Similarity in Ca 2+-induced changes between troponin-C and protein activator of 3′:5′-cyclic nucleotide phosphodiesterase and their tryptic fragments

Abstract

1. 1.|The Ca 2+-dependent protein activator of 3′:5′-cyclic nucleotide phosphodiesterase (3′:5′-cyclic-nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) has been isolated from bovine brain and its properties have been compared with those of troponin-C. It has been shown that bovine brain does not contain troponin-C, but only the protein activator of phosphodiesterase. Its molecular weight is 16 500 as compared with 18 000 for troponin-C. 2. 2.|Similarly to troponin-C, calcium has a pronounced effect on the rate of cleavage of protein activator by trypsin and on the obtained peptide pattern. In the presence of 0.1 mM CaCl 2 two large peptides are formed, essentially resistant to further splitting, whereas upon removal of calcium by chelators a fast cleavage of protein activator into small peptides takes place. 3. 3.|Similarly to troponin-C, tyrosine fluorescence intensity of protein activator is markedly enhanced in the presence of Ca 2+. The apparent binding constant for Ca 2+ calculated from the transition midpoint of fluorescence changes is about 1 · 10 7 M −1. During digestion of protein activator with trypsin in the presence of CaCl 2 essentially no change of fluorescence intensity takes place and the subsequent decrease upon removal of calcium is reversible. 4. 4.|Two large peptides that accumulate during digestion by trypsin in the presence of Ca 2+ of both troponin-C and protein activator have been isolated and their properties have been compared with those of corresponding parent molecules. One of the peptides obtained from both proteins retains the ability of the intact molecular to change the mobility on the polyacrylamide gel electrophoresis in the presence of urea depending on concentration of Ca 2+ and to interacted with the inhibitory component of troponin in the presence of Ca 2+. This peptide also shows Ca 2+-dependent fluorescence changes, characteristic for parent molecule. 5. 5.|On the basis of similarity between the two proteins and their tryptic fragments it is suggested that in the presence of Ca 2+ protein activator is cleaved in the same area as troponin-C. The peptide obtained from protein activator, which shows Ca 2+-dependent changes characteristics for intact molecules, corresponds to the troponin-C peptide containing calcium binding sites 3 and 4. All these results furnish new pieces of evidence for a pronounced structural similarity between troponin-C and protein activator of cyclic nucleotide phosphodiesterase.