Tropomyosin extractability

Fig. 29. Variation of su, X 10 of carp tropomyosin and nucleotropomyosin with concentration. Lower curve tropomyosin extracted at / < 0.6 soluble at low ionic strength. Median curve nucleotropomyosin. Upper curve carp myosin (see Fig. 26). The two circles correspond to rabbit tropomyosin (bailey, gut-FREUND, and OGSTON, 1948). (after hamoib, 1951a)...

Tropomyosin extracted at ij. 1, soluble at low ionic strength. When the mixture of Fig. 15 is diluted to p 0.05, tropomyosin can be isolated from the supernatant by the usual procedure. In this case, however, the acidification has been carried out only as far as pH 5.5 instead of 4.6. The fraction examined thus corresponds to a part of the tropomyosin soluble at low ionic strength present in the extracts. Its electrophoretic picture is similar to that of Fig. 13. When the preparation is precipitated at pH 4.6 and at relatively high salt concentration (p 1), it is profoundly transformed the fast electrophoretic component which corresponds to Bailey s tropomyosin increases notably at the expense of the slow one. The same transformation is observed by ultracentrifugation components sedimenting at rates varying between those of tropomyosin and nucleotropomyosin become less important while the slow tropomyosin peak increases (Fig. 30). As the nucleic acid present in the preparations does not precipitate with tropomyosin at p 1 and pH 4.6, the question arises if this transformation is not due to its removal. As the amounts present are. 

Fig. 30. Ultraoentrifugal patterns of the carp tropomyosin extracted at /i 1 soluble at low ionic strength before (full line) and after (broken line) precipitation at M 1 and pH 4.6 (after hamoir, 1955).

Tropomyosin extract prepared according to Bailey (1948) contains proteins of the myogen group and denatured G-actin (Dubuisson, 1950e). For complete purification a series of precipitations and crystallization are necessary (Bailey, 1948). 

Fig. 24. Electronmicrograph of crystals formed from 2 molar CaClj extracts o( Mercenaria mercenaria following removal of salt. Negatively stained with Kf uranyl acetate (x 50000). A characteristic staining pattern is developed with well-defined periodicities. This material represents the MM. The crystals and staining pattern closely resemble tropomyosin-troponin paracrys-tals,93>...

Tropomyosin and troponin. Tropomyosin is apparently the most stable of the fish fibrillar proteins during frozen storage. It can be extracted long after actin and myosin become inextract-able however, it does denature gradually (90). 

Fig. 13. Electrophoretic pattern of tropomyosin isolated from an extract obtained at (li 1 and pH 5.2 (see Fig. 11) (after hamoir, 1955).

The various structural proteins of Fig. 11 and 12 may thus be isolated. Their behavior is very similar to the corresponding components of rabbit muscle, but their extractibility is quite different. Fish actomyosin goes easily into solution and rabbit actomyosin dissolves with difficulty. Fish tropomyosin can be selectively extracted at fi 1 and pH 5.2, whereas similar experiments on rabbit muscle reveal only a very small solubilization of this protein (Van de Bergh, unpublished results). Fish muscle is fundamentally similar to other striated muscles but seems to be characterized by a looser association of its structure components. 

A stepwise degradation of the tropomyosin complex thus occurs in the course of the extraction, leading to a progressive increase of solubility and allowing the identification of more or less different fractions. The particle described by Bailey (1948) corresponds to the final step of this transformation. Tropomyosin may actually exist in fish muscle as a ribonucleoprotein but as the ultracentrifugal behavior typical of nucleotropomyosin is ob-... 

FIGURE 3 Silver-stained mini two-dimensional NEPHGE. Sample pig bladder whole muscle extract. Resolved pi values range from 4.7 (tropomyosin) to 9.82 (mammalian calponin a-variant). 

Proteins of the contractile apparatus, extractable with concentrated salt solutions (actomyosin, together with tropomyosin and troponin). 

---