Purification and amino acid microsequencing of alkaline phosphodiesterase I from calf kidney

Alkaline phosphodiesterase I was purified from calf kidney following extraction with 0.5% Triton X-100 and then fractionation with a Rotfor cell and high-pressure liquid chromatography. The isoelectric point of alkaline phosphodiesterase I was 5.9. Both the native and the subunit molecular weight of alkaline phosphodiesterase I was determined to be 125 kDa. The N-terminus of its amino acid sequence appeared to be blocked. The immobilized protein, separated by gel electrophoresis and transblotted to a polyvinylidene difluoride membrane, was digested on the membrane by proteases and separated by reverse-phase high-performance liquid chromatography. Eighty one amino acids were sequenced. One of the internal sequences was identical with that of an active-site-containing fragment from calf intestinal enzyme, as reported by Culp et al. (1985).