RABBIT SKELETAL-MUSCLE ALPHA-ALPHA-TROPOMYOSIN EXPRESSED IN BACULOVIRUS-INFECTED INSECT CELLS POSSESSES THE AUTHENTIC N-TERMINUS STRUCTURE AND FUNCTIONS

When expressed in E, coli, skeletal muscle alpha-tropomyosin has an un acetylated N-terminus. Unacetylated alpha-tropomyosin lacks important functions; this is non-polymerizable and has a low affinity to actin. In the present work, in order to obtain fully functional recombinant a lpha-tropomyosin, rabbit skeletal muscle alpha-tropomyosin (alpha-trop omyosin(BV)) has been expressed in baculovirus-infected insect cells. alpha-Tropomyosin(BV) was not distinguishable from the authentic tropo myosin, not only in functional properties but also in blocked N-termin us. To know the N terminus structure of alpha-tropomyosin(BV), the N-t erminal segment six amino acids long, MDAIKK, has been specifically an d efficiently removed from alpha-tropomyosin(BV) by use of an immobili zed proteolytic enzyme system based on E, coli cell bodies which carry the ompT gene product, a proteolytic enzyme localized on the outer ce ll wall of E, coli. The structure of recombinant alpha-tropomyosin(BV) was shown to be identical to the authentic protein by electrospray ma ss spectrometry and protein sequencing analysis. Additionally, electro spray mass spectometry indicated a single phosphorylation not only in alpha- but also beta-tropomyosin chains in the rabbit skeletal muscle. The differentiated susceptibilities of potential ompT cleavage sites are indicative of a non-coiled-coil conformation of the N-terminus of alpha-tropomyosin.