M-lines proteins

The C-protein (thick filaments), myomesin (M-line protein), and a-actinin (Z-line protein)110113114 each provide 2% of the protein in the myofibril. Less than 1% each of 11 or more other proteins may also be present within the sarcomere.86115 Several of these, including the cytoskeletal proteins desmin and vimentin, and synemin surround the Z-discs.116/116a... 

In the center of the A-band, the thick filaments are linked by a network of proteins forming the M-line. The pattern of cross-linking of these proteins, such as my-omesin, and their ability to bind the thick filament protein myosin probably explains the hexagonal arrangement of the thick filaments. Cytosolic creatine kinase activity also localizes extensively to the M-line region and may actually be a property of one of the myosin cross-linking M-line proteins. 

Troponin C Troponin I Troponin T Minor M protein 18 21 31 165 2 M line Ca binding Inhibits actin-myosin interaction Binds to tropomyosin Binds to myosin... 

Titin Reaches from the Z line to the M line Largest protein in body. Role in relaxation of muscle. 

Fig. 1. (opposite page) Distribution of FITC-conjugated BSA in various fibroblast cell lines under different fixation/permeabilization regimes. (A-D) Protein distribution in living cells (A) PtKj, (B) CHO, (C) 3T3, and (D) HeLa cells. The protein is excluded from the nuclei of all cells. (E-H) Protein distribution in cells extracted for 10 min with 0.1% Triton X-100 before fixation for 30 min with 3.7% formaldehyde (E) PtKi, (F) CHO, (G) 3T3, and (H) HeLa cells. Nuclear fluorescence is seen in (E) PtKj and (G) 3T3 cells. (I-L) Protein distribution in cells extracted for 10 min with 1% Triton X-100 before fixation for 30 min with 3.7% formaldehyde (I) PtKj, (J) CHO, (K) 3T3, and (L) HeLa cells. No fluorescence is detected in the cells with the exception of some nuclear fluorescence seen in (L) HeLa cells. (M-P) Protein distribution in cells fixed for 30 min with 3.7% paraformaldehyde before permeabilization for 10 min with 0.1% Triton X-100. Fluorescence is seen primarily in the cytoplasm with the exception that nuclear fluorescence is seen in (M) PtKi and (N) CHO cells. (Q-T) Protein distributions in cells fixed for 5 min with 90% methanol, 50 vaM EGTA at -20°C (Q) PtKj, (R) CHO, (S) 3T3, and (T) HeLa cells. All cells show an overall low fluorescence, fibrous-textured cytoplasmic fluorescence, and bright staining at the periphery of the nucleus. 10 mm per scale division (black bar). (Reproduced with permission from ref. 6.)... 

Even if relatively new, HF FIFFF has been used to separate supramicrometer particles, proteins, water-soluble polymers, and synthetic organic-soluble polymers. Particle separation in HF FIFFF has recently been improved, reaching the level of efficiency normally achieved by conventional, rectangular FIFFF channels. With these channel-optimized HF FIFFF systems, separation speed and the resolution of nanosized particles have been increased. HF FIFFF has recently been examined as a means for off-line and on-line protein characterization by using the mass spectrometry (MS) through matrix-assisted laser desorption ionization time-of-flight mass spectrometry (M ALDl-TOF MS) and electrospray ionization (ESl)-TOF MS, as specific detectors. On-line HF FIFFF and ESl-TOF MS analysis has demonstrated the viability of fractionating proteins by HF FIFFF followed by direct analysis of the protein ions in MS [38]. 

Under the electron microscope titin appears as a flexible beaded string 4 nm in diameter. Most of the molecule is made up of repetitive domains of two types. In human cardiac titin there are 132 folded domains that resemble type III fibronectin repeats and 112 immunoglobulin-like domains.98 In a "PEVK region," between residues 163 and 2174, 70% of the residues are Pro, Glu, Val, or Lys. The titin molecule may be organized as polyproline helices in this elastic region.1023 At the C terminus of titin 800 residues, including a Ser / Thr protein kinase domain, are found within the M-line. 

Central l-band and M-line titin-binding proteins... 

