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Purification and Characterization of a Phthalate Ester Hydrolyzing Enzyme from Artemia

  • David S. Miller
  • Patricia A. Healy
  • Roger A. Acey
Part of the NATO ASI Series book series (NSSA, volume 174)

Abstract

Previous studies have established the embryotoxicity of various dialkyl phthalate esters on developing Artemia, most notably di-n-butylphthalate (DBP). Hudson and coworkers [1] have reported the partial purification of an enzyme (DBPase) responsible for the hydrolysis of DBP and suggested that the observed embryotoxicity might be the result of DBP preventing DBPase from performing its normal biological function. We now report on the purification of this enzyme to near homogeneity and its partial biochemical characterization. The assay for DBPase involves incubation of appropriate samples with radiolabeled 14C-carbonyl-DBP and quantifying the amount of monobutyl ester found during a 2 hr incubation period. Free swimming nauplii were collected and homogenized in 50 mM Tris, 0.2 mM EDTA, 0.1 mM DTT, pH 7.5 (TED). Cytosol was made 50% (w/v) in ammonium sulfate and stored at −20°C to precipitate the enzyme. The pellet was dissolved in TED, desalted, and fractionated on Bio-Gel P-100, Mr=51,000. Peak fractions were pooled, applied to Bio-Gel DEAE, and eluted with a linear salt gradient (0.0–1.2 M KCl) in TED. Subsequently the eluted DBPase activity was fractionated by high performance liquid chromatography (HPLC) on a TSK-GW 3000 gel permeation column. Electrophoretic analysis on a 12% sodium dodecylsulfate polyacrylamide gel of the preparation revealed two polypeptides, Mr=51,000 and 98,000, respectively. The enzyme has a neutral pH optimum. In addition, the enzyme is unaffected by soybean trypsin inhibitor but its activity is almost completely inhibited by phenylmethyl sulfonylfluoride. Furthermore, when either EDTA or dithiothreitol are excluded from the purification protocol, DBPase activity is significantly diminished.

Keywords

High Performance Liquid Chromatography High Performance Liquid Chromatography Ammonium Sulfate Brine Shrimp Partial Purification 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Reference

  1. 1.
    R. A. Hudson, T. Giancarlo II, C. F. Austerberry and J. C. Bagshaw, Isolation and partial purification of phthalate ester hydrolyzing enzyme(s) from the brine shrimp, Artemia, Toxicol. Letters 10:389 (1982).CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1989

Authors and Affiliations

  • David S. Miller
    • 1
  • Patricia A. Healy
    • 1
  • Roger A. Acey
    • 1
  1. 1.Department of ChemistryCalifornia State UniversityLong BeachUSA

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