Purification and characterization of a 5′-nucleotidase from Zea mays microsomes

Abstract

A 5′-nucleotidase (EC 3.1.3.5) from Zea mays seedling shoot microsomes has been purified 125-fold to apparent homogeneity. The enzyme is competitively inhibited by cAMP ( K i = 5.2 μM) and is also inhibited by adenosine in a noncompetitive manner ( K i ⪕ 57 μM). The inhibition by adenosine allowed us to distinguish the specific 5′-nucleotidase activity from that of nonspecific acid phosphatase activity in crude tissue homogenates. When an assay base on that observation is used, about half of the total 5′-nucleotidase activity in a crude homogenate is estimated to be associated with the microsomal membranes. The microsomal enzyme has been solubilized and purified. Estimates from gel filtration and from gel electrophoresis in sodium dodecylsulphate suggest that the enzyme is composed of two subunits of M r 24 500 and 25 500. The purified enzyme is specific for nucleoside monophosphates, with the activity assayed with p-nitrophenyl phosphates, α- and β-glycerol phosphates and ribose-5-phosphate ranging from 0 to 3% of that with 5′-AMP. K m values for purine nucleotides are somewhat lower than for pyrimidine nucleotides. Divalent cations had no effect on the enzyme activity. A possible role for the enzyme in nucleotide pool size regulation is discussed.