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Purification and characterization of a troponin C-like phosphodiesterase activator from bovine thyroid
Abstract:
A troponin C-like phosphodiesterase activator from bovine thyroid has been purified to homogeneity. The overall purification was about 9,800-fold with a yield of 8%. Bovine thyroid activator protein is identical in biologic properties to that isolated from bovine brain. They have the same specific activity regarding stimulation of bovine brain cyclic nucleotide phosphodiesterase. Both proteins form a Ca2+-dependent complex with heart muscle troponin I which is stable in 6M urea-polyacrylamide gel and which is similar, but not identical, to the troponin C-troponin I complex. The physiochemical properties of bovine thyroid activator protein are identical with those of bovine brain and other phosphodiesterase activator proteins and are similar to heart muscle and skeletal muscle troponin C as follows: (A) they bind 3-4 exchangeable calcium ions/mol with dissociation constants between 10(-5) and 10(-6) M, (B) they are highly acidic with a high content of aspartic and glutamic acids and isoelectric points of approximately 4.1, (C) these proteins have an unusual ultraviolet absorption spectrum with six discrete maxima between 250 and 284 nm which are characteristic of phenylalanine and tyrosine, and (D) these proteins have a low content of cysteine, histidine, tyrosine and proline. The tryptic peptide maps of bovine thyroid and brain activator protein are very similar. However, despite a very similar amino acid composition, the peptide map of bovine heart muscle troponin C is significantly different from that of the other two proteins. The molecular weight of thyroid and brain activator protein is 16,500, while that of heart troponin C is 18,500. Thyroid and brain activator protein, as well as heart troponin C, appear to undergo significant Ca2+-dependent conformational changes, as measured by the difference in the circular dichroism spectrum and electrophoretic mobility observed in the presence and absence of calcium ion.