiles of the recovered myosin heavy chain and actin after 2 h incubation with TGase both resembled those of the control sample. The results suggest that the intermediary polypeptides originated from myosin and actin probably due to structural rearrangements, and that TGase catalyzed reversible intramolecular association under the conditions used in the study. The exact molecular mechanism, however, remains to be elucidated. The results should contribute to the understanding of the kinetics of TGase-catalyzed muscle protein cross-linking reactions.
Session 76D, Muscle Foods II
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