Purification and properties of native titin.
A procedure has been developed for the extraction and purification of the massive myofibrillar protein titin without exposing it to denaturing conditions. The form of the molecule that has been isolated is soluble at high ionic strength and alkaline pH, but precipitates in low salt or at pH values below 7. Sedimentation velocity experiments indicate that titin is a highly asymmetric molecule with a sedimentation coefficient of 13.4 S. This asymmetry is confirmed by electron microscopy of rotary-shadowed specimens, which shows string-like structures of diameter 40 A and lengths up to 8000 A. Significant differences were observed depending on whether the electron microscope specimens were prepared by spraying or by layering of the titin onto a mica substrate; we tentatively attribute these differences to elasticity in the titin, revealed by the high shearing forces that accompany spraying. In accord with this, the circular dichroism spectrum of titin indicates that its secondary structure is largely random coil, a conformation characteristic of elastic proteins such as elastin. Negative staining of titin again shows long string-like structures, but these can now be seen to have an appearance similar to a string of beads, where the spacing between successive beads is about 40 A. Very similar beaded strings have been observed also associated with negatively stained separated native thick filaments; these are found running alongside the cross-bridge regions and in coils near the filament ends. Since the periodicity of the strings is similar to that of end-filaments, recently identified structures at the tips of thick filaments, it is likely that end-filaments are formed from titin. Titin comprises approximately 9% of the myofibrillar mass, which means that it is the third most abundant protein in muscle. The possible role of titin in forming elastic filaments within myofibrils is discussed.
D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002942:Circular Dichroism; D064211:Connectin; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D009124:Muscle Proteins / Q000302:isolation & purification*; D009210:Myofibrils / Q000032:analysis; D011494:Protein Kinases; D011817:Rabbits; D012995:Solubility; D014461:Ultracentrifugation
D064211:Connectin; D009124:Muscle Proteins; D011494:Protein Kinases