5?-Nucleotidase: Solubilization, radiochemical analysis, and electrophoresis

Abstract

5'-Nucleotidase (5'-NT, E.C. 3.1.3.5) of cultured human and rodent cells was rendered soluble using the zwitterionic detergent Zwittergent 314. Optimal activity of 5'-NT was obtained when sonicated cells were incubated in solutions containing 0.75% (w/v) Zwittergent. A method was developed for the determination of the activity of 5'-NT in which the unutilized substrate, [14C]-AMP, was precipitated with lanthanum chloride and the soluble [14C]-adenosine was measured by scintillation counting. 5'-NT isozymes were separated using agarose gel electrophoresis and isoelectric focusing in polyacrylamide gel. The zones of enzyme activity were established by precipitation of unutilized [14C]-AMP with LaCl3, removal of soluble [14C)-adenosine by washing gels in water, and autoradiography. The zones of 5'-NT appeared as clear zones on darkened X-ray film. When analyzed by agarose gel electrophoresis, fibroblasts derived from human skin and rat liver produced a single zone of 5'-NT activity. The 5'-NT isozyme of rat cells migrated faster than that of human cells and was easy to distinguish. The presence of detergent in the sample and in the gel enhanced enzymatic activity and improved the separation of the isozymes. Isoelectric focusing resolved 5'-NT of human fibroblasts into two molecular forms, one of which focused in the region of pH 6 and the other at pH5.