Purification and Characterization of Acidic Adenylate Kinase in Porcine Heart

Porcine heart adenylate kinase can be separated into acidic and basic components by electrofocusing, and the latter is identical with the skeletal muscle adenylate kinase. The acidic adenylate kinase was purified approximately 3600‐fold from porcine heart extract in an overall yield of 59% by DEAE‐cellulose chromatography, substrate elution from a column of phosphocellulose, affinity chromatography on a column of blue dextran‐Sepharose 4B, and gel filtration. The purified acidic enzyme had a specific activity of 1100 units per mg of enzyme, and was found to be a homogeneous protein in all the procedures used, i.e. disc and sodium dodecylsulfate electrophoresis, and gel filtration. However, electrophoretic variants were observed on isoelectrofocusing and this variance may depend on the formation of protein‐ampholine complexes. The purified acidic enzyme had a molecular weight of 26 900 and an optimum pH of 5.8 in the forward reaction (ATP + AMP → 2ADP). The amino acid composition of the acidic enzyme was found to be 22 aspartic acid or asparagine, 13 threonine, 14 serine, 29 glutamic acid or glutamine, 17 proline, 17 glycine; 22 alanine, 4 half‐cystine, 13 valine, 8 methionine, 13 isoleucine, 24 leucine, 4 tyrosine, 7 phenylalanine, 5 histidine, 19 lysine, 14 arginine and no tryptophan, totaling 245 residues. The enzyme had two – SS – bonds but no free – SH, and was not inhibited at all by 5,5′‐dithio‐bis(2‐nitrobenzoic acid). From these results, it may be concluded that the acidic adenylate kinase of porcine heart is similar to liver mitochondrial adenylate kinase.