Cleavage points of rabbit skeletal myosin light chains selectively modified in situ by limited proteolysis: structural characteristics of the neoformed isozymes

FEBS Letters

Ghelis, Charis²  Cardinaud, Robert¹  Burgat, Jean-Marc¹ 

[1] Laboratoire de Biologie Physico-chimique, Université Paris-Sud, Bat. 433, F91405 Orsay, France;Laboratoire de Physico-chimie des proteines, Unité de Recherches Associée au Centre National de la Recherche Scientifique 1131, Université Paris-Sud, 91405 Orsay, France

关键词: Myosin light chain (rabbit fast skeletal);    Artificial myosin isozyme;    Limited proteolysis;    N-terminal sequence;    LC1 variability;    ATPase;    Electric birefringence;   

DOI  :  10.1016/0014-5793(95)00743-S

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The functional significance of myosin light chains in vertebrate striated muscle is an issue of interest and myosin species selectivity modified by papain or trypsin in their LC1 and LC2 light chains are potentially useful for further investigation. We therefore determined the cleavage sites resulting in the (T)-LC1′, (P)-LC1′ and (T)-LC2′ species. Sequence analysis of (T)LC1′ indicated that the cleavage point in LC1 is at Lys⁷. 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 cleavage occurred at either Ala¹¹, Ala¹² or Ala¹³, the Ala¹¹ cleavage being the most frequent. Trypsin was used to produce a myosin species with a regulatory light chain 2 specifically truncated of a short N-terminal segment. The cleavage was specific at Arg⁸ with no indication of other significant cleavage sites in this LC2. The effects of trypsin and papain on myosin light chains are different, indicating different proteolytic specificities. None of these modifications, including (CT)-LC2″ cleavage at Phe¹⁹, changed the K⁺-EDTA- and Ca²⁺-ATPase activities of monomeric myosin significantly, indicating that LC1 and LC2 N-termini have little or no direct influence on the active site. An electric birefringence study also showed that these modified species retained their average shape and flexibility. These observations are essential in showing that the role of light chain extremities is expressed only in the presence of a minimum of structural organization (filament or acto-myosin complex).

【 授权许可】

Unknown