Circular dichroic, infrared and other studies on the protein component of pig brain thromboplastin.
1. A four-step procedure used to isolate the protein component (apoprotein III) of pig brain thromboplastin yielded approximately 25 mg from 500g of brain. 2. In the absence of detergent, apoprotein III had an apparent mol.wt. of 360 000 by gel-filtration, and, after electrophoresis on polyacrylamide gels in the presence of sodium docecyl sulphate, it appeared as a major protein band of mol.wt.59 000, suggesting the existence of polymeric and monomeric forms. 3. Chemical analyses of apoprotein III revealed that hydrophilic and hydrophobic amino acids were present in a ratio of 3:2, together with approx, 9% (w/w) of carbohydrate. 4. The far-u.v.c.d. and i.r. spectral data indicated that, like other membrane proteins, apoprotein III has a high percentage of unordered structure with lesser amounts of alpha and beta-forms. 5. Relipidation of apoprotein III to restore clotting activity caused no extensive alteration in the c.d. and i.r. spectra, indicating that the phospholipid associates with a comparatively small hydrophobic segment. The constrained unordered conformation, which makes the major contribution to the c.d. spectrum, probably forms a separate domain in the aqueous phase. The absence of any increase in the amplitude of both negative c.d. extrema, following relipidation, contrasted with the substantial increase observed in a helix-forming solvent and raises the possibility that the more stable polymeric form of apoprotein III is retained as the active form in the lipid phase. 6. We suggest that as a consequence of cell membrane damage, the recognition and activation of factor VII may involve minor changes of conformation that are dependent upon the flexibility inherent in an unordered secondary structure.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001059:Apoproteins / Q000302:isolation & purification; D001923:Brain Chemistry; D002241:Carbohydrates / Q000032:analysis; D002942:Circular Dichroism; D006358:Hot Temperature; D010743:Phospholipids; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared; D013552:Swine; D013925:Thromboplastin
D000596:Amino Acids; D001059:Apoproteins; D002241:Carbohydrates; D010743:Phospholipids; D013925:Thromboplastin