A Calcium-Citro-Phosphate Technique for the Histochemical Localization of Myosin ATPase

Abstract

A simple and sensitive calcium precipitation method is described for the histochemical demonstration of myosin ATPase in striated muscle. In this technique inorganic pyrophosphate is incorporated in the fixative to protect the sites of myosin ATPase activity, thus minimizing enzymatic inactivation during fixation. The incubation medium used contains Ca⁺⁺ (70 mM) as the capturing agent, ATP (4 mM) as substrate, citric acid (7.5 mM) and gelatin. During incubation, the enzyme catalyzes the hydrolysis of ATP to ADP and inorganic phosphate. The liberated phosphate is precipitated as “calcium-citro-phosphate”. The latter is then converted to black cobalt sulphide. The calcium-citro-phosphate is rapidly formed during incubation (within 10 min) and can not be washed off the tissue sections.