E-C Muller, M Schumann, A Rickers, K Bommert, B Wittmann-Liebold, A Otto. Study of Burkitt lymphoma cell line proteins by high resolution two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry. Electrophoresis 20 320-330, 1999. 

Figure 8.9 Ultrastructure of the myofibril indicating the various bands observed microscopically. The I bands are largely the thin filaments, whereas the A bands are the thick filaments with partially overlapping thin filaments. The thick filaments are about 1600 nm in length, whereas the thin filaments are about 1000 nm in length. The Z line is a transverse tubule that serves to bind the thin filaments together. Thick filaments are bound together by a protein seen as a dark line (M line) in the middle of the H zone. (Reproduced by permission from Snell RS. Clinical and Functional Histology for Medical Students. Boston Little, Brown, 1984,...

The thick filament system, which comprises myosin protein, connected from the M-line to the Z-disc by titin (connectin), and myosin-binding protein C, which binds at one end to the thick filament and at the other to actin. 

Fig. 9. Preparative chromatography of bovine ehymotrypsinogen B (105). CM-cellulose column (3.0 X 9.0 cm) equilibrated with 0.05 M citrate, pH 4.2. Arrow indicates change to 0.05 M citrate, pH 4.6. Solid line, potential activity. Dotted line, proteins. The values along the peaks indicate the specific activity of some fractions. I. Chromatography of the precipitate obtained in 0.4 saturated ammonium sulfate (specific activity, 0.9). II. Second chromatography of the fractions having the lowest activity in diagram I. Ordinates, activity or proteins found in each fraction (1 ml) and expressed in per cent of the total activity and total proteins introduced into the column. Abscissas, volume of eluate in milliliters.

Fig. 11. Second chromatography of porcine chymotrypsinogen A and trypsinogen (85). Both elutions are performed with buffers of constant composition (equilibrium chromatography). On the left chymotrypsinogen A (specific activity, 2.9). CM-cellu-lose column equilibrated and eluted with 0.03 M citrate, pH 5.0. On the right trypsinogen (specific activity, 0.35-0.37). CM-cellulose column equilibrated and eluted with pH 6.0 buffer 0.015 M in citrate and 10 < M in DFP. Ordinates and abscissas are the same as in Figs. 9 and 10. Solid line, activity. Dotted line, proteins.

Fig. 16. Electrophoretic separation of the lipase and esterase activities of porcine pancreas (146, 147). Starch columns equilibrated with 0.025 M acetate buffer, pH 5.25. The activities of the fractions have been determined (o) on emulsions of triolein and tributyrin (black circles), methyl oleate, methyl laurate, and p-nitro-phenyllaurate (black triangles), (b) On solutions of methyl butyrate and p-nitro-phenylacetate (crosses). White circles and dotted line, protein background. Figures along the first peak give the specific activity (lipase) of some fractions, determined against triolein emulsion. Ordinates and abscissas are the same as in Fig. 14.

Mass analysis of peptide fragments frx>m a protein of "known sequence is the method of choice for speed and accuracy. Howevo, at least three major options are available for mass analysis, each varying in their merits. The three most widely used techniques are LSIMS, M/JLDI-TOF-MS, and ESI-MS. In LSIMS and MALDI, individual peptide fractions are analyzed by mixing with a matrix followed by ionization and mass analysis, while in ESI the samples can be separated on-line by LC, eliminating the need fcH individual p collecticMi. In this report we compare each of the techniques for the analysis of the heavy and light chains of the anti-CEA antibody CEA.11 H5 (1). 

Bergfors, T. M. 1999. Protein Crystallization Techniques, Strategies and Tips. International University Line, La Jolla, CA. 

During the preparation of C protein. Offer et al. (1973) obtained F protein (121 kDa) as a by-product. Miyahara et al. (1980) characterized F protein and showed its binding to myosin. The H protein (74 kDa) has been purified and its specific location closer to the M line than to the C-protein zone has been shown by Yamamoto (1984). The I protein (50 kDa), which inhibits the ATPase activity of myosin, is localized in the edge regions of the A band, but is easily translocated to near the Z lines in aged myofibrils (Ohashi and Maruyama, 1985). The structural roles of F, H, and I proteins have not yet been clarified. 

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