Qualitative analysis of skeletal myosin as substrate of Ca2+-activated neutral protease: comparison of filamentous and soluble, native, and L2-deficient myosin.

Abstract

Ca2+-activated neutral protease (CAF) was capable of degrading myosin over a 200-fold range of protease concentrations. CAF selected the heavy chain of myosin, although either prolonged exposure to or high concentrations of the protease degraded the L1, but not the L2 or L3, light chains of myosin. The following results indicated that during the first hour of digestion, under conditions where native myosin was the substrate, CAF selected for the "head" region of the myosin heavy chain: (a)large heavy chain fragments of identical molecular weight were produced from filamentous and from soluble myosin; (b) light meromyosin was not a substrate; (c) agents known to bind to the head of myosin (actin, MgATP, and L2) had both a qualitative and quantitative effect on degradation; and (d) similar cleavage sites could be demonstrated for myosin and for heavy meromyosin (HMM) despite the fact that HMM was a much poorer substrate than myosin. This observation is interpreted as an indication that the conformation of myosin heavy chain is altered in the preparation of HMM. The principal cleavage sites on the heavy chain of myosin were 20,000, 35,000 and 50,000 D from the Nterminus, producing large fragments with molecular weights of 180,000, 165,000, and 150,000 which comprised a "nicked" species of myosin. This nicked species retained both normal solubility properties and normal hydrolytic activities. For this reason, it is concluded that "nicked myosin" is an important pathophysiological species.

Parasites, proteomics and performance: effects of gregarine gut parasites on dragonfly flight muscle composition and function

“…Three of the bands are likely to be in vitro calpain degradation products of purified dragonfly MHC, with a prominent ~156·kDa band showing molecular mass similarity to our Spot 6 (Fig.·1). The numerous lower molecular mass bands observed in Fig.·3B appear to be the result of calpain autodegradation, as observed previously (Pemrick and Grebenau, 1984). …”

“…In addition, we putatively identified a calpain 80·kDa subunit, and other bands as calpain autodegradation products [i.e. similar to results reported by Pemrick and Grebenau (Pemrick and Grebenau, 1984)]. (Schilder and Marden, 2006).…”

“…Kim et al, 2005). Pemrick and Grebenau reported that proteolytic activity by calcium-activated neutral protease (CANP; also known as calpain) in the head region of ~200·kDa MHC from rabbit muscle produced 180, 165 and 150·kDa sized MHC peptides (Pemrick and Grebenau, 1984). Similarly, digestion of 200·kDa Octopus MHC by endogenous calpain-like proteinase CaDP results in a 155·kDa proteolytic product (Hatzizisis et al, 2000).…”

The Calpain System

“…Myosin Very slow degradation of undenatured myosin; the 210-kDa large subunit is degraded to fragments of 150, 165, and 180 kDa; slow degradation of LC2 light chain (347). Nebulin…”

Characterization of the Intracellular Proteolytic Cleavage of Myocilin and Identification of Calpain II as a Myocilin-processing Protease

“…It has been suggested that calpains play an important role in retinal cell death induced by ischemic reperfusion (29) or by hypoxia in cultured retina (30). Common calpain targets include fibronectin (31), cytoskeletal proteins, various muscle proteins, and neurofilament proteins (32)(33)(34)(35). The enzymatic activity of calpain is also regulated by calpastatin, an endogenous inhibitory protein (36).…”