Purification and properties of a quinone-dependent p-nitrophenylphosphatase from Clostridium sticklandii

A highly specialized phosphatase that depends on both a quinone (e.g., 2-methyl-1,4-napthoquinone) and a sulfhydryl compound for activity was purified to homogeneity from extracts of Clostridium sticklandii. Selective adsorption to Cibacron Blue-Sepharose 4B followed by elution with p-nitrophenylphosphate was an effective enrichment procedure. An affinity matrix containing vitamin K5 (4-amino-2-methyl-1-naphthol) covalently attached to Sepharose 4B selectively retained the enzyme and was also used in its purification. The only known substate for the enzyme, p-nitrophenylphosphate, is hydrolyzed to equivalent amounts of orthophosphate and p-nitrophenol. Although a protein phosphotyrosine residue seemed a likely candidate as the natural substrate, the enzyme failed to hydrolyze 32P-labeled phosphotyrosine residues in casein, in vinculin, or in denatured glutamine synthetase. Also, free O-phosphotyrosine and numerous phosphate esters that serve as substrates for common phosphomonoesterases were not hydrolyzed. The molecular weight of the native enzyme, estimated by Sephacryl-S-200 gel chromatography, is 27,600. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed a single component with a molecular weight of 28,600. From the amino acid composition, a minimum molecular weight of 28,000 was calculated.