CLEAVAGE POINTS OF RABBIT SKELETAL MYOSIN LIGHT-CHAINS SELECTIVELY MODIFIED IN-SITU BY LIMITED PROTEOLYSIS - STRUCTURAL CHARACTERISTICS OF THE NEOFORMED ISOZYMES

The functional significance of myosin light chains in vertebrate stria ted muscle is an issue of interest and myosin species selectivity modi fied by papain or trypsin in their LC1 and LC2 light chains are potent ially useful for further investigation. We therefore determined the cl eavage sites resulting in the (T)-LC1', (P)-LC1' and (T)-LC2' species. Sequence analysis of (T)-LC1' indicated that the cleavage point in LC 1 is at Lys(7). Under appropriate conditions papain rapidly cleaves a short N-terminal segment from myosin light chain 1 and produces a new isozyme specifically modified in its essential light chain 1. The clea vage occurred at either Ala(11), Ala(12) or Ala(13), the Ala(11) cleav age being the most frequent. Trypsin was used to produce a myosin spec ies with a regulatory light chain 2 specifically truncated of a short N-terminal segment. The cleavage was specific at Arg(8) with no indica tion of other significant cleavage sites in this LC2. The effects of t rypsin and papain on myosin light chains are different, indicating dif ferent proteolytic specificities. None of these modifications, includi ng (CT)-LC2 '' cleavage at Phe(19), changed the K+-EDTA- and Ca2+-ATPa se activities of monomeric myosin significantly, indicating that LC1 a nd LC2 N-termini have little or no direct influence on the active site . An electric birefringence study also showed that these modified spec ies retained their average shape and flexibility. These observations a re essential in showing that the role of light chain extremities is ex pressed only in the presence of a minimum of structural organization ( filament or acto-myosin complex).