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.
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D009597:4-Nitrophenylphosphatase / Q000302:isolation & purification*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D016227:Benzoquinones; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification*; D019000:Phosphotyrosine; D011809:Quinones / Q000494:pharmacology*; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000596:Amino Acids; D016227:Benzoquinones; D011809:Quinones; D019000:Phosphotyrosine; C004532:quinone; D000249:Adenosine Monophosphate; D014443:Tyrosine; D000244:Adenosine Diphosphate; D010744:Phosphoric Monoester Hydrolases; D009597:4-Nitrophenylphosphatase