Myosin tryptic digestion

Myosin. Rabbit muscle myosin is a long, thin molecule (VI400 X 20-50 A) with a molecular weight of 5 X 10. It is composed of two heavy chains and four light chains as demonstrated by SDS-polyacrylamide disc gel electrophoresis. On tryptic digestion, myosin is split into the subunits, H-meromyosin (HMM) and L-mero-myosin (LMM). HMM is further split into S-l and S-2 subunits. While LMM is a rod of V)0% a-helical content, the a-helical content for HMM, S-l and S-2 fragments is 46%, 33% and 87%, respectively. The ATPase activity is localized in the S-l subunit (33,34). Although fish myosins appear to have the same structural profile (10,22,35-40) and similar amino acid composition as rabbit myosin (39,41,42), fish myosin is different from rabbit myosin in physicochemical properties such as solubility, viscosity and stability (10,22,35-40). 

Moreover, we have determined the false positive rate for this approach. Many tryptic peptides originated from different proteins can be attributed to a single mass (e.g. HQHPLQCVMEK 1364.63 Da and EADFINCVIWR 1364.65 Da AM < 20 ppm). Thereby false positive identification may occur. To evaluate the false positive rate, we have selected three common proteins which were not identified during the nanoLC-MS/MS analysis. These proteins (tubulin, actin and myosin) were digested in-silico, and the generated mass lists were compared to the LC-MALDI-MS peaklist. A total of only five masses were attributed to the three... 

Similar results were obtained by Connell (1900) in an examination of the kinetics of tryptic digestion of cod myosin. 

Peptide maps of the tryptic digest of myosin have been performed on thin-layer plates (20 x 20 cm) by successive TLC and electrophoresis. In the first-dimension TLC... 

Peptide maps of the tryptic digest of myosin have been performed on thin-layer plates (20 x 20 cm) by successive TLC and electrophoresis. In the first-dimension TLC with chloroform-methanol-ammonium hydroxide 34% (40 40 20, v/v) as eluent, the time was as long as 60 min. The second-dimension electrophoresis with pyridine-glacial acetic acid-water (1 10 489, v/v) buffer and 980 V, 30 mA current, the time was 1 hr. The peptide mixture is applied in amounts of 0.05 to 0.5 mg per peptide map. " In another paper, electrophoresis was applied in one direction on buffered adsorbents, followed by chromatography in the second dimension. Here, phosphate esters were separated by TLC development twice with n-propanol-ammonium hydroxide-water (60 30 1, v/v), and electrophoresis was carried out in 0.28 M acetate buffer (pH 3.6), 1000 V, 35 mA, and 16 min. 

A tryptic fragment of connectin, the 400-kDa peptide, has been demonstrated to cause aggregation of myosin filaments. Further digested fragments of chain weights less than 40 kDa lost this ability (Kimura et al., 1984b). 

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